Khosrow Adeli

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

The primary genetic, environmental, and metabolic factors responsible for causing insulin resistance and pancreatic β-cell failure and the precise sequence of events leading to the development of type 2 diabetes are not yet fully understood. Abnormalities of triglyceride storage and lipolysis in insulin-sensitive tissues are an early manifestation of conditions characterized by insulin resistance and are detectable before the development of postprandial or fasting hyperglycemia. Increased free fatty acid (FFA) flux from adipose tissue to nonadipose tissue, resulting from abnormalities of fat metabolism, participates in and amplifies many of the fundamental metabolic derangements that are characteristic of the insulin resistance syndrome and type 2 diabetes. It is also likely to play an important role in the progression from normal glucose tolerance to fasting hyperglycemia and conversion to frank type 2 diabetes in insulin resistant individuals. Adverse metabolic consequences of increased FFA flux, to be discussed in this review, are extremely wide ranging and include, but are not limited to: 1) dyslipidemia and hepatic steatosis, 2) impaired glucose metabolism and insulin sensitivity in muscle and liver, 3) diminished insulin clearance, aggravating peripheral tissue hyperinsulinemia, and 4) impaired pancreatic β-cell function. The precise biochemical mechanisms whereby fatty acids and cytosolic triglycerides exert their effects remain poorly understood. Recent studies, however, suggest that the sequence of events may be the following: in states of positive net energy balance, triglyceride accumulation in “fat-buffering” adipose tissue is limited by the development of adipose tissue insulin resistance. This results in diversion of energy substrates to nonadipose tissue, which in turn leads to a complex array of metabolic abnormalities characteristic of insulin-resistant states and type 2 diabetes. Recent evidence suggests that some of the biochemical mechanisms whereby glucose and fat exert adverse effects in insulin-sensitive and insulin-producing tissues are shared, thus implicating a diabetogenic role for energy excess as a whole. Although there is now evidence that weight loss through reduction of caloric intake and increase in physical activity can prevent the development of diabetes, it remains an open question as to whether specific modulation of fat metabolism will result in improvement in some or all of the above metabolic derangements or will prevent progression from insulin resistance syndrome to type 2 diabetes.

Obesity and type 2 diabetes are occurring at epidemic rates in the United States and many parts of the world. The "obesity epidemic" appears to have emerged largely from changes in our diet and reduced physical activity. An important but not well-appreciated dietary change has been the substantial increase in the amount of dietary fructose consumption from high intake of sucrose and high fructose corn syrup, a common sweetener used in the food industry. A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, perturbs glucose metabolism and glucose uptake pathways, and leads to a significantly enhanced rate of de novo lipogenesis and triglyceride (TG) synthesis, driven by the high flux of glycerol and acyl portions of TG molecules from fructose catabolism. These metabolic disturbances appear to underlie the induction of insulin resistance commonly observed with high fructose feeding in both humans and animal models. Fructose-induced insulin resistant states are commonly characterized by a profound metabolic dyslipidemia, which appears to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles. Thus, emerging evidence from recent epidemiological and biochemical studies clearly suggests that the high dietary intake of fructose has rapidly become an important causative factor in the development of the metabolic syndrome. There is an urgent need for increased public awareness of the risks associated with high fructose consumption and greater efforts should be made to curb the supplementation of packaged foods with high fructose additives. The present review will discuss the trends in fructose consumption, the metabolic consequences of increased fructose intake, and the molecular mechanisms leading to fructose-induced lipogenesis, insulin resistance and metabolic dyslipidemia.

Pediatric healthcare is critically dependent on the availability of accurate and precise laboratory biomarkers of pediatric disease, and on the availability of reference intervals to allow appropriate clinical interpretation. The development and growth of children profoundly influence normal circulating concentrations of biochemical markers and thus the respective reference intervals. There are currently substantial gaps in our knowledge of the influences of age, sex, and ethnicity on reference intervals. We report a comprehensive covariate-stratified reference interval database established from a healthy, nonhospitalized, and multiethnic pediatric population.Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer.Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers.The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

The metabolic syndrome is a constellation of common metabolic disorders that is associated with cardiovascular disease. Insulin resistance has a central role in the pathophysiology of metabolic syndrome. It is now commonly accepted that chronic inflammation associated with visceral obesity induces insulin resistance in the liver. Chronic inflammation is characterized by the production of abnormal adipokines and cytokines such as TNF-α, FFA, IL-1, IL-6, leptin and resistin. These factors inhibit insulin signalling in hepatocytes by activating SOCS proteins, several kinases such as JNK, IKK-β and PKC and protein tyrosine phosphatases such as PTP1B and PTEN, that in turn impair insulin signalling at insulin receptor and insulin receptor substrate (IRS) level. Hepatic insulin resistance in turn causes impaired suppression of glucose production by insulin in hepatocytes leading to hyperglycemia. An important and early complication of hepatic insulin resistance is the induction of hepatic VLDL production, via changes in the rate of apoB synthesis and degradation and de novo lipogenesis, or increased FFA flux from adipose tissue into the liver. Insulin resistance also stimulates the production of CRP and PAI-1, both markers of an inflammatory state. All metabolic abnormalities related to hepatic insulin resistance have been shown to directly or indirectly promote atherosclerosis. Hyperglycemia induces a series of alterations including endothelial dysfunction, cellular proliferation, changes in extracellular matrix conformation and impairment of LDL receptor-mediated uptake decreasing the in vivo clearance of LDL. Small dense LDLs associated with high circulating VLDL have higher affinity to the intimal proteoglycans leading to the penetration of more LDL particles into the arterial wall. CRP can also accelerate atherosclerosis by increasing the expression of PAI-1 and adhesion molecules in endothelial cells, inhibition of nitric oxide formation and increasing LDL uptake into macrophages. Overall, growing evidence suggests that hepatic insulin resistance is sufficient to induce several components of the metabolic syndrome and promote progression to cardiovascular disease. Many unresolved questions remain however on the molecular and cellular mechanisms that trigger hepatic insulin resistance and promote the development of clinical metabolic syndrome.

As dietary exposure to fructose has increased over the past 40 years, there is growing concern that high fructose consumption in humans may be in part responsible for the rising incidence of obesity worldwide. Obesity is associated with a host of metabolic challenges, collectively termed the metabolic syndrome. Fructose is a highly lipogenic sugar that has profound metabolic effects in the liver and has been associated with many of the components of the metabolic syndrome (insulin resistance, elevated waist circumference, dyslipidemia, and hypertension). Recent evidence has also uncovered effects of fructose in other tissues, including adipose tissue, the brain, and the gastrointestinal system, that may provide new insight into the metabolic consequences of high-fructose diets. Fructose feeding has now been shown to alter gene expression patterns (such as peroxisome proliferator-activated receptor-γ coactivator-1α/β in the liver), alter satiety factors in the brain, increase inflammation, reactive oxygen species, and portal endotoxin concentrations via Toll-like receptors, and induce leptin resistance. This review highlights recent findings in fructose feeding studies in both human and animal models with a focus on the molecular and biochemical mechanisms that underlie the development of insulin resistance, hepatic steatosis, and the metabolic syndrome.

Insulin resistant states are commonly associated with an atherogenic dyslipidemia that contributes to significantly higher risk of atherosclerosis and cardiovascular disease. Indeed, disorders of carbohydrate and lipid metabolism co-exist in the majority of subjects with the “metabolic syndrome” and form the basis for the definition and diagnosis of this complex syndrome. The most fundamental defect in these patients is resistance to cellular actions of insulin, particularly resistance to insulin-stimulated glucose uptake. Insulin insensitivity appears to cause hyperinsulinemia, enhanced hepatic gluconeogenesis and glucose output, reduced suppression of lipolysis in adipose tissue leading to a high free fatty acid flux, and increased hepatic very low density lipoprotein (VLDL) secretion causing hypertriglyceridemia and reduced plasma levels of high density lipoprotein (HDL) cholesterol. Although the link between insulin resistance and dysregulation of lipoprotein metabolism is well established, a significant gap of knowledge exists regarding the underlying cellular and molecular mechanisms. Emerging evidence suggests that insulin resistance and its associated metabolic dyslipidemia result from perturbations in key molecules of the insulin signaling pathway, including overexpression of key phosphatases, downregulation and/or activation of key protein kinase cascades, leading to a state of mixed hepatic insulin resistance and sensitivity. These signaling changes in turn cause an increased expression of sterol regulatory element binding protein (SREBP) 1c, induction of de novo lipogensis and higher activity of microsomal triglyceride transfer protein (MTP), which together with high exogenous free fatty acid (FFA) flux collectively stimulate the hepatic production of apolipoprotein B (apoB)-containing VLDL particles. VLDL overproduction underlies the high triglyceride/low HDL-cholesterol lipid profile commonly observed in insulin resistant subjects.

Glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors attenuate postprandial lipaemia through mechanisms that remain unclear. As dyslipidaemia is a contributing risk factor for cardiovascular disease in type 2 diabetes, we examined the mechanisms linking pharmacological and physiological regulation of GLP-1 action to control of postprandial lipid metabolism.Postprandial lipid synthesis and secretion were assessed in normal and fructose-fed hamsters and in wild-type mice that were treated with or without sitagliptin. Apolipoprotein B-48 (ApoB-48) synthesis and secretion were also examined in primary enterocyte cultures. The importance of exogenous vs endogenous GLP-1R signalling for regulation of intestinal lipoprotein synthesis and secretion was assessed in mice and hamsters treated with the GLP-1R agonist exendin-4, the GLP-1R antagonist exendin(9-39) and in Glp1r (+/+) vs Glp1r (-/-) mice.Sitagliptin decreased fasting plasma triacylglycerol, predominantly in the VLDL fraction, as well as postprandial triacylglycerol-rich lipoprotein (TRL)-triacylglycerol, TRL-cholesterol and TRL-ApoB-48 in hamsters and mice. GLP-1R activation with exendin-4 alone also decreased plasma and TRL-ApoB-48 in hamsters and mice, and reduced secretion of ApoB-48 in hamster enterocyte cultures. Conversely, blockade of endogenous GLP-1R signalling by the antagonist exendin(9-39) or genetic elimination of GLP-1R signalling in Glp1r (-/-) mice enhanced TRL-ApoB-48 secretion in vivo. Co-administration of exendin(9-39) also abolished the hypolipidaemic effect of sitagliptin.Potentiation of endogenous incretin action via DPP-4 inhibition or pharmacological augmentation of GLP-1R signalling reduces intestinal secretion of triacylglycerol, cholesterol and ApoB-48. Moreover, endogenous GLP-1R signalling is essential for the control of intestinal lipoprotein biosynthesis and secretion.

We investigated the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the resistance of dyslipidemic hamsters to statin-induced LDL-cholesterol (LDL-C) reduction and the molecular mechanism by which statins modulated PCSK9 gene expression in vivo. We utilized the fructose diet-induced dyslipidemic hamsters as an in vivo model and rosuvastatin to examine its effects on liver PCSK9 and LDL receptor (LDLR) expression and serum lipid levels. We showed that rosuvastatin induced PCSK9 mRNA to a greater extent than LDLR mRNA in the hamster liver. The net result was that hepatic LDLR protein level was reduced. This correlated closely with an increase in serum LDL-C with statin treatment. More importantly, we demonstrated that in addition to an increase in sterol response element binding protein 2 (SREBP2) expression, rosuvastatin treatment increased the liver expression of hepatocyte nuclear factor 1 alpha (HNF1alpha), the newly identified key transactivator for PCSK9 gene expression. Our study suggests that the inducing effect of rosuvastatin on HNF1alpha is likely a underlying mechanism accounting for the higher induction of PCSK9 than LDLR because of the utilization of two transactivators (HNF1alpha and SREBP2) in PCSK9 transcription versus one (SREBP2) in LDLR transcription. Thus, the net balance is in favor of PCSK9-induced degradation of LDLR in the hamster liver, abrogating the effect of rosuvastatin on LDL-C lowering.

Laboratory investigations provide physicians with objective data to aid in disease diagnosis, clinical decision making, and patient follow up. Clinical interpretation of laboratory test results relies heavily on the availability of appropriate population-based reference intervals (i.e. normative values) or decision limits developed through clinical outcome studies. Although reference intervals are fundamental to accurate laboratory test interpretation, and thus critically important to healthcare, the need for sound evidence-based reference intervals has been largely overlooked, particularly in the pediatric population. In the field of pediatric laboratory medicine, accurate age- and sex-specific reference intervals established using samples from healthy children and adolescents have not been readily available, forcing many clinical laboratories to report adult reference intervals with pediatric test results. When pediatric reference intervals are available, they have often been established with a small sample size, inpatient or outpatient samples, outdated methodologies, and/or inappropriate statistical procedures. To address these unacceptable limitations, several national and global initiatives have begun to close the critical evidence gaps in pediatric reference intervals. Notably, the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) has made significant strides towards improving pediatric healthcare in Canada and globally. The present report is a white paper summarizing CALIPER, and provides a comprehensive compendium of the data generated through this project over the past decade as a single resource for clinical laboratory specialists, clinicians, and other healthcare workers. CALIPER launched an outreach campaign in 2008 to recruit healthy community children and adolescents, and developed a robust statistical algorithm, in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines, to develop accurate age- and sex-specific pediatric reference intervals. The first CALIPER direct reference interval study was published in 2012, with age- and sex-specific reference intervals reported for 40 common biochemical markers. To date, CALIPER has collected health information and blood samples from over 9700 community children and adolescents, and has established a comprehensive database of age- and sex-specific reference intervals for over 100 biomarkers of pediatric disease. CALIPER has also performed a series of transference and verification studies to expand the applicability of the CALIPER database to five major analytical platforms, including Abbott, Beckman, Ortho, Roche, and Siemens. Through novel knowledge translation initiatives, the CALIPER Reference Interval Database has been made freely available online ( www.caliperproject.ca ) as well as on a mobile application (CALIPER Reference App), and it is used by clinical laboratories across Canada, the United States, and globally. In addition to establishing this comprehensive pediatric reference interval database, CALIPER has also performed a series of sub-studies, including examining how reference intervals are affected by pre-analytical factors (i.e. sample stability at specific storage conditions, fasting status and time of sample collection), biological variation (i.e. intraindividual and interindividual biological variation, reference change values), and ethnicity and pubertal development stage. In this white paper, extensive tables of pediatric reference intervals are provided for easy reference for clinical laboratories worldwide. All data reported have been published in over 20 peer reviewed publications and are also available through the CALIPER Reference Interval Database as well as the CALIPER Reference App for mobile devices.

With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D3 (25(OH)D3). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D3 that has been shown to contribute significantly to 25(OH)D3 concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D3 methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D3 in infant, pediatric, and adult populations. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.

The global epidemiology of coronavirus disease 2019 (COVID-19) suggests a wide spectrum of clinical severity, ranging from asymptomatic to fatal. Although the clinical and laboratory characteristics of COVID-19 patients have been well characterized, the pathophysiological mechanisms underlying disease severity and progression remain unclear. This review highlights key mechanisms that have been proposed to contribute to COVID-19 progression from viral entry to multisystem organ failure, as well as the central role of the immune response in successful viral clearance or progression to death.

Abstract The global coronavirus disease 2019 (COVID-19) has presented major challenges for clinical laboratories, from initial diagnosis to patient monitoring and treatment. Initial response to this pandemic involved the development, production, and distribution of diagnostic molecular assays at an unprecedented rate, leading to minimal validation requirements and concerns regarding their diagnostic accuracy in clinical settings. In addition to molecular testing, serological assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now becoming available from numerous diagnostic manufacturers. In both cases, the lack of peer-reviewed data and regulatory oversight, combined with general misconceptions regarding their appropriate use, have highlighted the importance of laboratory professionals in robustly validating and evaluating these assays for appropriate clinical use. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 has been established to synthesize up-to-date information on the epidemiology, pathogenesis, and laboratory diagnosis and monitoring of COVID-19, as well as to develop practical recommendations on the use of molecular, serological, and biochemical tests in disease diagnosis and management. This review summarizes the latest evidence and status of molecular, serological, and biochemical testing in COVID-19 and highlights some key considerations for clinical laboratories operating to support the global fight against this ongoing pandemic. Confidently this consolidated information provides a useful resource to laboratories and a reminder of the laboratory’s critical role as the world battles this unprecedented crisis.

Gut microbiota play an important role in maintaining intestinal health and are involved in the metabolism of carbohydrates, lipids, and amino acids. Recent studies have shown that the central nervous system (CNS) and enteric nervous system (ENS) can interact with gut microbiota to regulate nutrient metabolism. The vagal nerve system communicates between the CNS and ENS to control gastrointestinal tract functions and feeding behavior. Vagal afferent neurons also express receptors for gut peptides that are secreted from enteroendocrine cells (EECs), such as cholecystokinin (CCK), ghrelin, leptin, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1 (GLP-1), and 5-hydroxytryptamine (5-HT; serotonin). Gut microbiota can regulate levels of these gut peptides to influence the vagal afferent pathway and thus regulate intestinal metabolism via the microbiota-gut-brain axis. In addition, bile acids, short-chain fatty acids (SCFAs), trimethylamine-N-oxide (TMAO), and Immunoglobulin A (IgA) can also exert metabolic control through the microbiota-gut-liver axis. This review is mainly focused on the role of gut microbiota in neuroendocrine regulation of nutrient metabolism via the microbiota-gut-brain-liver axis.

Objectives: To summarize new knowledge surrounding the physiological activity of tocotrienol, a natural analogue of tocopherol. Results: The biological activity of vitamin E has generally been associated with its well-defined antioxidant property, specifically against lipid peroxidation in biological membranes. In the vitamin E group, α-tocopherol is considered to be the most active form. However, recent research has suggested tocotrienol to be a better antioxidant. Moreover, tocotrienol has been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein B and lipoprotein(a) plasma levels. In addition, tocotrienol has been suggested to have an anti-thrombotic and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer. Conclusion: The physiological activities of tocotrienol suggest it to be superior than α-tocopherol in many situations. Hence, the role of tocotrienol in the prevention of cardiovascular disease and cancer may have significant clinical implications. Additional studies on its mechanism of action, as well as, long-term intervention studies, are needed to clarify its function. From the pharmacological point-of-view, the current formulation of vitamin E supplements, which is comprised mainly of α-tocopherol, may be questionable.

A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster, was employed to investigate the mechanisms mediating the overproduction of very low density lipoprotein (VLDL) in the insulin resistant state. Fructose feeding for a 2-week period induced significant hypertriglyceridemia and hyperinsulinemia, and the development of whole body insulin resistance was documented using the euglycemic-hyperinsulinemic clamp technique. In vivo Triton WR-1339 studies showed evidence of VLDL-apoB overproduction in the fructose-fed hamster. Fructose feeding induced a significant increase in cellular synthesis and secretion of total triglyceride (TG) as well as VLDL-TG by primary hamster hepatocytes. Increased TG secretion was accompanied by a 4.6-fold increase in VLDL-apoB secretion. Enhanced stability of nascent apoB in fructose-fed hepatocytes was evident in intact cells as well as in a permeabilized cell system. Analysis of newly formed lipoprotein particles in hepatic microsomes revealed significant differences in the pattern and density of lipoproteins, with hepatocytes derived from fructose-fed hamsters having higher levels of luminal lipoproteins at a density of VLDL versus controls. Immunoblot analysis of the intracellular mass of microsomal triglyceride transfer protein, a key enzyme involved in VLDL assembly, showed a striking 2.1-fold elevation in hepatocytes derived from fructose-fed versus control hamsters. Direct incubation of hamster hepatocytes with various concentrations of fructose failed to show any direct stimulation of its intracellular stability or extracellular secretion, further supporting the notion that the apoB overproduction in the fructose-fed hamster may be related to the fructose-induced insulin resistance in this animal model. In summary, hepatic VLDL-apoB overproduction in fructose-fed hamsters appears to result from increased intracellular stability of nascent apoB and an enhanced expression of MTP, which act to facilitate the assembly and secretion of apoB-containing lipoprotein particles. A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster, was employed to investigate the mechanisms mediating the overproduction of very low density lipoprotein (VLDL) in the insulin resistant state. Fructose feeding for a 2-week period induced significant hypertriglyceridemia and hyperinsulinemia, and the development of whole body insulin resistance was documented using the euglycemic-hyperinsulinemic clamp technique. In vivo Triton WR-1339 studies showed evidence of VLDL-apoB overproduction in the fructose-fed hamster. Fructose feeding induced a significant increase in cellular synthesis and secretion of total triglyceride (TG) as well as VLDL-TG by primary hamster hepatocytes. Increased TG secretion was accompanied by a 4.6-fold increase in VLDL-apoB secretion. Enhanced stability of nascent apoB in fructose-fed hepatocytes was evident in intact cells as well as in a permeabilized cell system. Analysis of newly formed lipoprotein particles in hepatic microsomes revealed significant differences in the pattern and density of lipoproteins, with hepatocytes derived from fructose-fed hamsters having higher levels of luminal lipoproteins at a density of VLDL versus controls. Immunoblot analysis of the intracellular mass of microsomal triglyceride transfer protein, a key enzyme involved in VLDL assembly, showed a striking 2.1-fold elevation in hepatocytes derived from fructose-fed versus control hamsters. Direct incubation of hamster hepatocytes with various concentrations of fructose failed to show any direct stimulation of its intracellular stability or extracellular secretion, further supporting the notion that the apoB overproduction in the fructose-fed hamster may be related to the fructose-induced insulin resistance in this animal model. In summary, hepatic VLDL-apoB overproduction in fructose-fed hamsters appears to result from increased intracellular stability of nascent apoB and an enhanced expression of MTP, which act to facilitate the assembly and secretion of apoB-containing lipoprotein particles. very low density lipoprotein apolipoprotein B cytoskeletal buffer free fatty acids low density lipoprotein microsomal triglyceride transfer protein polyacrylamide gel electrophoresis triglyceride Insulin resistance is an extremely common pathophysiological trait that is implicated in the development of a number of important human diseases including Type 2 diabetes, atherosclerosis, hypertension, and dyslipidemia (1.Moller D.E. Flier J.S. N. Engl. J. Med. 1991; 325: 938-948Crossref PubMed Scopus (768) Google Scholar, 2.Reaven G.M. Diabetes. 1988; 37: 1595-1607Crossref PubMed Google Scholar). Many studies have suggested that insulin resistance may be a factor in causing dyslipidemia (3.Garg A. Helderman J.H. Koffler M. 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Biochem. J. 1989; 261: 83-88Crossref PubMed Scopus (27) Google Scholar), primary rat hepatocytes (18.Patsch W. Franz S. Schonfeld G. J. Clin. Invest. 1983; 71: 1161-1174Crossref PubMed Scopus (100) Google Scholar, 19.Sparks C.E. Sparks J.D. Bolognino M. Salhanick A. Strumph P.S. Amatruda J.M. Metabolism. 1986; 35: 1128-1136Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 20.Sparks J.D. Sparks C.E. J. Biol. Chem. 1990; 265: 8854-8862Abstract Full Text PDF PubMed Google Scholar), human hepatocytes (21.Salhanick A.I. Schwartz S.I. Amatruda J.M. Metabolism. 1991; 40: 275-279Abstract Full Text PDF PubMed Scopus (39) Google Scholar), as well as in human subjects in vivo (22.Lewis G.F. Uffelman K.D. Szeto L.W. Steiner G. Diabetes. 1993; 42: 833-842Crossref PubMed Scopus (0) Google Scholar, 23.Lewis G.F. Uffelman K.D. Szeto L.W. Weller B. Steiner G. J. Clin. Invest. 1995; 95: 158-166Crossref PubMed Google Scholar) (reviewed in Refs. 15.Sparks J.D. Sparks C.E. Biochim. Biophys. 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Biol. 1997; 17: 1454-1464Crossref PubMed Scopus (166) Google Scholar, 26.Malmstrom R. Packard C.J. Caslake M. Bedford D. Stewart P. Yki-Jarvinen H. Shepherd J. Taskinen M.R. Diabetes. 1998; 47: 779-787Crossref PubMed Scopus (161) Google Scholar). Primary rat hepatocytes, incubated in vitro with high concentrations of insulin for 3 days, no longer respond to insulin suppression of VLDL apoB secretion, and secrete higher basal levels of VLDL-apoB (27.Bjornsson O.G. Duerden J.M. Bartlett S.M. Sparks J.D. Sparks C.E. Gibbons G.F. Biochem. J. 1992; 281: 381-386Crossref PubMed Scopus (59) Google Scholar). A similar resistance to the acute suppressive effects of insulin has been observed in HepG2 cells (28.Dashti N. Williams D.L. Alaupovic P. J. Lipid Res. 1989; 30: 1365-1373Abstract Full Text PDF PubMed Google Scholar). Secretion of VLDL by hepatocytes from hypertriglyceridemic and hyperinsulinemic Zucker fatty rats (fa/fa), is also resistant to the inhibitory effect of insulin (28.Dashti N. Williams D.L. Alaupovic P. J. Lipid Res. 1989; 30: 1365-1373Abstract Full Text PDF PubMed Google Scholar, 29.Sparks J.D. Sparks C.E. Biochem. Biophys. Res. Commun. 1994; 205: 417-422Crossref PubMed Scopus (40) Google Scholar). Insulin regulates hepatic synthesis and secretion of apoB, either directly or indirectly, by its effects on lipid availability (15.Sparks J.D. Sparks C.E. Biochim. Biophys. Acta. 1994; 1215: 9-32Crossref PubMed Scopus (198) Google Scholar). Acute insulin exposure reduces the synthesis of apoB in cultured hepatocytes (20.Sparks J.D. Sparks C.E. J. Biol. Chem. 1990; 265: 8854-8862Abstract Full Text PDF PubMed Google Scholar) and increases the rate of apoB degradation (20.Sparks J.D. Sparks C.E. J. Biol. Chem. 1990; 265: 8854-8862Abstract Full Text PDF PubMed Google Scholar). Studies in our laboratory, using cell-free translation systems, have shown that insulin attenuates the rate of apoB mRNA translation (30.Theriault A. Cheung R. Adeli K. Clin. Biochem. 1992; 25: 321-323Crossref PubMed Scopus (23) Google Scholar, 31.Adeli K. Theriault A. Biochem. Cell Biol. 1992; 70: 1301-1312Crossref PubMed Scopus (55) Google Scholar). It has also been suggested that apoB availability may become a limiting factor in VLDL assembly and secretion in insulin-treated hepatocytes (32.Wiggins D. Gibbons G.F. Biochem. J. 1992; 284: 457-462Crossref PubMed Scopus (201) Google Scholar). Recent studies by Sparks and co-workers (33.Sparks J.D. Phung T.L. Bolognino M. Sparks C.E. Biochem. J. 1996; 313: 567-574Crossref PubMed Scopus (81) Google Scholar, 34.Phung T.L. Roncone A. de Mesy Jensen K.L. Sparks C.E. Sparks J.D. J. Biol. Chem. 1997; 272: 30693-30702Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) have suggested that insulin inhibits apoB secretion through activation of phosphoinositide 3-kinase. Phosphoinositide 3-kinase activity which phosphorylates phosphoinositol in the 3′-position of the inositol ring (35.Whitman M. Downes C.P. Keeler M. Keller T. Cantley L. Nature. 1988; 332: 644-646Crossref PubMed Scopus (740) Google Scholar) appears to be necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes (33.Sparks J.D. Phung T.L. Bolognino M. Sparks C.E. Biochem. J. 1996; 313: 567-574Crossref PubMed Scopus (81) Google Scholar, 34.Phung T.L. Roncone A. de Mesy Jensen K.L. Sparks C.E. Sparks J.D. J. Biol. Chem. 1997; 272: 30693-30702Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Insulin acts by causing the activation and localization of phosphoinositide 3-kinase to the site of apoB synthesis (34.Phung T.L. Roncone A. de Mesy Jensen K.L. Sparks C.E. Sparks J.D. J. Biol. Chem. 1997; 272: 30693-30702Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In the present study, we have employed a new animal model of chronic hyperinsulinemia and insulin resistance, namely, the fructose-fed Syrian golden hamster. The Syrian golden hamster has been used with increasing frequency in recent years to study hepatic lipid metabolism (36.Jackson B. Gee A.N. Martinez-Cayuela M. Suckling K.E. Biochim. Biophys. Acta. 1990; 1045: 21-28Crossref PubMed Scopus (32) Google Scholar, 37.Ontko J.A. Cheng Q. Yamamoto M. J. Lipid Res. 1990; 31: 1983-1992Abstract Full Text PDF PubMed Google Scholar, 38.Hoang V.Q. Botham K.M. Benson G.M. Eldredge E.E. Jackson B. Pearce N. Suckling K.E. Biochim. Biophys. Acta. 1993; 1210: 73-80Crossref PubMed Scopus (20) Google Scholar). Hamsters develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis in response to a modest increase in dietary cholesterol and saturated fat (39.Nistor A. Bulla A. Filip D.A. Radu A. Atherosclerosis. 1987; 68: 159-173Abstract Full Text PDF PubMed Scopus (187) Google Scholar, 40.Sullivan M.P. Cerda J.J. Robbins F.L. Burgin C.W. Beatty R.J. Lab. Anim. Sci. 1993; 43: 575-578PubMed Google Scholar). The hamster has attracted increasing attention as a model for lipoprotein research since its lipoprotein metabolism appears to closely resemble that in humans. The main plasma cholesterol carrier in the hamster is LDL (40.Sullivan M.P. Cerda J.J. Robbins F.L. Burgin C.W. Beatty R.J. Lab. Anim. Sci. 1993; 43: 575-578PubMed Google Scholar, 41.Simionescu N. Sima A. Dobrian A. Tirziu D. Simionescu M. Curr. Top. Pathol. 1993; 87: 1-45PubMed Google Scholar). Furthermore, hamster liver produces VLDL containing only apoB-100 with a density close to that of human VLDL (42.Arbeeny C.M. Meyers D.S. Bergquist K.E. Gregg R.E. J. Lipid Res. 1992; 33: 843-851Abstract Full Text PDF PubMed Google Scholar, 43.Liu G.L. Fan L.M. Redinger R.N. Comp. Biochem. Physiol. A. 1991; 99: 223-228Crossref PubMed Scopus (52) Google Scholar), unlike the rat, which has been used extensively for studies of VLDL metabolism and the effects of insulin resistance, and whose liver secretes both apoB-48 and apoB-100. Carbohydrate induced insulin resistance in rodents has been previously well documented. Reaven and colleagues (44.Wright D.W. Hansen R.I. Mondon C.E. Reaven G.M. Am. J. Clin. Nutr. 1983; 38: 879-883Crossref PubMed Scopus (94) Google Scholar, 45.Sleder J. Chen Y.D. Cully M.D. Reaven G.M. Metabolism. 1980; 29: 303-305Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 46.Tobey T.A. Mondon C.E. Zavaroni I. Reaven G.M. Metabolism. 1982; 31: 608-612Abstract Full Text PDF PubMed Scopus (190) Google Scholar, 47.Zavaroni I. Sander S. Scott S. Reaven G.M. Metabolism. 1980; 29: 970-973Abstract Full Text PDF PubMed Scopus (201) Google Scholar) were among the first groups to use sucrose or fructose feeding to induce insulin resistance in rats. Hamsters can also be made obese, hyperinsulinemic, hypertriglyceridemic, and insulin-resistant by fructose feeding (48.Kasim-Karakas S.E. Vriend H. Almario R. Chow L.C. Goodman M.N. J. Lab. Clin. Med. 1996; 128: 208-213Abstract Full Text PDF PubMed Scopus (93) Google Scholar). Fructose feeding appears to interfere with glucose utilization in vivo, inducing an insulin resistant state (48.Kasim-Karakas S.E. Vriend H. Almario R. Chow L.C. Goodman M.N. J. Lab. Clin. Med. 1996; 128: 208-213Abstract Full Text PDF PubMed Scopus (93) Google Scholar). It thus appears feasible to induce insulin resistance in the hamster and use the insulin-resistant hamster model to study the mechanisms controlling hepatic VLDL-apoB secretion. Male Syrian golden hamsters (Mesocricetus auratus) were purchased from Charles River (Montreal, PQ). Fetal bovine serum (certified grade), liver perfusion medium, hepatocyte wash medium, liver digest medium, and hepatocyte attachment medium were obtained from Life Technologies (Grand Island, NY). Rabbit anti-hamster apoB antiserum was prepared commercially by Lampire Biological Laboratories (Pipersville, PA) using hamster LDL prepared in our laboratory. Specificity of this commercial preparation of anti-apoB polyclonal antibody and lack of any cross-reactivity to other hamster apolipoproteins (apoA-I or apoE) was confirmed by immunoblotting analysis of purified plasma lipoprotein fractions. Anti-bovine MTP antibody was generously provided by Dr. David Gordon (Bristol-Meyers Squibb). Anti-transferrin apoB antibody was obtained from Sigma. Anti-3-hydroxy-3-methylglutaryl-CoA reductase antibody (polyclonal anti-peptide antibody) was generously provided by Dr. S. P. Tam, Queen's University. Male Syrian golden hamsters were obtained from Charles River Canada (Montreal, PQ). All animals were housed in pairs and were given free access to food and water. After blood collection, animals were placed on either the control diet (normal chow) or fructose-enriched diet (hamster diet with 60% fructose, pelleted, Dyets Inc., Bethlehem, PA). The diet was continued for 2 weeks and hamster weight was monitored every 2 days. Plasma glucose, TG, and cholesterol levels were determined on an automated clinical chemistry analyzer (Hitachi 705). Plasma insulin levels were determined by radioimmunoassay using a rat insulin kit from Linco Research (St. Louis, MO). This assay has 100% cross-reactivity to hamster insulin and the intra- and interassay coefficient of variation were 6.8 and 10.6%, respectively. At the end of the 2-week feeding period anesthesia was induced using isoflurane (4% in 100% oxygen followed by 2% isoflurane with O2 enriched by mask throughout the surgical procedure), and catheters (PE 10 tubing) were inserted into the femoral vein (for infusion) and into the femoral artery (for blood sampling). The animals were allowed to awaken from anesthesia and were unrestrained in their cage. They were fasted from 6:00 p.m. that evening. Catheters were kept patent overnight with 1% citrate solution. At 9:00 a.m. two baseline blood samples (0.25 ml) were drawn at 10-min intervals followed by a primed-constant intravenous infusion of human biosynthetic insulin (Humulin R, Eli Lilly, Toronto, ON, Canada) (180 milliunits/kg bolus followed by 18 milliunits·kg−1 min−1 in 0.9% NaCl and 0.1% bovine serum albumin solution) for 2 h. The blood glucose level was maintained at the baseline value throughout the study by adjusting a 10% dextrose infusion according to frequent plasma glucose monitoring (approximately 0.1 ml every 10 min). Blood samples (0.25 ml) were drawn at 90, 100, 110, and 120 min to assess the steady state glucose and insulin levels. There was no significant decline in hematocrit throughout the study. In order to determine whether 2-week fructose feeding is associated with an in vivo increase in VLDL-apoB secretion, catheters were inserted in the femoral vein and artery of fructose (n = 6) and control fed animals (n = 7) of the same weight (119 ± 5 versus 121 ± 6 g, respectively,p = 0.79) as described above and VLDL-apoB and VLDL-TG levels were measured in the fasting state (12 h) at 1, 30, 60, and 90 min after an intravenous bolus (600 mg/kg) of a 20% (w/v) solution of Triton WR-1339 (Sigma) in normal saline (NaCl 0.9%). Because Triton WR-1339 effectively blocks the activity of lipoprotein lipase in vivo and therefore blocks the VLDL particle clearance, the secretion rate of VLDL-apoB and VLDL-TG is proportional to the rate of increase in VLDL-apoB and VLDL-TG concentration over time (49.Steiner G. Haynes F.J. Yoshino G. Vranic M. Am. J. Physiol. 1984; 246: E187-E192Crossref PubMed Google Scholar, 50.Kazumi T. Vranic M. Steiner G. Endocrinology. 1985; 117: 1145-1150Crossref PubMed Scopus (27) Google Scholar, 51.Kazumi T. Vranic M. Steiner G. Am. J. Physiol. 1986; 250: E325-E330Crossref PubMed Google Scholar, 52.Yoshino G. Hirano T. Maeda E. Murata Y. Naka Y. Nagata K. Kazumi T. Urayama T. Atherosclerosis. 1997; 129: 33-39Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, 53.Boisfer E. Lambert G. Atger V. Tran N.Q. Pastier D. Benetollo C. Trottier J.F. Beaucamps I. Antonucci M. Laplaud M. Griglio S. Chambaz J. Kalopissis A.D. J. Biol. Chem. 1999; 274: 11564-11572Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). This method has been previously used in the hamster by others (42.Arbeeny C.M. Meyers D.S. Bergquist K.E. Gregg R.E. J. Lipid Res. 1992; 33: 843-851Abstract Full Text PDF PubMed Google Scholar, 54.Sugiyama Y. Odaka H. Itokawa S. Ishikawa E. Tomari Y. Ikeda H. Atherosclerosis. 1995; 118: 145-153Abstract Full Text PDF PubMed Scopus (21) Google Scholar,55.Eisele B. Budzinski R. Muller P. Maier R. Mark M. J. Lipid Res. 1997; 38: 564-575Abstract Full Text PDF PubMed Google Scholar). The total blood volume of the samples drawn was less than 1.5 ml/animal during the experiment. Calculation of the in vivo VLDL-apoB and VLDL-TG secretion rates was performed by multiplying the slope of the concentration increase of VLDL-apoB (in μg/ml/min) and VLDL-TG (in μmol/ml/min), respectively, over time by the VLDL distribution volume estimated as 3.8 ml/100 g body weight (56.Buyer's Guide Lab. Anim. 1993; 27: 1-22Crossref PubMed Scopus (162) Google Scholar). Linearity of the increase in VLDL-apoB and VLDL-TG concentration was assessed by the linear regressionR squared value. A two-tailed unpaired t test was used to compare the slope of the VLDL-apoB and VLDL-TG concentration increase and the VLDL-apoB and VLDL-TG secretion rates between fructose-fed and control-fed hamsters. VLDL (d < 1.006 g/ml) in the in vivostudies was isolated by ultracentrifugation of plasma samples at 110,000 rpm for 3 h at 16 °C in a TI 110 rotor with a Beckman Optima TLX ultracentrifuge. VLDL-TGs were measured using a Roche Molecular Biochemicals colorimetric kit. VLDL-apoB was quantified by electroimmunoassay as described (57.Reardon M.F. Poapst M.E. Uffelman K.D. Steiner G. Clin. Chem. 1981; 27: 892-895Crossref PubMed Scopus (32) Google Scholar), with an anti-hamster apoB rabbit polyclonal antibody and a standard curve performed on each plate. ApoB standards were prepared by isolation of hamster LDL and protein quantification using Lowry's method (58.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Triton WR-1339 addedin vitro at concentration in the range of those usedin vivo (∼15 mg/ml) to hamster samples did not interfere with this assay (data not shown). The CV of this assay is 10%. At the end of the 2-week feeding period, hamsters were fasted overnight and blood samples were collected for measurement of a number of analytes in plasma. Hamsters were then fed for another day and were sedated and anesthetized by intramuscular injection of acepromazine (1 mg/kg) and intrapretoneal injection of a mixture of ketamine (200 mg/kg) and xylazine (10 mg/kg). After achieving complete general anesthesia, lidocaine (10 units in 4 divided doses) was injected subcutaneously in the mid-abdominal line of the animal before surgical incision. The liver was perfused as described (59.Miller L.L. Miller L.L. Isolated Liver Perfusion and Its Applications. Raven Press, New York1973: 11-52Google Scholar) with small modifications. Released hepatocytes from digested liver tissue were washed three times in hepatocyte wash medium and eventually transferred into culture medium (hepatocyte attachment medium containing 5% fetal bovine serum, 1.0 μg/ml insulin, 1× penicillin-streptomycin) and seeded in collagen-coated plates (1.5 × 106cells/35-mm plate). After 4 h or overnight incubation at 37 °C, 5% CO2, attached cells were used to carry out the experiments. Primary hepatocytes were pulsed for 3 or 18 h with 5 μCi/ml [3H]acetate to assess the rates of synthesis and secretion of cholesterol, cholesteryl ester, and phospholipids. TG synthesis and secretion were monitored by labeling cells for 3–5 h with 5 μCi/ml [3H]oleate bound to bovine serum albumin. Following labeling, cells were extracted with hexane/isopropyl alcohol (3:2) and the total lipid extract was dried, suspended in hexane, and applied to a thin layer chromatogram. The TLC plates were developed using a two-solvent system to separate polar lipids with chloroform/methanol/acetic acid/formic acid/H2O (70:30:12:4:2) and neutral lipids with petroleum ether/ethyl ether/acetic acid (90:10:1). The lipids were stained with iodine vapor and identified based on the use of a set of known lipid standards (Sigma). The spots identified on the TLC plates were cut and counted using a scintillation counter. Primary hamster hepatocytes were preincubated in methionine-free minimal essential medium at 37 °C for 1 h and pulsed with 75–100 μCi/ml [35S]methionine for 45–60 min. Following the pulse, the cells were washed twice and chased in hepatocyte attachment medium supplemented with 10 mmmethionine. At various chase times duplicate or triplicate dishes were harvested, and cells were lysed in solubilization buffer (phosphate-buffered saline containing 1% Nonidet P-40, 1% deoxycholate, 5 mm EDTA, 1 mm EGTA, 2 mm phenylmethylsulfonyl fluoride, 0.1 mmleupeptin, 2 μg/ml N-acetyl-leucyl-leucyl-norleucinal). The lysates were centrifuged for 10 min at 4 °C in a microcentrifuge, and supernatants were collected for immunoprecipitation. Primary hamster hepatocytes cultured in 35-mm dishes were depleted of methionine by incubation in methionine-free minimal essential medium for 1 h at 37 °C under 5% CO2. Cells were pulsed with 100 μCi/ml [35S]methionine for 45–60 min and then permeabilized as described (60.Adeli K. J. Biol. Chem. 1994; 269: 9166-9175Abstract Full Text PDF PubMed Google Scholar, 61.Macri J. Adeli K. J. Biol. Chem. 1997; 272: 7328-7337Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). At various intervals, duplicate or triplicate dishes were washed, solubilized, and subjected to immunoprecipitation. Isolation of the microsomal fraction and the separation of the luminal and membrane components by carbonate extraction and ultracentrifugation was performed as described (62.Boren J. Wettesten M. Sjoberg A. Thorlin T. Bondjers G. Wiklund O. Olofsson S.O. J. Biol. Chem. 1990; 265: 10556-10564Abstract Full Text PDF PubMed Google Scholar, 63.Adeli K. Wettesten M. Asp L. Mohammadi A. Macri J. Olofsson S.O. J. Biol. Chem. 1997; 272: 5031-5039Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Membrane and luminal fractions were then diluted with 800 μl of a solubilization buffer containing 360 μl of 5×C buffer (250 mm Tris-HCl, pH 7.4, 750 mm NaCl, 25 mm EDTA, 5 mm phenylmethylsulfonyl fluoride, 5% Triton X-100) and 410 μl of phosphate-buffered saline supplemented with 450 KIU/ml Trasylol, 5 mm phenylmethylsulfonyl fluoride, and subjected to immunoprecipitation, SDS-PAGE, and fluorography. Cell samples were subjected to chemiluminescent immunoblotting for the MTP 97-kDa subunit. Samples were analyzed by SDS-PAGE using a 10% polyacrylamide mini-gel (8 × 5 cm). Following SDS-PAGE the proteins were transferred electrophoretically overnight at 4 °C onto nitrocellulose membranes using a Bio-Rad Wet Transfer System. The membranes were blocked with 5% solution of fat-free dry milk powder, incubated with a rabbit anti-hamster MTP antiserum, washed, and then incubated with a secondary antibody conjugated to peroxidase. Membranes were then incubated in an ECL detection reagent for 60 s and exposed to Hyperfilm. Films were then developed and quantitative analysis was performed using an Imaging Densitometer. Immunoprecipitation was performed as described previously (60.Adeli K. J. Biol. Chem. 1994; 269: 9166-9175Abstract Full Text PDF PubMed Google Scholar). Immunoprecipitates were washed with wash buffer (10 mm Tris-HCl, pH 7.4, 2 mm EDTA, 0.1% SDS, 1% Triton X-100) and were prepared for SDS-PAGE by suspending and boiling in 100 μl of electrophoresis sample buffer. SDS-PAGE was performed essentially as described (64.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). The gels were fixed, stained and soaked in Amplify (Amersham Pharmacia Biotech), before being dried, and exposed to Dupont autoradiographic film at −80 °C for 1–4 days. ApoB bands were excised from the gel, digested in hydrogen peroxide/perchloric acid, and the radioactivity was determined by scintillation counting. All the values are reported as mean ± S.

Insulin-resistant states are characterized by hypertriglyceridemia, predominantly because of overproduction of hepatic very low density lipoprotein particles. The additional contribution of intestinal lipoprotein overproduction to the dyslipidemia of insulin-resistant states has not been previously appreciated. Here, we have investigated intestinal lipoprotein production in a fructose-fed hamster model of insulin resistance previously documented to have whole body and hepatic insulin resistance, and hepatic very low density lipoprotein overproduction. Chronic fructose feeding for 3 weeks induced significant oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins in the fasting state and during steady state fat feeding, based on (<i>a</i>)<i>in vivo</i> Triton WR1339 studies of apoB48 production as well as (<i>b</i>) <i>ex vivo</i> pulse-chase labeling of intestinal enterocytes from fasted and fed hamsters. ApoB48 particle overproduction was accompanied by increased intracellular apoB48 stability, enhanced lipid synthesis, higher abundance of microsomal triglyceride transfer protein mass, and a significant shift toward the secretion of larger chylomicron-like particles. ApoB48 particle overproduction was not observed with short-term fructose feeding or<i>in vitro</i> incubation of enterocytes with fructose. Secretion of intestinal apoB48 and triglyceride was closely linked to intestinal enterocyte <i>de novo</i> lipogenesis, which was up-regulated in fructose-fed hamsters. Inhibition of fatty acid synthesis by cerulenin, a fatty acid synthase inhibitor, resulted in a dose-dependent decrease in intestinal apoB48 secretion. Overall, these findings further suggest that intestinal overproduction of apoB48 lipoproteins should also be considered as a major contributor to the fasting and postprandial dyslipidemia observed in response to chronic fructose feeding and development of an insulin-resistant state.

A fructose-fed hamster model of insulin resistance was previously documented to exhibit marked hepatic very low density lipoprotein (VLDL) overproduction.Here, we investigated whether VLDL overproduction was associated with down-regulation of hepatic insulin signaling and insulin resistance.Hepatocytes isolated from fructose-fed hamsters exhibited significantly reduced tyrosine phosphorylation of the insulin receptor and insulin receptor substrates 1 and 2. Phosphatidylinositol 3-kinase activity as well as insulin-stimulated Akt-Ser 473 and Akt-Thr 308 phosphorylation were also significantly reduced with fructose feeding.Interestingly, the protein mass and activity of protein-tyrosine phosphatase-1B (PTP-1B) were significantly higher in fructose-fed hamster hepatocytes.Chronic ex vivo exposure of control hamster hepatocytes to high insulin also appeared to attenuate insulin signaling and increase PTP-1B.Elevation in PTP-1B coincided with marked suppression of ER-60, a cysteine protease postulated to play a role in intracellular apoB degradation, and an increase in the synthesis and secretion of apoB.Sodium orthovanadate, a general phosphatase inhibitor, partially restored insulin receptor phosphorylation and significantly reduced apoB secretion.In summary, we hypothesize that fructose feeding induces hepatic insulin resistance at least in part via an increase in expression of PTP-1B.Induction of hepatic insulin resistance may then contribute to reduced apoB degradation and enhanced VLDL particle assembly and secretion.

Fructose, a naturally found sugar in many fruits, is now commonly used as an industrial sweetener and is excessively consumed in Western diets. High fructose intake is increasingly recognized as causative in development of prediabetes and metabolic syndrome. The mechanisms underlying fructose-induced metabolic disturbances are unclear but are beginning to be unravelled. This review presents recent findings in this field and an overall mechanistic insight into the metabolic effects of dietary fructose and its role in metabolic syndrome.Recent animal studies have confirmed the link between fructose feeding and increased plasma uric acid, a potentially causative factor in metabolic syndrome. Advanced glycation end products are also implicated because of their direct protein modifications and indirect effects on inflammation and oxidative stress. Human studies have demonstrated fructose's ability to change metabolic hormonal response, possibly contributing to decreased satiety.There is much evidence from both animal models and human studies supporting the notion that fructose is a highly lipogenic nutrient that, when consumed in high quantities, contributes to tissue insulin insensitivity, metabolic defects, and the development of a prediabetic state. Recently evidence has helped to decipher the mechanisms involved in these metabolic changes.

Excessive postprandial lipemia is a prevalent condition that results from intestinal oversecretion of apolipoprotein B48 (apoB48)-containing lipoproteins. Glucagon-like peptide-2 (GLP-2) is a gastrointestinal-derived intestinotropic hormone that links nutrient absorption to intestinal structure and function. We investigated the effects of GLP-2 on intestinal lipid absorption and lipoprotein production.Intestinal lipid absorption and chylomicron production were quantified in hamsters, wild-type mice, and Cd36(-/-) mice infused with exogenous GLP-2. Newly synthesized apoB48 was metabolically labelled in primary hamster jejunal fragments. Fatty acid absorption was measured, and putative fatty acid transporters were assessed by immunoblotting.Human GLP-2 increased secretion of the triglyceride (TG)-rich lipoprotein (TRL)-apoB48 following oral administration of olive oil to hamsters; TRL and cholesterol mass each increased 3-fold. Fast protein liquid chromatography profiling indicated that GLP-2 stimulated secretion of chylomicron/very low-density lipoprotein-sized particles. Moreover, GLP-2 directly stimulated apoB48 secretion in jejunal fragments cultured ex vivo, increased expression of fully glycosylated cluster of differentiation 36/fatty acid translocase (CD36), and induced intestinal absorption of [(3)H]triolein. The ability of GLP-2 to increase intestinal lipoprotein production was lost in Cd36(-/-) mice.GLP-2 stimulates intestinal apoB48-containing lipoprotein secretion, possibly through increased lipid uptake, via a pathway that requires CD36. These findings suggest that GLP-2 represents a nutrient-dependent signal that regulates intestinal lipid absorption and the assembly and secretion of TRLs from intestinal enterocytes.

Insulin plays a central role in regulating energy metabolism, including hepatic transport of very low-density lipoprotein (VLDL)-associated triglyceride. Hepatic hypersecretion of VLDL and consequent hypertriglyceridemia leads to lower circulating high-density lipoprotein levels and generation of small dense low-density lipoproteins characteristic of the dyslipidemia commonly observed in metabolic syndrome and type 2 diabetes mellitus. Physiological fluctuations of insulin modulate VLDL secretion, and insulin inhibition of VLDL secretion upon feeding may be the first pathway to become resistant in obesity that leads to VLDL hypersecretion. This review summarizes the role of insulin-related signaling pathways that determine hepatic VLDL production. Disruption in signaling pathways that reduce generation of the second messenger phosphatidylinositide (3,4,5) triphosphate downstream of activated phosphatidylinositide 3-kinase underlies the development of VLDL hypersecretion. As insulin resistance progresses, a number of pathways are altered that further augment VLDL hypersecretion, including hepatic inflammatory pathways. Insulin plays a complex role in regulating glucose metabolism, and it is not surprising that the role of insulin in VLDL and lipid metabolism will prove equally complex.

Abstract While considerable research has examined diminished insulin responses within peripheral tissues, comparatively little has been done to examine the effects of this metabolic disruption upon the CNS. The present study employed biochemical and electrophysiological assays of acutely prepared brain slices to determine whether neural insulin resistance is a component of the metabolic syndrome observed within the fructose‐fed (FF) hamster. The tyrosine phosphorylation levels of the insulin receptor (IR) and insulin receptor substrate 1 (IRS‐1) in response to insulin were significantly reduced within FF hamsters. Also, insulin‐mediated phosphorylation of both residues necessary for activation of the serine‐threonine kinase Akt/PKB, a key effector of insulin signaling, was markedly decreased. Elevated levels of the protein tyrosine phosphatase 1B, which dephosphorylates the IR and IRS‐1, were also observed within the cerebral cortex and hippocampus of FF hamsters. Examination of whether a nutritionally induced compromise of neural insulin signaling altered synaptic function revealed a significant attenuation of insulin‐induced long‐term depression, but no effect upon either paired‐pulse facilitation or electrically induced long‐term potentiation. Collectively, our results demonstrate, for the first time, that nutritionally induced insulin resistance significantly affects the neural insulin signaling pathway, and suggest that brain insulin resistance may contribute to cognitive impairment.

An important complication of insulin-resistant states, such as obesity and type 2 diabetes, is an atherogenic dyslipidemia profile characterized by hypertriglyceridemia, low plasma high-density lipoproteins (HDL) cholesterol and a small, dense low-density lipoprotein (LDL) particle profile. The physiological basis of this metabolic dyslipidemia appears to be hepatic overproduction of apoB-containing very low-density lipoprotein (VLDL) particles. This has focused attention on the mechanisms that regulate VLDL secretion in insulin-resistant states. Recent studies in animal models of insulin resistance, particularly the fructose-fed hamster, have enhanced our understanding of these mechanisms, and certain key factors have recently been identified that play important roles in hepatic insulin resistance and dysregulation of the VLDL secretory process. This review focuses on these recent developments as well as on the hypothesis that an interaction between enhanced flux of free fatty acids from peripheral tissues to liver, chronic up-regulation of de novo lipogenesis by hyperinsulinemia and attenuated insulin signaling in the liver may be critical to the VLDL overproduction state observed in insulin resistance. It should be noted that the focus of this review is on molecular mechanisms of the hypertriglyceridemic state associated with insulin resistance and not that observed in association with insulin deficiency (e.g., in streptozotocin-treated animals), which appears to have a different etiology and is related to a catabolic defect rather than secretory overproduction of triglyceride-rich lipoproteins.

Peer review has been defined as a process of subjecting an author's scholarly work, research or ideas to the scrutiny of others who are experts in the same field. It functions to encourage authors to meet the accepted high standards of their discipline and to control the dissemination of research data to ensure that unwarranted claims, unacceptable interpretations or personal views are not published without prior expert review. Despite its wide-spread use by most journals, the peer review process has also been widely criticised due to the slowness of the process to publish new findings and due to perceived bias by the editors and/or reviewers. Within the scientific community, peer review has become an essential component of the academic writing process. It helps ensure that papers published in scientific journals answer meaningful research questions and draw accurate conclusions based on professionally executed experimentation. Submission of low quality manuscripts has become increasingly prevalent, and peer review acts as a filter to prevent this work from reaching the scientific community. The major advantage of a peer review process is that peer-reviewed articles provide a trusted form of scientific communication. Since scientific knowledge is cumulative and builds on itself, this trust is particularly important. Despite the positive impacts of peer review, critics argue that the peer review process stifles innovation in experimentation, and acts as a poor screen against plagiarism. Despite its downfalls, there has not yet been a foolproof system developed to take the place of peer review, however, researchers have been looking into electronic means of improving the peer review process. Unfortunately, the recent explosion in online only/electronic journals has led to mass publication of a large number of scientific articles with little or no peer review. This poses significant risk to advances in scientific knowledge and its future potential. The current article summarizes the peer review process, highlights the pros and cons associated with different types of peer review, and describes new methods for improving peer review.

Reference intervals are indispensable in evaluating laboratory test results; however, appropriately partitioned pediatric reference values are not readily available. The Canadian Laboratory Initiative for Pediatric Reference Intervals (CALIPER) program is aimed at establishing the influence of age, sex, ethnicity, and body mass index on biochemical markers and developing a comprehensive database of pediatric reference intervals using an a posteriori approach.A total of 1482 samples were collected from ethnically diverse healthy children ages 2 days to 18 years and analyzed on the Abbott ARCHITECT i2000. Following the CLSI C28-A3 guidelines, age- and sex-specific partitioning was determined for each analyte. Nonparametric and robust methods were used to establish the 2.5th and 97.5th percentiles for the reference intervals as well as the 90% CIs.New pediatric reference intervals were generated for 14 biomarkers, including α-fetoprotein, cobalamin (vitamin B12), folate, homocysteine, ferritin, cortisol, troponin I, 25(OH)-vitamin D [25(OH)D], intact parathyroid hormone (iPTH), thyroid-stimulating hormone, total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4), and free triiodothyronine. The influence of ethnicity on reference values was also examined, and statistically significant differences were found between ethnic groups for FT4, TT3, TT4, cobalamin, ferritin, iPTH, and 25(OH)D.This study establishes comprehensive pediatric reference intervals for several common endocrine and immunochemical biomarkers obtained in a large cohort of healthy children. The new database will be of global benefit, ensuring appropriate interpretation of pediatric disease biomarkers, but will need further validation for specific immunoassay platforms and in local populations as recommended by the CLSI.

In a collaboration between the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER) and the Canadian Health Measures Survey (CHMS), we determined reference value distributions using an a priori approach and created a comprehensive database of age- and sex-stratified reference intervals for clinically relevant hematologic parameters in a large household population of children and adults.The CHMS collected data and blood samples from 11 999 respondents aged 3-79 years. Hematology markers were measured with either the Beckman Coulter HmX or Siemens Sysmex CA-500 Series analyzers. After applying exclusion criteria and removing outliers, we determined statistically relevant age and sex partitions and calculated reference intervals, including 90% CIs, according to CSLI C28-A3 guidelines.Hematology marker values showed dynamic changes from childhood into adulthood as well as between sexes, necessitating distinct partitions throughout life. Most age partitions were necessary during childhood, reflecting the hematologic changes that occur during growth and development. Hemoglobin, red blood cell count, hematocrit, and indices (mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration) increased with age, but females had lower hemoglobin and hematocrit starting at puberty. Platelet count gradually decreased with age and required multiple sex partitions during adolescence and adulthood. White blood cell count remained relatively constant over life, whereas fibrinogen increased slightly, requiring distinct age and sex partitions.The robust dataset generated in this study has allowed observation of dynamic biological profiles of several hematology markers and the establishment of comprehensive age- and sex-specific reference intervals that may contribute to accurate monitoring of pediatric, adult, and geriatric patients.

To investigate the association of plasma levels of methylglyoxal (MG) and markers of inflammation/endothelial dysfunction with diabetic nephropathy (DN). Plasma levels of MG, cytokines, and adhesion molecules were measured in type 2 diabetic patients (T2DM), T2DM patients with DN, and the controls. Plasma MG levels in DN were significantly higher than those in T2DM and the controls (312 ± 135 vs. 212 ± 73 and 312 ± 135 vs. 147 ± 78 nmol/L, respectively, P < 0.001). The plasma levels of MG were positively correlated with the fasting glucose, HbA1c, and urinary albumin/creatinine ratio (r = 0.754, P < 0.05). Plasma levels of IL-6, TNF-α, and adhesion molecules were markedly increased in DN compared to T2DM patients and the controls. Increased plasma levels of MG, cytokines, and adhesion molecules are associated with DN. These markers may be useful in predicting the development of DN.

Excessive postprandial lipemia is highly prevalent in obese and insulin-resistant/type 2 diabetic individuals and substantially increases the risk of atherosclerosis and cardiovascular disease. This article will review our current understanding of the link between insulin resistance and intestinal lipoprotein overproduction and highlight some of the key recent findings in the field.Emerging evidence from several animal models of insulin resistance as well as insulin-resistant humans clearly supports the link between insulin resistance and aberrant intestinal lipoprotein metabolism. In insulin-resistant states, elevated free fatty acid flux into the intestine, downregulation of intestinal insulin signaling and upregulation of microsomal triglyceride transfer protein all appear to stimulate intestinal lipoprotein production. Gut peptides, GLP-1 and GLP-2, may be important regulators of intestinal lipid absorption and lipoprotein production.Available evidence in humans and animal models strongly favors the concept that the small intestine is not merely an absorptive organ but rather plays an active role in regulating the rate of production of triglyceride-rich lipoproteins. Metabolic signals in insulin resistance and type 2 diabetes and in some cases an aberrant intestinal response to these factors all contribute to the enhanced formation and secretion of triglyceride-rich lipoproteins.

To examine outcomes at age 4.5 years and compare to earlier ages in children with fetal antiepileptic drug (AED) exposure.The NEAD Study is an ongoing prospective observational multicenter study, which enrolled pregnant women with epilepsy on AED monotherapy (1999-2004) to determine if differential long-term neurodevelopmental effects exist across 4 commonly used AEDs (carbamazepine, lamotrigine, phenytoin, or valproate). The primary outcome is IQ at 6 years of age. Planned analyses were conducted using Bayley Scales of Infant Development (BSID at age 2) and Differential Ability Scale (IQ at ages 3 and 4.5).Multivariate intent-to-treat (n = 310) and completer (n = 209) analyses of age 4.5 IQ revealed significant effects for AED group. IQ for children exposed to valproate was lower than each other AED. Adjusted means (95% confidence intervals) were carbamazepine 106 (102-109), lamotrigine 106 (102-109), phenytoin 105 (102-109), valproate 96 (91-100). IQ was negatively associated with valproate dose, but not other AEDs. Maternal IQ correlated with child IQ for children exposed to the other AEDs, but not valproate. Age 4.5 IQ correlated with age 2 BSID and age 3 IQ. Frequency of marked intellectual impairment diminished with age except for valproate (10% with IQ <70 at 4.5 years). Verbal abilities were impaired for all 4 AED groups compared to nonverbal skills.Adverse cognitive effects of fetal valproate exposure persist to 4.5 years and are related to performances at earlier ages. Verbal abilities may be impaired by commonly used AEDs. Additional research is needed.

Emerging evidence suggests that increased dietary consumption of fructose in Western society may be a potentially important factor in the growing rates of obesity and the metabolic syndrome. This review will discuss fructose-induced perturbations in cell signaling and inflammatory cascades in insulin-sensitive tissues. In particular, the roles of cellular signaling molecules including nuclear factor kappa B (NFkB), tumor necrosis factor alpha (TNF-alpha), c-Jun amino terminal kinase 1 (JNK-1), protein tyrosine phosphatase 1B (PTP-1B), phosphatase and tensin homolog deleted on chromosome ten (PTEN), liver X receptor (LXR), farnesoid X receptor (FXR), and sterol regulatory element-binding protein-1c (SREBP-1c) will be addressed. Considering the prevalence and seriousness of the metabolic syndrome, further research on the underlying molecular mechanisms and preventative and curative strategies is warranted.

Current evidence implicates autophagy in the regulation of lipid stores within the two main organs involved in maintaining lipid homeostasis, the liver and adipose tissue. Critical to this role in hepatocytes is the breakdown of cytoplasmic lipid droplets, a process referred to as lipophagy. Conversely, autophagy is required for adipocyte differentiation and the concurrent accumulation of lipid droplets. Autophagy also affects lipid metabolism through contributions to lipoprotein assembly. A number of reports have now implicated autophagy in the degradation of apolipoprotein B, the main structural protein of very-low-density-lipoprotein. Aberrant autophagy may also be involved in conditions of deregulated lipid homeostasis in metabolic disorders such as the metabolic syndrome. First, insulin signalling and autophagy activity appear to diverge in a mechanism of reciprocal regulation, suggesting a role for autophagy in insulin resistance. Secondly, upregulation of autophagy may lead to conversion of white adipose tissue into brown adipose tissue, thus regulating energy expenditure and obesity. Thirdly, upregulation of autophagy in hepatocytes could increase breakdown of lipid stores controlling triglyceride homeostasis and fatty liver. Taken together, autophagy appears to play a very complex role in lipid homeostasis, affecting lipid stores differently depending on the tissue, as well as contributing to pathways of lipoprotein metabolism.

This trial aimed to evaluate the effects of zinc sulfate in comparison with placebo on markers of insulin resistance, oxidative stress, and inflammation in a sample of obese prepubescent children.This triple-masked, randomized, placebo-controlled, crossover trial was conducted among 60 obese Iranian children in 2008. Participants were randomly assigned to two groups of equal number; one group received 20 mg of elemental zinc and the other group received placebo on a regular daily basis for 8 weeks. After a 4-week washout period, the groups were crossed over. In addition to anthropometric measures and blood pressure, fasting plasma glucose, lipid profile, insulin, apolipoproteins A-1 (ApoA-I) and B, high-sensitivity C-reactive protein (hs-CRP), leptin, oxidized low-density lipoprotein (ox-LDL), and malondialdehyde were determined at all four stages of the study.Irrespective of the order of receiving zinc and placebo, in both groups, significant decrease was documented for Apo B/ApoA-I ratio, ox-LDL, leptin and malondialdehyde, total and LDL-cholesterol after receiving zinc without significant change after receiving placebo. In groups, hs-CRP and markers of insulin resistance decreased significantly after receiving zinc, but increased after receiving placebo. In both groups, the mean body mass index (BMI) Z-score remained high, after receiving zinc, the mean weight, BMI, BMI Z-score decreased significantly, whereas these values increased after receiving placebo.These results are particularly important in light of the deleterious consequences of childhood obesity and early changes in markers of inflammatory and oxidative stress. We suggest exploring the direct clinical application of zinc supplementation in childhood obesity in future studies.

Biological covariates such as age and sex can markedly influence biochemical marker reference values, but no comprehensive study has examined such changes across pediatric, adult, and geriatric ages. The Canadian Health Measures Survey (CHMS) collected comprehensive nationwide health information and blood samples from children and adults in the household population and, in collaboration with the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER), examined biological changes in biochemical markers from pediatric to geriatric age, establishing a comprehensive reference interval database for routine disease biomarkers.The CHMS collected health information, physical measurements, and biosamples (blood and urine) from approximately 12 000 Canadians aged 3-79 years and measured 24 biochemical markers with the Ortho Vitros 5600 FS analyzer or a manual microplate. By use of CLSI C28-A3 guidelines, we determined age- and sex-specific reference intervals, including corresponding 90% CIs, on the basis of specific exclusion criteria.Biochemical marker reference values exhibited dynamic changes from pediatric to geriatric age. Most biochemical markers required some combination of age and/or sex partitioning. Two or more age partitions were required for all analytes except bicarbonate, which remained constant throughout life. Additional sex partitioning was required for most biomarkers, except bicarbonate, total cholesterol, total protein, urine iodine, and potassium.Understanding the fluctuations in biochemical markers over a wide age range provides important insight into biological processes and facilitates clinical application of biochemical markers to monitor manifestation of various disease states. The CHMS-CALIPER collaboration addresses this important evidence gap and allows the establishment of robust pediatric and adult reference intervals.

The liver and intestine have complementary and coordinated roles in lipoprotein metabolism. Despite their highly specialized functions, assembly and secretion of triglyceride-rich lipoproteins (TRL; apoB-100-containing VLDL in the liver and apoB-48-containing chylomicrons in the intestine) are regulated by many of the same hormonal, inflammatory, nutritional, and metabolic factors. Furthermore, lipoprotein metabolism in these two organs may be affected in a similar fashion by certain disorders. In insulin resistance, for example, overproduction of TRL by both liver and intestine is a prominent component of and underlies other features of a complex dyslipidemia and increased risk of atherosclerosis. The intestine is gaining increasing recognition for its importance in affecting whole body lipid homeostasis, in part through its interaction with the liver. This review aims to integrate recent advances in our understanding of these processes and attempts to provide insight into the factors that coordinate lipid homeostasis in these two organs in health and disease.

The glucagon-like peptides (GLP-1 and GLP-2) are processed from the proglucagon polypeptide and secreted in equimolar amounts but have opposite effects on chylomicron (CM) production, with GLP-1 significantly reducing and GLP-2 increasing postprandial chylomicronemia. In the current study, we evaluated the apparent paradoxical roles of GLP-1 and GLP-2 under physiological conditions in the Syrian golden hamster, a model with close similarity to humans in terms of lipoprotein metabolism. A short (30-min) intravenous infusion of GLP-2 resulted in a marked increase in postprandial apolipoprotein B48 (apoB48) and triglyceride (TG) levels in the TG-rich lipoprotein (TRL) fraction, whereas GLP-1 infusion decreased lipid absorption and levels of TRL-TG and apoB48. GLP-1 and GLP-2 coinfusion resulted in net increased lipid absorption and an increase in TRL-TG and apoB48. However, prolonged (120-min) coinfusion of GLP-1 and GLP-2 decreased postprandial lipemia. Blocking dipeptidyl peptidase-4 activity resulted in decreased postprandial lipemia. Interestingly, fructose-fed, insulin-resistant hamsters showed a more pronounced response, including possible hypersensitivity to GLP-2 or reduced sensitivity to GLP-1. In conclusion, under normal physiological conditions, the actions of GLP-2 predominate; however, when GLP-1 activity is sustained, the hypolipidemic action of GLP-1 predominates. Pharmacological inhibition of GLP-1 degradation tips the balance toward an inhibitory effect on intestinal production of atherogenic CM particles.

BACKGROUND Pediatric endocrinopathies are commonly diagnosed and monitored by measuring hormones of the hypothalamic-pituitary-gonadal axis. Because growth and development can markedly influence normal circulating concentrations of fertility hormones, accurate reference intervals established on the basis of a healthy, nonhospitalized pediatric population and that reflect age-, gender-, and pubertal stage–specific changes are essential for test result interpretation. METHODS Healthy children and adolescents (n = 1234) were recruited from a multiethnic population as part of the CALIPER study. After written informed parental consent was obtained, participants filled out a questionnaire including demographic and pubertal development information (assessed by self-reported Tanner stage) and provided a blood sample. We measured 7 fertility hormones including estradiol, testosterone (second generation), progesterone, sex hormone–binding globulin, prolactin, follicle-stimulating hormone, and luteinizing hormone by use of the Abbott Architect i2000 analyzer. We then used these data to calculate age-, gender-, and Tanner stage–specific reference intervals according to Clinical Laboratory Standards Institute C28-A3 guidelines. RESULTS We observed a complex pattern of change in each analyte concentration from the neonatal period to adolescence. Consequently, many age and sex partitions were required to cover the changes in most fertility hormones over this period. An exception to this was prolactin, for which no sex partition and only 3 age partitions were necessary. CONCLUSIONS This comprehensive database of pediatric reference intervals for fertility hormones will be of global benefit and should lead to improved diagnosis of pediatric endocrinopathies. The new database will need to be validated in local populations and for other immunoassay platforms as recommended by the Clinical Laboratory Standards Institute.

Both environmental and genetic factors contribute to relative species abundance and metabolic characteristics of the intestinal microbiota. The intestinal microbiota and accompanying microbial metabolites differ substantially in those who are obese or have other metabolic disorders. Accumulating evidence from germ-free mice and antibiotic-treated animal models suggests that altered intestinal gut microbiota contributes significantly to metabolic disorders involving impaired glucose and lipid metabolism. This review will summarize recent findings on potential mechanisms by which the microbiota affects intestinal lipid and lipoprotein metabolism including microbiota dependent changes in bile acid metabolism which affects bile acid signaling by bile acid receptors FXR and TGR5. Microbiota changes also involve altered short chain fatty acid signaling and influence enteroendocrine cell function including GLP-1/GLP-2-producing L-cells which regulate postprandial lipid metabolism.

The CALIPER program recently established a comprehensive database of age- and sex-stratified pediatric reference intervals for 40 biochemical markers. However, this database was only directly applicable for Abbott ARCHITECT assays. We therefore sought to expand the scope of this database to biochemical assays from other major manufacturers, allowing for a much wider application of the CALIPER database.Based on CLSI C28-A3 and EP9-A2 guidelines, CALIPER reference intervals were transferred (using specific statistical criteria) to assays performed on four other commonly used clinical chemistry platforms including Beckman Coulter DxC800, Ortho Vitros 5600, Roche Cobas 6000, and Siemens Vista 1500. The resulting reference intervals were subjected to a thorough validation using 100 reference specimens (healthy community children and adolescents) from the CALIPER bio-bank, and all testing centers participated in an external quality assessment (EQA) evaluation.In general, the transferred pediatric reference intervals were similar to those established in our previous study. However, assay-specific differences in reference limits were observed for many analytes, and in some instances were considerable. The results of the EQA evaluation generally mimicked the similarities and differences in reference limits among the five manufacturers' assays. In addition, the majority of transferred reference intervals were validated through the analysis of CALIPER reference samples.This study greatly extends the utility of the CALIPER reference interval database which is now directly applicable for assays performed on five major analytical platforms in clinical use, and should permit the worldwide application of CALIPER pediatric reference intervals.

Abstract Platelet αIIbβ3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet β3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbβ3 integrin, and the direct apoA-IV-αIIbβ3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbβ3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbβ3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.

Abstract Serological testing for the detection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is emerging as an important component of the clinical management of patients with coronavirus disease 2019 (COVID-19) as well as the epidemiological assessment of SARS-CoV-2 exposure worldwide. In addition to molecular testing for the detection of SARS-CoV-2 infection, clinical laboratories have also needed to increase testing capacity to include serological evaluation of patients with suspected or known COVID-19. While regulatory approved serological immunoassays are now widely available from diagnostic manufacturers globally, there is significant debate regarding the clinical utility of these tests, as well as their clinical and analytical performance requirements prior to application. This document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 provides interim guidance on: (A) clinical indications and target populations, (B) assay selection, (C) assay evaluation, and (D) test interpretation and limitations for serological testing of antibodies against SARS-CoV-2 infection. These evidence-based recommendations will provide practical guidance to clinical laboratories in the selection, verification, and implementation of serological assays and are of the utmost importance as we expand our pandemic response from initial case tracing and containment to mitigation strategies to minimize resurgence and further morbidity and mortality.

Two common features of dietary polyphenols have hampered our mechanistic understanding of their beneficial effects for decades: targeting multiple organs and extremely low bioavailability. We show here that resveratrol intervention (REV-I) in high-fat diet (HFD)-challenged male mice inhibits chylomicron secretion, associated with reduced expression of jejunal but not hepatic scavenger receptor class B type 1 (SR-B1). Intestinal mucosa-specific SR-B1-/- mice on HFD-challenge exhibit improved lipid homeostasis but show virtually no further response to REV-I. SR-B1 expression in Caco-2 cells cannot be repressed by pure resveratrol compound while fecal-microbiota transplantation from mice on REV-I suppresses jejunal SR-B1 in recipient mice. REV-I reduces fecal levels of bile acids and activity of fecal bile-salt hydrolase. In Caco-2 cells, chenodeoxycholic acid treatment stimulates both FXR and SR-B1. We conclude that gut microbiome is the primary target of REV-I, and REV-I improves lipid homeostasis at least partially via attenuating FXR-stimulated gut SR-B1 elevation.

Postprandial dyslipidemia is recognized as an important complication of insulin-resistant states, and recent evidence implicates intestinal lipoprotein overproduction as a causative factor. The mechanisms linking intestinal lipoprotein overproduction and aberrant insulin signaling in intestinal enterocytes are currently unknown. Intestinal insulin sensitivity and lipid metabolism were studied in a fructose-fed hamster model of insulin resistance and metabolic dyslipidemia. Intestinal lipoprotein production in chow-fed hamsters was responsive to the inhibitory effects of insulin, and a decrease in circulating levels of triglyceride-rich apolipoprotein (apo)B48-containing lipoproteins occurred 60 min after insulin administration. However, fructose-fed hamster intestine was not responsive to the insulin-induced downregulation of apoB48-lipoprotein production, suggesting insulin insensitivity at the level of the intestine. Enterocytes from the fructose-fed hamster exhibited normal activity of the insulin receptor but reduced levels of insulin receptor substrate-1 phosphorylation and mass and Akt protein mass. Conversely, the protein mass of the p110 subunit of phosphatidylinositol 3-kinase, protein tyrosine phosphatase-1B, and basal levels of phosphorylated extracellular signal-related kinase (ERK) were significantly increased in the fructose-fed hamster intestine. Modulating the ERK pathway through in vivo inhibition of mitogen-activated protein/ERK kinase 1/2, the upstream activator of ERK1/2, we observed a significant decrease in intestinal apoB48 synthesis and secretion. Interestingly, enhanced basal ERK activity in the fructose-fed hamster intestine was accompanied by an increased activation of sterol regulatory element-binding protein. In summary, these data suggest that insulin insensitivity at the level of the intestine and aberrant insulin signaling are important underlying factors in intestinal overproduction of highly atherogenic apoB48-containing lipoproteins in the insulin-resistant state. Basal activation of the ERK pathway may be an important contributor to the aberrant insulin signaling and lipoprotein overproduction in this model.

Abstract Aim: To determine the prevalence of paediatric metabolic syndrome (MetS) and its best predictive anthropometric index. Methods: This national study was conducted among 4811 students (2248 boys and 2563 girls) aged 6–18 y. This is the first study of its kind in Iran and, to our knowledge, in Asia as well. Two definitions were used for the MetS: type A was defined based on criteria analogous to ATP III, and type B was defined according to the cut‐offs obtained from NHANES III. Both types A and B define high fasting blood sugar as &gt; 100 mg/dl and systolic/diastolic blood pressure as &gt; 90th percentile. Results: The mean (SD) age of students studied was 12.07±3.2 y. MetS type A was seven times more prevalent than type B (14% vs 2%, respectively, p&lt;0.0001), and had no significant gender difference. The most frequent components of both definitions of the MetS were low high‐density lipoprotein cholesterol (HDL‐C) and high triglyceride (TG). Waist circumference (WC) and waist‐to‐hip ratio (WHR) had the strongest and weakest associations, respectively, with the MetS. Conclusion: Establishment of a uniform set of criteria for the MetS in children is needed. Routine WC measurement in the paediatric population may be clinically useful.

For the first time in Iran, and to the best of our knowledge in Asia, we assessed the anthropometric indices most closely correlated to cardiovascular disease (CVD) risk factors in a large nationally representative sample of children and adolescents to be used as a simple tool for identifying those at risk.This multi-center study was performed among a representative sample of 4811 school students (2248 boys and 2563 girls) aged 6-18 years, as part of the baseline survey of a national surveillance system. Anthropometric indices and CVD risk factors were measured using standard protocols, and their correlation was analyzed by using Receiver Operator Characteristic (ROC) curves and partial correlation.The most prevalent CVD risk factors were low HDL-C (28%), followed by hypertriglyceridemia (20.1%), and overweight (17%). The ROC analyses showed that among boys, all anthropometric indices had the same association with CVD risk factors in 6-9.9-year-age group, while in the 10-13.9 and 14-18-year-age groups, respectively waist circumference (WC) and body mass index (BMI) were the best in distinguishing CVD risk factors. Among girls, these indices were respectively BMI and waist to stature ratio (WSR); WC and WSR; and WC. In the partial correlation analysis, in boys, the highest coefficient was found for BMI; BMI and WC; and for WC and WSR; in girls, these indices were BMI; WC and WSR; and BMI respectively.In the present study, BMI, WC and WSR were the most appropriate in predicting CVD risk factors. It may be clinically useful in the pediatric population to routinely measure WC and WSR in addition to BMI as a screening tool to identify high-risk youth.

A digitonin-permeabilized HepG2 cell system has been developed and used to characterize the pathway responsible for apolipoprotein B100 (apoB) degradation. Degradation of 35S-labeled apoB occurred after an initial lag period and resulted in the loss of approximately 75.8% of apoB after 2 h of chase. The degradation rate in permeabilized cells was slower than that in intact cells, but approximately the same percentage of apoB was lost over 2 h of chase. The loss of intact apoB in permeabilized cells coincided with the appearance of a number of degradation fragments, including 335- and 70-kDa fragments. The detection of a 70-kDa fragment was a sensitive indicator of degradation. ApoB degradation was inhibited at temperatures below 37 degrees C and maximally activated at 42 degrees C. Degradation was also pH-dependent, with inhibition at pH > or = 7.5. ApoB decay was stimulated by ATP supplementation but not GTP and was inhibited in the presence of energy inhibitors. Degradation was not significantly affected by cycloheximide. However, the stability of the 70-kDa fragment was prolonged with cycloheximide. Preincubation of cells with brefeldin A and nocodazole, as well as monensin, did not diminish the degradation of intact apoB or the appearance of the 70-kDa fragment, suggesting a lack of requirement for intracellular transport from the endoplasmic reticulum. Among the various protease inhibitors tested, degradation was most sensitive to N-acetylleucylleucylnorleucinal (ALLN), which abolished the generation of the 70-kDa fragment in a dose-dependent manner. ALLN-sensitive degradation of apoB was unaffected by the calcium ionophore, A23187. Interestingly, degradation of unglycosylated apoB, detected in tunicamycin-pretreated cells, occurred earlier, resulted in generation of additional fragments, and was largely uninhibited by ALLN. In summary, apoB degradation occurs in permeabilized HepG2 cells by a temperature- and pH-sensitive, pre-Golgi degradation system and is catalyzed by a calcium-independent, ALLN-sensitive protease. Specificity of the initial apoB cleavage may require the proper N-linked glycosylation of the protein in the endoplasmic reticulum.

Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload-induced ER stress has been implicated in the development of insulin resistance. Here, evidence is provided for a molecular link between hepatic apolipoprotein B100 (apoB100), induction of ER stress, and attenuated insulin signaling. First, in vivo upregulation of hepatic apoB100 by a lipogenic diet was found to be closely associated with ER stress and attenuated insulin signaling in the liver. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further supported the link between excessive apoB100 expression and hepatic ER stress. Human apoB100 transgenic mice exhibited hypertriglyceridemia and hyperglycemia. In vitro, accumulation of cellular apoB100 by free fatty acid (oleate) stimulation or constant expression of wild-type or N-glycosylation mutant apoB50 in hepatic cells induced ER stress. This led to perturbed activation of glycogen synthase kinase 3 and glycogen synthase by way of the activation of c-Jun N-terminal kinase and suppression of insulin signaling cascade, suggesting that dysregulation of apoB was sufficient to disturb ER homeostasis and induce hepatic insulin resistance. Small interfering (si)RNA-mediated attenuation of elevated apoB level in the apoB50-expressing cells rescued cells from lipid-induced ER stress and reversed insulin insensitivity.These findings implicate apoB100 as a molecular link between lipid-induced ER stress and hepatic insulin resistance.

To assess whether patients after Kawasaki disease (KD) have increased risk factors and abnormalities suggestive of early atherosclerosis in systemic arteries.In a case-control study, we compared 52 patients after typical Kawasaki disease with varying coronary artery involvement (67% males; mean time from illness episode 11.2 +/- 3.7 years) studied between 10 and 20 years of age with 60 healthy control subjects (50% males). Brachial artery reactivity (BAR) was assessed using vascular ultrasonography, and atherosclerosis risk assessment was performed. Differences between cases and controls and factors associated with endothelial function in cases were determined.Case patients had lower resting systolic blood pressure (P < .001), lower apolipoprotein AI levels (P < .05), and higher levels of glycosylated hemoglobin (P = .007). There were no significant differences in BAR between case patients and control subjects in response to increased flow (P = .60) and nitroglycerine (P = .93). For case patients, significant factors in multivariable analysis for lower flow-mediated BAR included higher fasting triglyceride levels (P = .04) and lower free fatty acid levels (P < .001). No significant relationship was noted with past or current coronary artery involvement.Patients with KD have some abnormalities for risk factors for atherosclerosis, but systemic arterial endothelial dysfunction is not present in the long term.

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine307 phosphorylation of IRS-1. IRS-1 Serine307 phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IκB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKα/β, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10 mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.

There is growing evidence suggesting intestinal insulin resistance and overproduction of apolipoprotein (apo) B48–containing chylomicrons in insulin-resistant states. In the current study, we investigated the potential role of the inflammatory cytokine tumor necrosis factor-α (TNF-α) in the development of insulin resistance and aberrant lipoprotein metabolism in the small intestine in a Syrian golden hamster model. TNF-α infusion decreased whole-body insulin sensitivity, based on in vivo euglycemic clamp studies in chow-fed hamsters. Analysis of intestinal tissue in TNF-α–treated hamsters indicated impaired phosphorylation of insulin receptor-β, insulin receptor substrate-1, Akt, and Shc and increased phosphorylation of p38, extracellular signal–related kinase-1/2, and Jun NH2-terminal kinase. TNF-α infusion also increased intestinal production of total apoB48, triglyceride-rich lipoprotein apoB48, and serum triglyceride levels in both fasting and postprandial (fat load) states. The effects of TNF-α on plasma apoB48 levels could be blocked by the p38 inhibitor SB203580. Ex vivo experiments using freshly isolated enterocytes also showed TNF-α–induced p38 phosphorylation and intestinal apoB48 overproduction, effects that could be blocked by SB203580. Interestingly, TNF-α increased the mRNA and protein mass of intestinal microsomal triglyceride transfer protein without altering apoB mRNA levels. Enterocytes were found to have detectable levels of both TNF-α receptor types (p55 and p75), and antibodies against either of the two TNF-α receptors partially blocked the stimulatory effect of TNF-α on apoB48 production and p38 phosphorylation. In summary, these data suggest that intestinal insulin resistance can be induced in hamsters by TNF-α infusion, and it is accompanied by intestinal overproduction of apoB48-containing lipoproteins. TNF-α–induced stimulation of intestinal lipoprotein production appears to be mediated via TNF-α receptors and the p38 mitogen-activated protein kinase pathway.

To date, research on the influence of environmental factors on metabolic syndrome (MS) among youths is limited. This study was conducted to investigate for the first time the association of these factors with MS in a large national, representative sample of children from a non-Western population.The study population comprised of 4811 students (2248 boys and 2563 girls) aged 6-18 years, living in six different provinces in Iran. MS, defined based on criteria analogous to those of the Adult Treatment Panel III, was detected in 14.1% of participants. A birth weight of >4000 g in boys and <2500 g in girls increased the risk of having the MS [OR, 95% CI: 1.4 (1.007, 2.05) and 1.2 (1.1, 1.4), respectively]. Poorly educated parents and a positive parental history of chronic disease were other risks factors associated with MS. Low levels of physical activity significantly increased the risk of having MS [boys: 1.3 (1.1, 1.7); girls: 1.4 (1.2, 1.6)]. The risk of MS increased in-line with the consumption of solid hydrogenated fat [boys: 1.2 (1.07, 1.3); girls, 1.3 (1.1, 1.5)] and bread made with white flour [boys: 1.6 (1.3, 2.1); girls, 1.4 (1.1, 1.7)]. In contrast, an increased frequency of consumption of fruits and vegetable, as well as dairy products decreased the risk of having MS.Considering the effect of modifiable lifestyle habits and birth weight on MS in youths, urgent public health approaches should be directed towards primordial and primary prevention of this rapidly growing problem.

Insulin resistance is strongly associated with metabolic dyslipidemia, which is largely a postprandial phenomenon. Though previously regarded as a consequence of delayed triglyceride-rich lipoprotein clearance, emerging evidence present intestinal overproduction of apoB-48-containing lipoproteins as a major contributor to postprandial dyslipidemia. The majority of mechanistic information is however derived from animal models, namely the fructose-fed Syrian Golden hamster, and extension to human studies to date has been limited. Work in our laboratory has established that aberrant insulin signalling exists in the enterocyte, and that inflammation appears to induce intestinal insulin resistance. The intestine is a major site of lipid synthesis in the body, and upregulated intestinal de novo lipogenesis and cholesterogenesis have been noted in insulin resistant and diabetic states. There is also enhanced dietary lipid absorption attributable to changes in ABCG5/8, NPC1L1, CD36/FAT, and FATP4. Proteins that are involved in chylomicron assembly and secretion, including MTP, MGAT, DGAT, apoAI-V, and Sar1 GTPase, show evidence of increased expression and activity levels. Increased circulating free fatty acids, typically observed in insulin resistant states, may serve to deliver lipid substrates to the intestine for enhanced chylomicron assembly and secretion. To compound the dysregulation of intestinal lipid metabolism, there are changes in the secretion of gut-derived peptides, which include GLP-1, GLP-2, and GIP. Thus, accumulating evidence presents intestinal lipoprotein secretion as a highly regulated process that is sensitive to perturbations in whole body energy homeostasis, and is severely perturbed in insulin resistant states.

Purpose of review MicroRNAs (miRNAs) regulate gene expression by binding to target mRNAs and control a wide range of biological functions. Recent reports have identified specific miRNAs as major regulators of fatty acid and cholesterol homeostasis. This review examines the biological function of various miRNAs and the emerging evidence linking specific miRNAs to critical pathways in lipid metabolism. Recent findings Disruption of lipid balance can lead to metabolic disturbances and thus tight regulation is required to maintain lipid homeostasis. Recent studies have shown key roles for miR-33 and miR-122 in regulation of lipid metabolism, and further evidence implicates miR-370 in regulation of miR-122. In addition, miRNAs involved in adipogenesis (miR-378/378* and miR-27) as well as newly discovered miRNAs such as miR-613, miR-302a, and miR-168 have now been implicated in regulation of lipid metabolism. Summary Growing evidence support key roles for miRNAs in regulating both cholesterol and fatty acid metabolism, leading to considerable interest in miRNAs as potential drug targets to modulate lipid and lipoprotein metabolism. MiRNA-based therapeutics hold considerable promise in the fight to curtail the growing epidemic of obesity and type 2 diabetes and the associated risk of atherosclerosis.

Although the atherogenic role of dietary cholesterol has been well established, its diabetogenic potential and associated metabolic disturbances have not been reported. Diet-induced hamster models of insulin resistance and dyslipidemia were employed to determine lipogenic and diabetogenic effects of dietary cholesterol. Metabolic studies were conducted in hamsters fed diets rich in fructose (40%), fat (30%), and cholesterol (0.05–0.25%) (FFC) and other test diets. Short-term feeding of the FFC diet induced insulin resistance, glucose intolerance, hypertriglyceridemia, and hypercholesterolemia. Prolonged feeding (6–22 wk) of the FFC diet led to severe hepatic steatosis, glucose intolerance, and mild increases in fasting blood glucose, suggesting progression toward type 2 diabetes, but did not induce β-cell dysfunction. Metabolic changes induced by the diet, including dyslipidemia and insulin resistance, were cholesterol concentration dependent and were only markedly induced on a high-fructose and high-fat dietary background. There were significant increases in hepatic and plasma triglyceride with FFC feeding, likely due to a 10- to 15-fold induction of hepatic stearoyl-CoA desaturase compared with chow levels ( P &lt; 0.03). Hepatic insulin resistance was evident based on reduced tyrosine phosphorylation of the insulin receptor-β, IRS-1, and IRS-2 as well as increased protein mass of protein tyrosine phosphatase 1B. Interestingly, nuclear liver X receptor (LXR) target genes such as ABCA1 were upregulated on the FFC diet, and dietary supplementation with an LXR agonist (instead of dietary cholesterol) worsened dyslipidemia, glucose intolerance, and upregulation of target mRNA and proteins similar to that of dietary cholesterol. In summary, these data clearly implicate dietary cholesterol, synergistically acting with dietary fat and fructose, as a major determinant of the severity of metabolic disturbances in the hamster model. Dietary cholesterol appears to induce hepatic cholesterol ester and triglyceride accumulation, and diet-induced LXR activation (via cholesterol-derived oxysterols) may possibly be one key underlying mechanism.

Reference intervals provided on laboratory reports are essential for appropriate interpretation of test results, and can significantly impact clinical decision-making and the quality of patient care. Careful determination and/or validation of reference intervals by the laboratory for use in the patient population it serves are therefore important to ensure their proper utility. Unfortunately, critical gaps currently exist in accurate and up-to-date pediatric reference intervals for accurate interpretation of laboratory tests performed in children and adolescents. These critical gaps in the available pediatric laboratory reference intervals have the clear potential of contributing to erroneous diagnosis or misdiagnosis of many diseases of childhood and adolescence. Most of the available “normal” ranges for laboratory tests were determined over 2 decades ago on older instruments and technologies, and are no longer relevant considering the current testing technology used by clinical laboratories. It is thus critical and of utmost urgency that a more acceptable and comprehensive database be established. In the present review, we discuss the considerations and challenges faced when generating and validating reference intervals in accordance to the current guidelines published by the Clinical Laboratory Standards Institute (CLSI). We raise particular attention to the present-day deficiencies in available pediatric reference intervals, and highlight the special issues and unique difficulties that are additionally faced when establishing reference intervals in children. Finally, we highlight a recent Canadian initiative, the CALIPER project, whose mandate is to establish and maintain a database of comprehensive and up-to-date pediatric reference intervals to be eventually made available to all clinical laboratories worldwide.

Fasting dyslipidemia is commonly observed in insulin resistant states and mechanistically linked to hepatic overproduction of very low density lipoprotein (VLDL). Recently, the incretin hormone glucagon-like peptide-1 (GLP-1) has been implicated in ameliorating dyslipidemia associated with insulin resistance and reducing hepatic lipid stores. Given that hepatic VLDL production is a key determinant of circulating lipid levels, we investigated the role of both peripheral and central GLP-1 receptor (GLP-1R) agonism in regulation of VLDL production.The fructose-fed Syrian golden hamster was employed as a model of diet-induced insulin resistance and VLDL overproduction. Hamsters were treated with the GLP-1R agonist exendin-4 by intraperitoneal (ip) injection for peripheral studies or by intracerebroventricular (ICV) administration into the 3rd ventricle for central studies. Peripheral studies were repeated in vagotomised hamsters.Short term (7-10 day) peripheral exendin-4 enhanced satiety and also prevented fructose-induced fasting dyslipidemia and hyperinsulinemia. These changes were accompanied by decreased fasting plasma glucose levels, reduced hepatic lipid content and decreased levels of VLDL-TG and -apoB100 in plasma. The observed changes in fasting dyslipidemia could be partially explained by reduced respiratory exchange ratio (RER) thereby indicating a switch in energy utilization from carbohydrate to lipid. Additionally, exendin-4 reduced mRNA markers associated with hepatic de novo lipogenesis and inflammation. Despite these observations, GLP-1R activity could not be detected in primary hamster hepatocytes, thus leading to the investigation of a potential brain-liver axis functioning to regulate lipid metabolism. Short term (4 day) central administration of exendin-4 decreased body weight and food consumption and further prevented fructose-induced hypertriglyceridemia. Additionally, the peripheral lipid-lowering effects of exendin-4 were negated in vagotomised hamsters implicating the involvement of parasympathetic signaling.Exendin-4 prevents fructose-induced dyslipidemia and hepatic VLDL overproduction in insulin resistance through an indirect mechanism involving altered energy utilization, decreased hepatic lipid synthesis and also requires an intact parasympathetic signaling pathway.

Despite growing evidence for a causal role of environmental factors in autoimmune diseases including the rise in disease frequencies over the past several decades we lack an understanding of how particular environmental exposures modify disease risk. In addition, many autoimmune diseases display sex-biased incidence, with females being disproportionately affected but the mechanisms underlying this sex bias remain elusive. Emerging evidence suggests that both host metabolism and immune function is crucially regulated by the intestinal microbiome. Recently, we showed that in the non-obese diabetic (NOD) mouse model of Type 1 Diabetes (T1D), the gut commensal microbial community strongly impacts the pronounced sex bias in T1D risk by controlling serum testosterone and metabolic phenotypesCitation1. Here we present new data in the NOD model that explores the correlations between microbial phylogeny, testosterone levels, and metabolic phenotypes, and discuss the future of microbiome-centered analysis and microbe-based therapeutic approaches in autoimmune diseases.

To compare pediatric reference intervals calculated using hospital-based patient data with those calculated using samples collected from healthy children in the community as part of the CALIPER study.Hospital-based data for 13 analytes (calcium, phosphate, iron, ALP, cholesterol, triglycerides, creatinine, direct bilirubin, total bilirubin, ALT, AST, albumin and magnesium), measured on the Vitros 5600, collected between 2007 and 2011 were obtained. The data for each analyte were partitioned by age and gender as previously defined by the CALIPER study. Outliers in each partition were removed using the Tukey method. The cumulative distribution function (cdf) was then determined for each analyte value following which, the inverse cdf values of a standard Gaussian distribution were calculated. The analyte values were plotted against the inverse cdf of the standard Gaussian distribution. Piece-wise regression determined the linear portion of the resulting graph using the statistical software R. Linear regression determined an equation for the linear portion in each partition and reference intervals were calculated by extrapolating to identify the 2.5th and 97.5th centiles in each partition based on the inverse cdf values (which would correspond to the values -1.96 and 1.96 of the Gaussian distribution). Using the 90% confidence intervals for the reference intervals defined by CALIPER and the Reference Change Value (RCV) as the criteria, these calculated reference intervals were compared to those reported previously by CALIPER. Reference samples were also measured on the Vitros 5600 analyzer in an attempt to validate the calculated reference intervals.In general, the reference intervals calculated from hospital-based data were generally wider than those calculated by CALIPER. None of the reference intervals calculated using the Hoffmann approach fell completely within the 90% confidence intervals calculated by CALIPER.These results suggest that calculating pediatric reference intervals from hospital-based data may be useful, as a guide, in some cases but will likely not replace the need to establish reference intervals in healthy pediatric populations.

The intestinal overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles is a common feature of diabetic dyslipidemia and contributes to cardiovascular risk in insulin resistant states. We previously reported that glucagon-like peptide-2 (GLP-2) is a key endocrine stimulator of enterocyte fat absorption and chylomicron output in the postprandial state. GLP-2's stimulatory effect on chylomicron production in the postabsorptive state has been confirmed in human studies. The mechanism by which GLP-2 regulates chylomicron production is unclear, because its receptor is not expressed on enterocytes. We provide evidence for a key role of nitric oxide (NO) in mediating the stimulatory effects of GLP-2 during the postprandial and postabsorptive periods. Intestinal chylomicron production was assessed in GLP-2-treated hamsters administered the pan-specific NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), and in GLP-2-treated endothelial NOS knockout mice. L-NAME blocked GLP-2-stimulated apoB48 secretion and reduced triglycerides (TGs) in the TG-rich lipoprotein (TRL) fraction of the plasma in the postprandial state. Endothelial NOS-deficient mice were resistant to GLP-2 stimulation and secreted fewer large apoB48-particles. When TG storage pools were allowed to accumulate, L-NAME mitigated the GLP-2-mediated increase in TRL-TG, suggesting that NO is required for early mobilization and secretion of stored TG and preformed chylomicrons. Importantly, the NO donor S-nitroso-L-glutathione was able to elicit an increase in TRL-TG in vivo and stimulate chylomicron release in vitro in primary enterocytes. We describe a novel role for GLP-2-mediated NO-signaling as a critical regulator of intestinal lipid handling and a potential contributor to postprandial dyslipidemia.

Emerging evidence demonstrates a close interplay between disturbances in mitochondrial function and ER homeostasis in the development of the metabolic syndrome. The present investigation sought to advance our understanding of the communication between mitochondrial dysfunction and ER stress in the onset of hepatic steatosis in male rodents with defective peroxisome proliferator-activated receptor-α (PPARα) signaling. Genetic depletion of PPARα or perturbation of PPARα signaling by high-fructose diet compromised the functional activity of metabolic enzymes involved in mitochondrial fatty acid β-oxidation and induced hepatic mitochondrial stress in rats and mice. Inhibition of PPARα activity further enhanced the expression of apolipoprotein B (apoB) mRNA and protein, which was associated with reduced mRNA expression of the sarco/endoplasmic reticulum calcium ATPase (SERCA), the induction of hepatic ER stress, and hepatic steatosis. Restoration of PPARα activity recovered the metabolic function of the mitochondria and ER, alleviated systemic hypertriglyceridemia, and improved hepatic steatosis. These findings unveil novel roles for PPARα in mediating stress signals between hepatic subcellular stress-responding machinery and in the onset of hepatic steatosis under conditions of metabolic stress.

Nutrient sensing plays an important role in ensuring that appropriate digestive or hormonal responses are elicited following the ingestion of fuel substrates. Mechanisms of nutrient sensing in the oral cavity have been fairly well characterized and involve lingual taste receptors. These include heterodimers of G protein-coupled receptors (GPCRs) of the taste receptor type 1 (T1R) family for sensing sweet (T1R2-T1R3) and umami (T1R1-T1R3) stimuli, the T2R family for sensing bitter stimuli, and ion channels for conferring sour and salty tastes. In recent years, several studies have revealed the existence of additional nutrient-sensing mechanisms along the gastrointestinal tract. Glucose sensing is achieved by the T1R2-T1R3 heterodimer on enteroendocrine cells, which plays a role in triggering the secretion of incretin hormones for improved glycemic and lipemic control. Protein hydrolysates are detected by Ca2+-sensing receptor, the T1R1-T1R3 heterodimer, and G protein-coupled receptor 92/93 (GPR92/93), which leads to the release of the gut-derived satiety factor cholecystokinin. Furthermore, several GPCRs have been implicated in fatty acid sensing: GPR40 and GPR120 respond to medium- and long-chain fatty acids, GPR41 and GPR43 to short-chain fatty acids, and GPR119 to endogenous lipid derivatives. Aside from the recognition of fuel substrates, both the oral cavity and the gastrointestinal tract also possess T2R-mediated mechanisms of recognizing nonnutrients such as environmental contaminants, bacterial toxins, and secondary plant metabolites that evoke a bitter taste. These gastrointestinal sensing mechanisms result in the transmission of neuronal signals to the brain through the release of gastrointestinal hormones that act on vagal and enteric afferents to modulate the physiological response to nutrients, particularly satiety and energy homeostasis. Modulating these orally accessible nutrient-sensing pathways using particular foods, dietary supplements, or pharmaceutical compounds may have therapeutic potential for treating obesity and metabolic diseases.

Significance Sudden infant death syndrome (SIDS), the leading cause of postneonatal infant mortality, is defined as the sudden death of an infant less than 1 y of age that remains unexplained after a complete autopsy and death scene investigation. Although SIDS has been associated with deficiencies in central (brainstem) serotonin (5-hydroxytryptamine, 5-HT), there are no known peripheral biomarkers for SIDS. Here we demonstrate increased serum serotonin levels in a subset (31%) of SIDS infants compared with control infants. These findings suggest the potential of a high serum serotonin level as a forensic biomarker at autopsy to differentiate SIDS deaths with serotonergic defects from other causes of sudden death and, importantly, as evidence of a peripheral 5-HT abnormality in SIDS.

Reference intervals (RIs) are fundamental tools used by healthcare and laboratory professionals to interpret patient laboratory test results, ideally enabling differentiation of healthy and unhealthy individuals. Under optimal conditions, a laboratory should perform its own RI study to establish RIs specific for its method and local population. However, the process of developing RIs is often beyond the capabilities of an individual laboratory due to the complex, expensive and time-consuming process to develop them. Therefore, a laboratory can alternatively verify RIs established by an external source. Common RIs can be established by large, multicenter studies and can subsequently be received by local laboratories using various verification procedures. The standard approach to verify RIs recommended by the Clinical Laboratory Standards Institute (CLSI) EP28-A3c guideline for routine clinical laboratories is to collect and analyze a minimum of 20 samples from healthy subjects from the local population. Alternatively, "data mining" techniques using large amounts of patient test results can be used to verify RIs, considering both the laboratory method and local population. Although procedures for verifying RIs in the literature and guidelines are clear in theory, gaps remain for the implementation of these procedures in routine clinical laboratories. Pediatric and geriatric age-groups also continue to pose additional challenges in respect of acquiring and verifying RIs. In this article, we review the current guidelines/approaches and challenges to RI verification and provide a practical guide for routine implementation in clinical laboratories.

Abstract The diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection globally has relied extensively on molecular testing, contributing vitally to case identification, isolation, contact tracing, and rationalization of infection control measures during the coronavirus disease 2019 (COVID-19) pandemic. Clinical laboratories have thus needed to verify newly developed molecular tests and increase testing capacity at an unprecedented rate. As the COVID-19 pandemic continues to pose a global health threat, laboratories continue to encounter challenges in the selection, verification, and interpretation of these tests. This document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 provides interim guidance on: (A) clinical indications and target populations, (B) assay selection, (C) assay verification, and (D) test interpretation and limitations for molecular testing of SARS-CoV-2 infection. These evidence-based recommendations will provide practical guidance to clinical laboratories worldwide and highlight the continued importance of laboratory medicine in our collective pandemic response.

Abstract Coronavirus disease 2019 (COVID-19) is the third coronavirus outbreak that has emerged in the past 20 years, after severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). One important aspect, highlighted by many global health organizations, is that this novel coronavirus outbreak may be especially hazardous to healthcare personnel, including laboratory professionals. Therefore, the aim of this document, prepared by the COVID-19 taskforce of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), is to provide a set of recommendations, adapted from official documents of international and national health agencies, on biosafety measures for routine clinical chemistry laboratories that operate at biosafety levels 1 (BSL-1; work with agents posing minimal threat to laboratory workers) and 2 (BSL-2; work with agents associated with human disease which pose moderate hazard). We believe that the interim measures proposed in this document for best practice will help minimazing the risk of developing COVID-19 while working in clinical laboratories.

Abstract Routine biochemical and hematological tests have been reported to be useful in the stratification and prognostication of pediatric and adult patients with diagnosed coronavirus disease (COVID-19), correlating with poor outcomes such as the need for mechanical ventilation or intensive care, progression to multisystem organ failure, and/or death. While these tests are already well established in most clinical laboratories, there is still debate regarding their clinical value in the management of COVID-19, particularly in pediatrics, as well as the value of composite clinical risk scores in COVID-19 prognostication. This document by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Task Force on COVID-19 provides interim guidance on: (A) clinical indications for testing, (B) recommendations for test selection and interpretation, (C) considerations in test interpretation, and (D) current limitations of biochemical/hematological monitoring of COVID-19 patients. These evidence-based recommendations will provide practical guidance to clinical laboratories worldwide, underscoring the contribution of biochemical and hematological testing to our collective pandemic response.

Abstract With an almost unremittent progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections all around the world, there is a compelling need to introduce rapid, reliable, and high-throughput testing to allow appropriate clinical management and/or timely isolation of infected individuals. Although nucleic acid amplification testing (NAAT) remains the gold standard for detecting and theoretically quantifying SARS-CoV-2 mRNA in various specimen types, antigen assays may be considered a suitable alternative, under specific circumstances. Rapid antigen tests are meant to detect viral antigen proteins in biological specimens (e.g. nasal, nasopharyngeal, saliva), to indicate current SARS-CoV-2 infection. The available assay methodology includes rapid chromatographic immunoassays, used at the point-of-care, which carries some advantages and drawbacks compared to more conventional, instrumentation-based, laboratory immunoassays. Therefore, this document by the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) Taskforce on COVID-19 aims to summarize available data on the performance of currently available SARS-CoV-2 antigen rapid detection tests (Ag-RDTs), providing interim guidance on clinical indications and target populations, assay selection, and evaluation, test interpretation and limitations, as well as on pre-analytical considerations. This document is hence mainly aimed to assist laboratory and regulated health professionals in selecting, validating, and implementing regulatory approved Ag-RDTs.

Measuring the level of protection conferred by anti-SARS-CoV-2 (trimeric) spike or RBD (receptor binding domain) antibodies (especially total and IgG) is a suitable and reliable approach for predicting biological protection against the risk of infection and severe coronavirus disease 2019 (COVID-19) illness. Nonetheless, SARS-CoV-2 has undergone a broad process of recombination since the identification of the prototype lineage in 2019, introducing a huge number of mutations in its genome and generating a vast array of variants of interest (VoI) and concern (VoC). Many of such variants developed several mutations in spike protein and RBD, with the new Omicron (B.1.1.529) clade displaying over 30 changes, 15 of which concentrated in the RBD. Besides their impact on virus biology, as well as on the risk of detection failure with some molecular techniques (i.e., S gene dropout), recent evidence suggests that these mutations may also jeopardize the reliability of currently available commercial immunoassays for detecting anti-SARS-CoV-2 antibodies. The antigen (either spike or RBD) and epitopes of the prototype SARS-CoV-2 coated in some immunoassays may no longer reflect the sequence of circulating variants. On the other hand, anti-SARS-CoV-2 antibodies elicited by highly mutated SARS-CoV-2 variants may no longer be efficiently recognized by the currently available commercial immunoassays. Therefore, beside the compelling need to regularly re-evaluate and revalidate all commercially available immunoassays against live virus neutralization assays based on emerging VoCs or VoIs, diagnostic companies may also consider to redevelop their methods, replacing former SARS-CoV-2 antigens and epitopes with those of the new variants.

The intestine represents the body's largest interface between internal organs and external environments except for its nutrient and fluid absorption functions. It has the ability to sense numerous endogenous and exogenous signals from both apical and basolateral surfaces and respond through endocrine and neuronal signaling to maintain metabolic homeostasis and energy expenditure. The intestine also harbours the largest population of microbes that interact with the host to maintain human health and diseases. Furthermore, the gut is known as the largest endocrine gland, secreting over 100 peptides and other molecules that act as signaling molecules to regulate human nutrition and physiology. Among these gut-derived hormones, glucagon-like peptide 1 (GLP-1) and -2 have received the most attention due to their critical role in intestinal function and food absorption as well as their application as key drug targets. In this review, we highlight the current state of the literature that has brought into light the importance of GLP-1 and GLP-2 in orchestrating intestine-microbiota-immune system crosstalk to maintain intestinal barrier integrity, inflammation, and metabolic homeostasis.

To determine whether reduction of insulin resistance could ameliorate fructose-induced very low density lipoprotein (VLDL) oversecretion and to explore the mechanism of this effect, fructose-fed hamsters received placebo or rosiglitazone for 3 weeks. Rosiglitazone treatment led to normalization of the blunted insulin-mediated suppression of the glucose production rate and to a ∼2-fold increase in whole body insulin-mediated glucose disappearance rate (p < 0.001). Rosiglitazone ameliorated the defect in hepatocyte insulin-stimulated tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 and the reduced protein mass of IRS-1 and IRS-2 induced by fructose feeding. Protein-tyrosine phosphatase 1B levels were increased with fructose feeding and were markedly reduced by rosiglitazone. Rosiglitazone treatment led to a ∼50% reduction of VLDL secretion rates (p < 0.05)in vivo and ex vivo. VLDL clearance assessed directly in vivo was not significantly different in the FR (fructose-fed + rosiglitazone-treated) versus F (fructose-fed + placebo-treated) hamsters, although there was a trend toward a lower clearance with rosiglitazone. Enhanced stability of nascent apolipoprotein B (apoB) in fructose-fed hepatocytes was evident, and rosiglitazone treatment resulted in a significant reduction in apoB stability. The increase in intracellular mass of microsomal triglyceride transfer protein seen with fructose feeding was reduced by treatment with rosiglitazone. In conclusion, improvement of hepatic insulin signaling with rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, is associated with reduced hepatic VLDL assembly and secretion due to reduced intracellular apoB stability. To determine whether reduction of insulin resistance could ameliorate fructose-induced very low density lipoprotein (VLDL) oversecretion and to explore the mechanism of this effect, fructose-fed hamsters received placebo or rosiglitazone for 3 weeks. Rosiglitazone treatment led to normalization of the blunted insulin-mediated suppression of the glucose production rate and to a ∼2-fold increase in whole body insulin-mediated glucose disappearance rate (p < 0.001). Rosiglitazone ameliorated the defect in hepatocyte insulin-stimulated tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 and the reduced protein mass of IRS-1 and IRS-2 induced by fructose feeding. Protein-tyrosine phosphatase 1B levels were increased with fructose feeding and were markedly reduced by rosiglitazone. Rosiglitazone treatment led to a ∼50% reduction of VLDL secretion rates (p < 0.05)in vivo and ex vivo. VLDL clearance assessed directly in vivo was not significantly different in the FR (fructose-fed + rosiglitazone-treated) versus F (fructose-fed + placebo-treated) hamsters, although there was a trend toward a lower clearance with rosiglitazone. Enhanced stability of nascent apolipoprotein B (apoB) in fructose-fed hepatocytes was evident, and rosiglitazone treatment resulted in a significant reduction in apoB stability. The increase in intracellular mass of microsomal triglyceride transfer protein seen with fructose feeding was reduced by treatment with rosiglitazone. In conclusion, improvement of hepatic insulin signaling with rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, is associated with reduced hepatic VLDL assembly and secretion due to reduced intracellular apoB stability. very low density lipoprotein apolipoprotein B fructose-fed + placebo-treated hamsters free fatty acids fructose-fed + rosiglitazone-treated hamsters microsomal triglyceride transfer protein insulin receptor insulin receptor substrate-1 protein-tyrosine phosphatase 1B endogenous glucose appearance rate insulin-mediated glucose disappearance rate specific activity triglyceride free fatty acid The typical dyslipidemia of insulin-resistant states and Type 2 diabetes consists of hypertriglyceridemia due to VLDL1 overproduction, low high density lipoprotein cholesterol, and small dense low density lipoprotein particles (1Lewis G.F. Steiner G. Diabetes Metab. Rev. 1996; 12: 37-56Crossref PubMed Google Scholar). Elevated plasma free fatty acid (FFA) flux from peripheral and intra-abdominal adipose tissue depots due to resistance to the insulin anti-lipolytic and esterification effect in adipose tissue is felt to play an important role in driving VLDL assembly and secretion in insulin resistant states (2Ginsberg H.N. J. Clin. Invest. 2000; 106: 453-458Crossref PubMed Scopus (916) Google Scholar, 3Lewis G.F. Carpentier A. Adeli K. Giacca A. Endocr. Rev. 2002; 23: 201-229Crossref PubMed Scopus (829) Google Scholar, 4Lewis G.F. Curr. Opin. Lipidol. 1997; 8: 146-153Crossref PubMed Scopus (244) Google Scholar). Nevertheless, previous studies in humans suggest that insulin also has an important direct effect on the liver in controlling VLDL secretion (5Lewis G.F. Uffelman K.D. Szeto L.W. Weller B. Steiner G. J. Clin. Invest. 1995; 95: 158-166Crossref PubMed Google Scholar, 6Malmstrom R. Packard C.J. Caslake M. Bedford D. Stewart P. YkiJarvinen H. Shepherd J. Taskinen M.R. Diabetes. 1998; 47: 779-787Crossref PubMed Scopus (159) Google Scholar, 7Carpentier A. Patterson B.W. Uffelman K. Giacca A. Vranic M. Cattral M.S. Lewis G.F. Diabetes. 2001; 50: 1402-1413Crossref PubMed Scopus (62) Google Scholar).Rat and mouse models of insulin resistance and type 2 diabetes have provided important insights into the molecular mechanisms of insulin resistance. These animal models may not, however, be ideal for the study of human lipoprotein disorders because, unlike humans, their livers secrete both apoB48 and apoB100-containing VLDL, and they do not necessarily develop VLDL oversecretion as the basis for their hypertriglyceridemia (8Hirano T. Mamo J. Poapst M. Steiner G. Am. J. Physiol. 1988; 255: E236-E240PubMed Google Scholar, 9Li X. Grundy S.M. Patel S.B. J. Lipid Res. 1997; 38: 1277-1288Abstract Full Text PDF PubMed Google Scholar). Unlike livers from rat or mouse, the liver of the golden Syrian hamster secretes only apoB100-containing VLDL, and its lipoprotein metabolism more closely resembles that of humans (10Taghibiglou C. Rudy D. VanIderstine S.C. Aiton A. Cavallo D. Cheung R. Adeli K. J. Lipid Res. 2000; 41: 499-513Abstract Full Text Full Text PDF PubMed Google Scholar). We have shown that insulin resistance in the fructose-fed golden Syrian hamster is associated with mild hypertriglyceridemia, VLDL-apoB oversecretion, increased intracellular apoB-containing lipoprotein particle stability, and increased expression of microsomal triglyceride transfer protein (MTP) (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The present studies were conducted to explore the effect of improving insulin sensitivity in this insulin-resistant animal model by treatment with rosiglitazone, a peroxisome proliferator-activated receptor γ agonist and insulin sensitizer and to gain further insight into the molecular mechanisms of VLDL oversecretion in insulin resistant states.EXPERIMENTAL PROCEDURESAnimals and Study ProtocolsMale Syrian golden hamsters (Charles River, Quebec, Canada) were housed in pairs and given free access to food and water. After 7 days acclimatization, animals were placed on a fructose-enriched diet (hamster diet with 60% fructose, pelleted, Dyets Inc., Bethlehem, PA) for 5 weeks. After 2 weeks of feeding with the fructose-enriched diet, the animals were randomized to receive either rosiglitazone (20 μmol/kg/day) (GlaxoSmithKline) diluted in waterversus water only given once daily by gavage for the remaining 3 weeks of the fructose feeding period. At the end of the 5 weeks, the fructose-fed (F) and fructose-fed + rosiglitazone-treated (FR) animals underwent either one of the three in vivoprotocols described below or isolation of hepatocytes for the ex vivo protocols. In addition, some animals remained on regular chow for 5 weeks to serve as normal controls.In Vivo ProtocolsEuglycemic Hyperinsulinemic Clamp StudiesStudies were performed as previously described (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) with the following modifications. Catheters were kept patent overnight with 4% heparin in normal saline (Hepalean, Organon Teknica, 1000 IU/ml). At 8:00 a.m. the morning after insertion of femoral vein and arterial catheters, a primed (10 μCi) constant (0.1 μCi/min) infusion of high performance liquid chromatography-purified [3-3H]glucose (PerkinElmer Life Sciences) was started (time, 90 min) (12Schwenk W.F. Butler P.C. Haymond M.W. Rizza R.A. Am. J. Physiol. 1990; 258: E228-E233PubMed Google Scholar). [3-3H]Glucose was added to the 20% dextrose infusate to minimize the decline in glucose specific activity during the clamp. After 75 min of equilibration at time 0 min, a primed (80 milliunits/kg) constant insulin infusion (8 milliunits/kg/min in 0.1% bovine serum albumin in normal saline) (Humulin R, Eli Lilly, Canada) was started, and a D20% infusion was adjusted at 10-min intervals to maintain blood glucose at base-line level. Blood samples (0.25 ml) were taken from the arterial line at times 15, 0, 90, 100, 110, and 120 min of the clamp for measurement of blood glucose, [3-3H]glucose specific activity (SA), and plasma insulin levels. There was no significant decline in hematocrit throughout the study. Endogenous glucose production (Ra) was calculated as the endogenous rate of appearance measured with [3-3H]glucose using a modified one-compartment model (13Finegood D.T. Bergman R.N. Vranic M. Diabetes. 1987; 36: 914-924Crossref PubMed Google Scholar). Insulin-mediated glucose disappearance (ΔRd) was the rate of disappearance measured with [3-3H]glucose during the clamp minus the mean base-line Rd level. Data were smoothed with the optimal segments routine (14Finegood D.T. Bergman R.N. Am. J. Physiol. 1983; 244: E472-E479PubMed Google Scholar) using the optimal error algorithm (15Bradley D.C. Steil G.M. Bergman R.N. Am. J. Physiol. 1993; 264: E902-E911PubMed Google Scholar). Because euglycemia was not maintained in one hamster of the FR group, this animal was not included in the analysis of these experiments.In Vivo VLDL Secretion StudiesOne day before these studies, catheters were inserted into the femoral vein and artery of F (n = 10) and FR animals (n = 9) of similar weight (134 ± 3 g versus 132 ± 2 g, respectively, p = 0.64) and chow-fed controls (n = 5) as previously described (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). VLDL-apoB and VLDL-triglyceride (TG) secretion rates were measured in the fasting state (12 h) after intravenous injection of Triton WR-1339 (Sigma) as previously described (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The total blood volume of the samples drawn was less than 1.5 ml per animal during the experiment, and there was no significant decline in hematocrit.In Vivo VLDL Clearance StudiesBecause the Triton method does not allow direct assessment of VLDL clearance, the following studies were performed after a 12-h fast in 7 F and 8 FR animals of similar weight (129 ± 6 g versus 126 ± 4 g, respectively, p = 0.68). Catheters were inserted the day before these studies into the femoral vein and artery. A bolus (20 μCi) of [2-3H]glycerol (PerkinElmer Life Sciences) was injected intravenously, and blood samples were collected at times 10, 15, 20, 25, 30, 35, 40, and 50 min after the injection to measure VLDL-TG levels and to determine the rate of decline of VLDL-TG [3-3H]glycerol SA. The fractional clearance rate of VLDL-TG (pool/min) was assessed by the slope of the natural logarithm of VLDL-TG [2-3H] glycerol SA over time, determined by linear regression over the linear portion of the down-slope, as previously described (16Lemieux S. Patterson B.W. Carpentier A. Lewis G.F. Steiner G. J. Lipid Res. 1999; 40: 2111-2117Abstract Full Text Full Text PDF PubMed Google Scholar).Ex Vivo ProtocolsLiver Perfusion and Isolation of Primary Hamster HepatocytesAfter an overnight fast, the liver of animals from the F and FR groups was perfused under anesthesia, and hepatocytes released from digested liver tissue were transferred into culture medium and seeded in collagen-coated plates as previously described (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Determination of ex Vivo Tyrosine Phosphorylation of Insulin Receptor, IRS-1, and IRS-2 in Primary Hamster HepatocytesTo detect tyrosine phosphorylation of insulin receptor β (IR) subunits, IRS-1 and IRS-2, hepatocytes derived from fructose-fed and fructose-fed rosiglitazone-treated hamsters were incubated for 5 h in serum and insulin-free media. Cells were then stimulated with 100 nminsulin for 10 min at room temperature. Cells were lysed with a buffer containing phosphatase inhibitor mixture (150 mm NaCl, 10 mm Tris (pH 7.4), 1 mm EDTA, 1 mmEGTA, 1% Triton X-100, 1% Nonidet P-40, 2 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 100 mm sodium fluoride, 10 mm sodium pyrophosphate, and 2 mm sodium orthovanadate and subjected to immunoprecipitation with specific polyclonal antibodies (against insulin receptor β subunit or IRS-1) or a specific mouse monoclonal antibody against IRS-2. Immunoprecipitates were used for immunoblotting with monoclonal antibody αPY (1:1000 dilution) using ECL chemiluminescence system as described below.Determination of ex Vivo VLDL-apoB Secretion in Primary Hepatocyte CulturesRadiolabeled VLDL-apoB prepared from collected media by ultracentrifugation was subjected to immunoprecipitation and SDS-PAGE, and apoB band was quantified by liquid scintillation counting as described (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar).Pulse-Chase of Primary Hamster Hepatocytes to Assess Nascent ApoB Particle StabilityWe employed pulse-chase labeling experiments to assess the stability of apoB in hepatocytes isolated from fructose-fed hamsters treated with rosiglitazone versusplacebo, as described previously (17Adeli K. J. Biol. Chem. 1994; 269: 9166-9175Abstract Full Text PDF PubMed Google Scholar).Chemiluminescent ImmunoblottingCell samples were subjected to chemiluminescent immunoblotting for the protein mass of the MTP 97-kDa subunit, as previously described (11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). A similar method was utilized to measure protein expression levels of IR, IRS-1, IRS-2, and PTP-1B.Other Laboratory MethodsMeasurement of glucose, insulin, FFA, TG, apoB, [3-3H]glucose SA, and VLDL isolation were performed as previously described (7Carpentier A. Patterson B.W. Uffelman K. Giacca A. Vranic M. Cattral M.S. Lewis G.F. Diabetes. 2001; 50: 1402-1413Crossref PubMed Scopus (62) Google Scholar, 11Taghibiglou C. Carpentier A. Rudy D. Aiton A. Lewis G.F. Adeli K. J. Biol. Chem. 2000; 275: 8416-8425Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). VLDL-TG [2-3H]glycerol SA (dpm/mg) was determined as previously described (5Lewis G.F. Uffelman K.D. Szeto L.W. Weller B. Steiner G. J. Clin. Invest. 1995; 95: 158-166Crossref PubMed Google Scholar).Statistical AnalysisAll the values are reported as mean ± S.E. unless otherwise stated. For the euglycemic clamp studies, two-way analysis of variance was used to compare the glucose, insulin, Ra, and ΔRd curves of the F, FR, and control chow-fed groups at base line and during the last 30 min of the clamp, and the difference between the three groups was assessed by post-hoc analysis using Scheffe test. A two-tailed unpaired homoscedastic t test was used to compare all the other quantitative parameters between F and FR hamsters and between F and control chow-fed hamsters. A p value less than 0.05 was considered to be significant.RESULTSEffect of Rosiglitazone Treatment on Body Weight, Plasma Insulin, FFA, TG, and Glucose (Table I)Because of constraints imposed by the small blood volume of the animals, not all variables were measured on each animal undergoing the various experiments. Fasting plasma insulin was significantly lower (p = 0.02) in the FR and control chow-fed group than in the F group. Total plasma TG levels tended to be lower (by ∼30%,p = 0.16) after rosiglitazone treatmentversus the fructose-fed hamsters and were identical to TG levels in the control chow-fed hamsters. All other variables were not significantly different.Table ICharacteristics of F vs. FR vs. chow-fed control hamsters (S.E.)FnFRnControlsnΔ Weight (g)34 (1)5136 (2)5230 (3)19FFA (mmol/liter)0.625 (0.061)130.550 (0.097)130.689 (0.139)9Total TG (mmol/liter)2.06 (0.36)111.40 (0.24)91.40 (0.24)9Insulin (pmol/liter)331 (38)26221 (25)1-ap < 0.05 vs.F group. F, fructose fed; FR, fructose fed + rosiglitazone-treated.24140 (25)1-ap < 0.05 vs.F group. F, fructose fed; FR, fructose fed + rosiglitazone-treated.14Glucose (mmol/liter)4.3 (0.3)233.9 (0.3)223.5 (0.1)141-a p < 0.05 vs.F group. F, fructose fed; FR, fructose fed + rosiglitazone-treated. Open table in a new tab Treatment of Fructose-fed Hamsters with Rosiglitazone Ameliorates Whole-body Insulin Sensitivity and Improves Hepatocyte Insulin SignalingEuglycemic Hyperinsulinemic Clamp StudiesPlasma glucose (Fig. 1 A) was higher in the Fversus FR animals at base line (4.4 ± 0.3versus 3.2 ± 0.2 mmol/liter, p = 0.03) and during the last 30 min of the clamp (4.0 ± 0.3 mmol/literversus 3.0 ± 0.1 mmol/liter, p < 0.001) but was kept constant by design throughout the clamp. Hamsters fed a normal chow diet had intermediate glucose levels at base line (3.7 ± 0.2 mmol/liter) and during the last 30 min of the clamp (3.4 ± 0.1 mmol/liter, p < 0.001versus the F group). The insulin levels (Fig. 1 B) were similar throughout the clamp in the F, FR, and the control chow-fed group. Glucose SA (not shown) remained constant in the last 30 min of the clamp in the three groups. The endogenous glucose production rate (Ra) (Fig. 1 C) was significantly higher in the Fversus FR animals at base line (80.6 ± 12.2versus 54.0 ± 11.1 μmol/kg/min, p < 0.001) and throughout the clamp (51.9 ± 14.3 versus10.7 ± 7.0 μmol/kg/min, p < 0.001). Treatment of the F animals with rosiglitazone resulted in normalization of Ra at base line and during the clamp (p = NSversus control chow-fed group) and also led to normalization of the level of suppression of Ra from base line (Ra was suppressed to 64.7 ± 15.6 of base line versus 19.1 ± 11.4versus 13.1 ± 8.4% of base line level during the clamp in the F, FR, and control chow-fed group respectively,p < 0.001 for the difference between F and the two other groups). The glucose infusion rate (not shown) was significantly lower in the F versus the FR group during the last 30 min of the clamp (64.7 ± 8.7 versus 121.7 ± 25.1 μmol/kg/min, p < 0.001). However, rosiglitazone treatment did not completely correct the glucose infusion rate and remained lower than the control chow-fed group (glucose infusion rate of control chow-fed group, 176.2 ± 3.0 μmol/kg/min,p < 0.001 versus FR group). Consequently, insulin-mediated glucose disappearance rate (ΔRd) (Fig.1 D) during the clamp was also significantly lower in the Fversus FR animals (29.4 ± 8.4 versus75.3 ± 20.8 μmol/kg/min, p < 0.001) but was not completely normalized by treatment with rosiglitazone (ΔRd of control chow-fed group, 119.6 ± 5.1 μmol/kg/min,p < 0.001 versus FR).Insulin Signaling in Hamster Primary Hepatocyte CulturesIn hepatocytes isolated from F, insulin-stimulated insulin receptor β subunit tyrosine phosphorylation was reduced to 34.1 ± 2.6% (n = 3, p = 0.033) of that in control hepatocytes derived from chow-fed hamsters, and this was restored to the control levels (98.3 ± 0.5%, n = 3,p = 0.01 versus F) after rosiglitazone treatment, indicating complete restoration of insulin receptor phosphorylation by the drug (Fig.2 A). Insulin receptor appears as a doublet on the gel. We have consistently observed this doublet in hamster hepatocytes. We do not believe that the second band is a result of degradation, since the addition of protease inhibitors does not prevent the detection of the doublet (data not shown). Insulin-stimulated IRS-1 phosphorylation versus basal was 184.3 ± 22.6% in the control chow-fed (n = 4,p = 0.002), 130.3 ± 5.3% in F (n= 4, p = 0.007), and 188.9 ± 8.5% in FR (n = 4, p = 0.001) (Fig. 2 B) groups, indicating improvement of IRS-1 phosphorylation to the control levels in hepatocytes isolated from FR (p < 0.001 Fversus FR and p = 0.49 for control chow-fedversus FR groups). The effect of insulin on phosphorylation of IRS-2 was similar to that of IRS-1, as shown in Fig. 2 C, indicating significant reduction (n = 3,p = 0.01 versus control chow-fed group) in insulin-stimulated IRS-2 phosphorylation with fructose feeding and a marked improvement (n = 3, p = 0.004versus F) after treatment with rosiglitazone. As shown in Fig. 3 A, fructose feeding had no significant effect on IR protein mass (100 ± 14.1% in the control chow-fed group versus 88.3 ± 29.6% in F,n = 4, p = 0.3). However, in FR hepatocytes, IR protein mass was increased more than 2-foldversus cells derived from control chow-fed and F animals (212.6 ± 47% of control chow-fed animals, n = 4,p = 0.001 versus F). Fructose feeding reduced the protein mass of IRS-1 (Fig. 3 B) by 77% from 359.7 ± 23.9 scanning units/mg of total protein in hepatocytes from control chow-fed animals to 80 ± 11.5 in hepatocytes from F animals (n = 3, p = 0.0002versus control chow-fed animals). Rosiglitazone treatment partially restored IRS-1 mass to 52.8 ± 11.1% that in control chow-fed animals (n = 3, p = 0.003versus F). IRS-2 protein mass in hepatocytes isolated from F hamsters was reduced to 57.8 ± 7.1% (p = 0.001) that of the levels in control chow-fed animals, whereas rosiglitazone treatment increased protein mass to 74.1 ± 8% that of control hepatocytes (n = 4, p = 0.002versus F) (Fig. 3 C). These data suggest that the observed change in IR, IRS-1, and IRS-2 phosphorylation in hepatocytes isolated from FR may be partially due to change in protein expression levels of these proteins.Figure 2Insulin-mediated phosphorylation of the IR , IRS-1 , and IRS-2. Each panel depicts a representative immunoblot along with combined densitometric quantitation of multiple experiments performed in duplicate or triplicate. Net intensity of the bands was normalized for the total protein content of the samples. Shown are insulin-mediated phosphorylation of the insulin receptor (n = 3) (A), IRS-1 (n = 3) (B), and IRS-2 (n = 4) (C) in hepatocytes from control hamsters fed regular chow and from fructose-fed hamsters treated with rosiglitazone versusplacebo (n = 3 to 4 per experiment). All data are shown as the mean ± S.D.View Large Image Figure ViewerDownload (PPT)Figure 3Protein mass of IR , IRS-1 , and IRS-2.Representative immunoblots along with combined densitometric quantitation of 3–4 experiments performed in duplicate or triplicate for IR (A), IRS-1 (B), IRS-2 (C), and PTP-1B (D), respectively. Net intensity of the bands was normalized for the total protein content of the samples and is either expressed as scanning unit/mg of total protein (panel B) or percent of control cells (panels A, C, andD). Solid, open, and gray bars represent IR, IRS-1, and IRS-2 protein mass in control chow-fed, fructose-fed, and fructose-fed + rosiglitazone-treated hepatocytes, respectively. All data are shown as the mean ± S.D.View Large Image Figure ViewerDownload (PPT)Interestingly, PTP-1B protein mass increased to 169.9 ± 13.2% (n = 3, p = 0.0002) that of controls with fructose feeding. FR had marked reduction of PTP-1B levels to 24.4 ± 12.9% that of control chow-fed animals (n= 3, p = 0.0004 versus F) (Fig.3 D).Treatment of Fructose-fed Hamsters with Rosiglitazone Ameliorates VLDL-apoB and VLDL-TG Oversecretion in Vivo and ex Vivo without Affecting VLDL ClearanceThe slope of the increase in VLDL-apoB (Fig.4 A) over time after the injection of Triton WR-1339 was significantly steeper in the Fversus FR group (2.42 ± 0.51 versus1.09 ± 0.27 μg/ml/min, p < 0.05) andversus the control chow-fed group (0.3 ± 0.1 μg/ml/min, p < 0.05). Consequently, the VLDL-apoB secretion rate was higher in the F versus FR group (12.4 ± 2.7 versus 5.5 ± 1.4 μg/min, respectively, p < 0.05) and versus the control chow-fed group (1.3 ± 0.3 μg/min, p < 0.05) (inset of Fig. 4 A). Similarly, VLDL-TG increase over time after the injection of Triton WR-1339 (Fig.4 B) was significantly higher in F versus FR hamsters (0.024 ± 0.004 versus 0.011 ± 0.004 μmol/ml/min, respectively, p < 0.05) andversus the control chow-fed group (0.009 ± 0.002 μmol/ml/min, p < 0.05). The VLDL-TG secretion rate (inset of Fig. 4 B) was higher in the F than in the FR group (0.12 ± 0.02 versus 0.06 ± 0.02 μmol/min respectively, p < 0.05) and higher than the control chow-fed group (0.04 ± 0.01 μmol/min, p< 0.05). As depicted in Fig. 4 C, rosiglitazone treatment significantly reduced ex vivo VLDL-apoB secretion to 38 ± 32% (mean ± S.D., n = 4, p < 0.001) that of fructose-fed hepatocytes, in keeping with the in vivo findings. In vivo VLDL-TG fractional clearance rate, as determined from the [2-3H]glycerol bolus studies, was not significantly different between the Fversus FR animals (0.034 ± 0.008 versus0.025 ± 0.004 min−1 respectively, p= 0.33), although clearance tended to be slightly delayed in the latter.Figure 4VLDL-apoB and VLDL-TG secretion rates.Shown are in vivo VLDL-apoB (A) and VLDL-TG (B) levels over time after intravenous injection of Triton WR-1339 (600 mg/kg) in fructose-fed hamsters treated with rosiglitazone (light gray circles, n = 10)versus placebo (closed circles, n= 9) and control chow-fed hamsters (dark gray squares,n = 5). Insets in A andB show VLDL-apoB and VLDL-TG secretion rate, respectively, in the rosiglitazone (light gray bars), placebo-treated group (closed bars), and in the control chow-fed group (dark gray bars). Ex vivo VLDL-apoB secretion rate (C) in hepatocytes derived from fructose-fed hamsters treated with rosiglitazone (open bars, n = 4) versus placebo (closed bars, n= 3). Data are shown as the mean ± S.D.View Large Image Figure ViewerDownload (PPT)Treatment of Fructose-fed Hamsters with Rosiglitazone Leads to Intracellular Destabilization of Nascent VLDL Particles and Correction of Enhanced Expression of MTPIn pulse-chase labeling experiments, after a 1-h chase, there was a significant reduction in the fraction of apoB secreted (Fig.5 A) in hepatocytes from Fversus FR animals (88 ± 3% versus 49 ± 6% respectively, p = 0.001). Decreased secretion was also accompanied with a significant decrease in total apoB recovered (Fig. 5 B). There was also a significant reduction in the fraction of labeled apoB secreted in the FR versus F animals after a 2-h chase (53 ± 7% versus 97 ± 1% in the FR versus F animals, respectively,p = 0.004), and similarly higher levels of total apoB were recovered, suggesting that rosiglitazone treatment led to destabilization and increased degradation of nascent apoB-containing particles. The cellular protein mass of MTP in hepatocytes from F was 153.3 ± 6.6% (n = 4, p = 0.0002) that of controls (Fig. 5 C). Rosiglitazone treatment led to normalization of cellular protein mass of MTP in fructose-fed hamsters to 107.0 ± 9.4% that of controls (n = 4,p < 0.005 versus F).Figure 5Pulse-chase labeling experiments to assess the stability of apoB in hepatocytes from fructose-fed hamsters treated with rosiglitazone. A, distribution of immunoprecipitable apoB in media (Secreted apoB). B, immunoprecipitable apoB remaining in cells + media (total apoB). The fructose-fed + rosiglitazone-treated (closed circles)versus fructose-fed + placebo-treated group (open circles) expressed as a percentage of radiolabeled apoB at time 0. *, significantly different from fructose-fed hepatocytes (secreted apoB; p = 0.001 at 1 h, p = 0.004 at 2 h). **, significantly different from fructose-fed hepatocytes (total apoB; p = 0.001 at 1 h, p = 0.0095 at 2 h) (n = 3). C, microsomal MTP expression. Data are shown for hepatocytes from control hamsters fed regular chow and from fructose-fed hamsters treated with rosiglitazone versus placebo as indicated (n= 4 per group, p < 0.005 for the difference between fructose-fed + rosiglitazone versus fructose-fed + placebo animals). The MTP bands were quantitated by densitometric scanning, and the mass of the 97-kDa MTP subunit detected was expressed as a percentage of the MTP mass detected in control cells. Please note that the blot shows the result of one representative experiment, whereas the graph displays the mean ± S.D. of four independent experiments. They are not therefore exactly the same. Data are the mean ± S.D.View Large Image Figure ViewerDownload (PPT)DISCUSSIONIn the present study we have demonstrated that treatment of fructose-fed insulin-resistant ham

Insulin resistance and type 2 diabetes are rapidly emerging as major disorders of childhood and adolescence. This appears to be closely linked to a rapid rise in the prevalence of obesity in the pediatric population. The development of insulin resistance appears to lead to a “metabolic syndrome” which includes a number of major complications such as dyslipidemia and hypertension. Childhood metabolic syndrome promotes the development of premature atherosclerosis and significantly increases cardiovascular disease risk early in life. The mechanisms linking obesity, insulin resistance, and metabolic dyslipidemia are not fully understood. This review will attempt to discuss some of the key mechanistic issues surrounding insulin resistance and its association with metabolic dyslipidemia. Most of the recent progress in this field has come from the use of genetic and diet-induced animal models of insulin resistance. New data from these animal studies particularly the fructose-fed hamster, a model of metabolic syndrome and dyslipidemia, will be reviewed. Evidence from both animal and human studies suggest a key role for insulin sensitive tissues such as adipose tissue, liver, and intestine in the development of an insulin resistant state and its associated lipid and lipoprotein disorders. The critical interaction of metabolic signals among these tissues appears to govern the transition from an insulin sensitive to an insulin resistant state that underlies dyslipidemic conditions.

We investigated whether intestinal lipoprotein overproduction in a fructose-fed, insulin-resistant hamster model is prevented with insulin sensitization. Syrian Golden hamsters were fed either chow, 60% fructose for 5 wk, chow for 5 wk with the insulin sensitizer rosiglitazone added for the last 3 wk, or 60% fructose plus rosiglitazone. In vivo Triton studies showed a 2- to 3-fold increase in the large (Svedberg unit > 400) and smaller (Sf 100-400) triglyceride-rich lipoprotein particle apolipoprotein B48 (apoB48) but not triglyceride secretion with fructose feeding in the fasted state (P < 0.01) and partial normalization with rosiglitazone in fructose-fed hamsters. Ex vivo pulse-chase labeling of enterocytes confirmed the oversecretion of apoB48 lipoproteins with fructose feeding. Intestinal lipoprotein oversecretion was associated with increased expression of microsomal triglyceride transfer protein expression. With rosiglitazone treatment of fructose-fed hamsters, there was approximately 50% reduction in apoB48 secretion from primary cultured enterocytes and amelioration of the elevated microsomal triglyceride transfer protein mass and activity in fructose-fed hamsters. In contrast, in the postprandial state, the major differences between nutritional and drug intervention protocols were evident in triglyceride-rich lipoprotein triglyceride and not apoB48 secretion rates. The data suggest that intestinal lipoprotein overproduction can be ameliorated with the insulin sensitizer rosiglitazone.

A simple, rapid method for isolating human DNA has been developed, which can be routinely used in clinical chemistry laboratories. The entire procedure takes less than 90 min, and as many as 12 blood samples can be handled in one cycle. One milliliter of EDTA-treated blood is lysed and centrifuged to yield a nuclear fraction. The nuclear pellet is treated with sodium dodecyl sulfate/urea and phenol/chloroform to remove contaminating proteins, then the crude DNA extract is purified by use of a Sephadex G-25 spin-column. Typical 260 nm/280 nm absorbance ratio (used to assess purity) and yield for DNA so purified were 1.84 and 24.5 micrograms/mL, respectively. Within- and between-day CVs for recovery of DNA from pooled blood were 8% and 11% respectively. Such DNA preparations were found quite suitable for digestion by a variety of restriction endonucleases and for restriction fragment length polymorphism analysis. We are using this method to isolate DNA from whole blood of myocardial infarction patients for studies on the apolipoprotein B gene.

The precise biochemical mechanisms underlying the reduction of HDL levels in hypertriglyceridemic states are currently not known. In humans, we showed that triglyceride (TG) enrichment of HDL, as occurs in hypertriglyceridemic states, enhances the clearance of HDL-associated apolipoprotein A-I (apoA-I) from the circulation. In the New Zealand White rabbit (an animal model naturally deficient in hepatic lipase [HL]), however, TG enrichment of HDL is not sufficient to alter the clearance of either the protein or lipid moieties of HDL. In the present study, therefore, we determined in the New Zealand White rabbit the combined effects of ex vivo TG enrichment and lipolytic transformation of HDL by HL on the subsequent metabolic clearance of HDL apoA-I. Results of the in vivo kinetic studies (n=18 animals) showed that apoA-I associated with TG-enriched rabbit HDL modified ex vivo by catalytically active HL was cleared 22% more rapidly versus TG-enriched HDL incubated with heat-inactivated HL, and 26% more rapidly than fasting (TG-poor) HDL incubated with active HL (P<0.05 for both). Furthermore, a strong correlation was observed between the HDL TG content and apoA-I fractional catabolic rate (0.59, P<0.05) in the combined active HL groups. These data establish that TG enrichment of HDL with subsequent lipolysis by HL enhances HDL apoA-I clearance, but neither TG enrichment of HDL without HL lipolysis nor HL lipolysis in the absence of previous TG enrichment of HDL is sufficient to enhance HDL clearance. These data further support the important interaction between HDL TG enrichment and HL action in the pathogenesis of HDL lowering in hypertriglyceridemic states.

Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway.HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins.Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

The present study evaluated the effect of green tea (Camellia sinensis L.) leaf extract on triglyceride and glucose homeostasis in a fructose-fed hypertriglyceridemic, insulin-resistant hamster model. There was a significant decrease in plasma triglyceride levels following supplementation of the green tea epigallocatechin gallate-enriched extract (42% at 150 mg/(kg day) to 62% at 300 mg/(kg day) for 4 weeks). Compared to baseline, the fructose control group at the end of the study showed elevated serum insulin and apolipoprotein B levels, and decreased serum adiponectin levels. The fructose/green tea extract group showed a reversal in all of these metabolic defects, including an improvement in glucose levels during a glucose tolerance test. Triglyceride content was also examined in various tissues and compared to the control fructose group; supplementation of the green tea extract (300 mg/kg) reduced triglyceride content in liver and heart tissues. There was molecular evidence of improved lipid and glucose homeostasis based on peroxisome proliferator-activated receptor (PPAR) protein expression. Compared to the control fructose group, supplementation of the green tea extract (300 mg/kg) significantly increased PPARalpha and PPARgamma protein expression. In summary, the data suggest that intake of the green tea extract ameliorated the fructose-induced hypertriglyceridemia and the insulin-resistant state in part through PPAR.

A comprehensive set of age- and gender-specific pediatric reference intervals is essential for accurate interpretation of laboratory tests in a pediatric setting.1459 serum/plasma from children attending select outpatient clinics and deemed to be metabolically stable, were collected from five age groups; 0-12 months, 1-5 years, 6-10 years, 11-14 years and 15-20 years. Samples were analyzed for 24 chemistries and 15 immunoassays on ARCHITECT ci8200.Reference intervals were established according to CLSI/IFCC C28-P3 guidelines by the Robust statistical method. The ranges reflect the central 95% confidence intervals for the population tested. Age and gender were partitioned using the Harris-Boyd method.While these intervals are ci8200 method specific, they not only provide robust intervals for users of this system but are also useful for any laboratory requiring pediatric intervals if they can be shown to be transferable and if validated for the local patient population.

Ghrelin has been proposed to be a regulator of energy balance, and its dysregulation may be important in obesity. The aims of this study were (i) to compare short- and long-term changes in circulating ghrelin concentration after increasing energy expenditure vs. its changes after decreasing energy intake, (ii) to determine factors associated with changes in ghrelin level, and (iii) to assess relationships of ghrelin concentration with metabolic syndrome (MetS) in prepubescent obese children.Randomized controlled trial.About 100 obese children aged 7-9 years.After baseline testing, children were randomly assigned to two interventional groups, either receiving dietary recommendations or engaging in physical training classes for 6 months. Ghrelin, insulin, leptin, fasting blood sugar, lipid profile and anthropometric indexes, as well as energy intake and expenditure were measured.Of the participants, 92 completed the 6-month trial, and 87 returned for the 1-year follow-up. Except ghrelin level, other biochemical variables had no significant change at 12- vs. 6-month follow-up. In both groups, ghrelin showed a progressive increase in the periods of time with significant reduction of overweight and negative energy balance; while after the end of the trial, when children regained weight, it decreased toward baseline levels. Baseline ghrelin had strong negative correlation with measures of central obesity. The odds of having the MetS were 12% lower in the middle and 37% lower in the highest tertile of ghrelin level. As the number of MetS components increased, there was a progressive decrease in ghrelin and quantitative insulin sensitivity check index (QUICKI), with a progressive increase in serum insulin, HOMA-R and leptin levels.Ghrelin increases in response to overweight reduction and negative energy balance resulting from either an exercise intervention or reduction in food intake in prepubescent obese children. It is unlikely to regulate long-term energy balance in young obese children.

This study was undertaken to determine the association of serum C-reactive protein (CRP) with generalized and abdominal obesity, body fat composition, the metabolic syndrome, and oxidative stress markers among young people.We conducted a population-based study of 512 young people, aged 10-18 years. We obtained anthropometric and blood pressure measurements. Fasting blood sugar, total cholesterol (TC), HDL-cholesterol, triglycerides, CRP, malondialdehyde (MDA), and conjugated diene (CDE) were quantified. LDL-cholesterol (LDL-C) was calculated for samples with TG < or =4.52 mmol/L RESULTS: Mean triglycerides, waist and hip circumferences, percentage body fat, subcutaneous fat, and systolic blood pressure increased significantly with increasing body mass index (BMI). In contrast, the mean LDL and TC were higher in underweight than normal weight individuals, and then increased significantly from normal to higher BMI categories. Mean HDL cholesterol significantly decreased with increasing BMI. Overall, CRP, MDA, and CDE were significantly correlated with measures of abdominal obesity. Serum CRP, MDA, and CDE significantly increased in the upper quartiles of waist circumference. Study participants with higher CRP concentrations were more likely to have metabolic syndrome and high oxidative stress markers.We found a significant positive association between CRP and oxidative stress markers in healthy young people, as well as an increase in these markers in the upper quartiles of waist circumference, but not BMI. Oxidative stress and CRP may interact in the early inflammatory processes of atherosclerosis.

Pediatric serum samples collected from healthy children in the CALIPER (Canadian Laboratory Initiative in Pediatric Reference Interval) project are stored at − 80 °C for various periods of time. This study aimed to determine the stability of chemistry, protein, and hormone analytes under these conditions. Serum samples collected from children of 0–18 years of age attending outpatient clinics were pooled into a single pool or into age-group specific pools. Following baseline measurement, each pool was aliquoted and kept frozen at − 80 °C until analysis. Samples were analyzed for 57 biochemical markers at monthly intervals over a 10–13 month period and each aliquot was subject to one freeze–thaw cycle before analysis. The analysis was performed on VITROS® Chemistry System, COBAS INTEGRA® 400 Plus and IMMULITE® 2500. Values obtained at monthly intervals were compared to baseline measurements and examined for trends over time. A majority of analytes measured in this study showed no significant time-dependent change relative to baseline or trend over time after up to 13 months of storage. PTH showed up to 27.2% decline after 10 months of storage with most of the decline evident after 2 months. Most analytes showed variability over time, which is thought to reflect assay variability rather than changes in analyte stability. The study shows stability for a majority of analytes stored in serum at − 80 °C after up to 13 months of storage. Samples do not require immediate testing for reference interval determination for the selected analytes with possible exception of PTH.

Induction of endoplasmic reticulum (ER) stress was previously shown to impair hepatic apolipoprotein B100 (apoB) production by enhancing cotranslational and posttranslational degradation of newly synthesized apoB. Here, we report the involvement of autophagy in ER stress–induced degradation of apoB and provide evidence for a significant role of autophagy in regulating apoB biogenesis in primary hepatocyte systems. Induction of ER stress following short-term glucosamine treatment of McA-RH7777 cells resulted in significantly increased colocalization of apoB with green fluorescent protein–microtubule-associated protein 1 light chain 3 (GFP-LC3), referred to as apoB-GFP-LC3 puncta, in a dose-dependent manner. Colocalization with this autophagic marker correlated positively with the reduction in newly synthesized apoB100. Treatment of McA-RH7777 cells with 4-phenyl butyric acid, a chemical ER stress inhibitor, prevented glucosamine- and tunicamycin-induced increases in GRP78 and phosphorylated eIF2α, rescued newly synthesized [35S]-labeled apoB100, and substantially blocked the colocalization of apoB with GFP-LC3. Autophagic apoB degradation was also observed in primary rat and hamster hepatocytes at basal conditions as well as upon the induction of ER stress. In contrast, this pathway was inactive in HepG2 cells under ER stress conditions, unless proteasomal degradation was blocked with N-acetyl-leucinyl-leucinyl-norleucinal and the medium was supplemented with oleate. Transient transfection of McA-RH7777 cells with a wild-type protein kinase R–like ER kinase (PERK) complementary DNA resulted in dramatic induction of apoB autophagy. In contrast, transfection with a kinase inactive mutant PERK gave rise to reduced apoB autophagy, suggesting that apoB autophagy may occur via a PERK signaling–dependent mechanism. Conclusion: Taken together, these data suggest that induction of ER stress leads to markedly enhanced apoB autophagy in a PERK-dependent pathway, which can be blocked with the chemical chaperone 4-phenyl butyric acid. ApoB autophagy rather than proteasomal degradation may be a more pertinent physiological mechanism regulating hepatic lipoprotein production in primary hepatocytes. (HEPATOLOGY 2011;)

Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and is now considered to be the hepatic manifestation of the metabolic syndrome. However, the role of steatosis per se and the precise factors required in the progression to steatohepatitis or insulin resistance remain elusive. The JAK-STAT pathway is critical in mediating signaling of a wide variety of cytokines and growth factors. Mice with hepatocyte-specific deletion of Janus kinase 2 (L-JAK2 KO mice) develop spontaneous steatosis as early as 2 weeks of age. In this study, we investigated the metabolic consequences of jak2 deletion in response to diet-induced metabolic stress. To our surprise, despite the profound hepatosteatosis, deletion of hepatic jak2 did not sensitize the liver to accelerated inflammatory injury on a prolonged high fat diet (HFD). This was accompanied by complete protection against HFD-induced whole-body insulin resistance and glucose intolerance. Improved glucose-stimulated insulin secretion and an increase in β-cell mass were also present in these mice. Moreover, L-JAK2 KO mice had progressively reduced adiposity in association with blunted hepatic growth hormone signaling. These mice also exhibited increased resting energy expenditure on both chow and high fat diet. In conclusion, our findings indicate a key role of hepatic JAK2 in metabolism such that its absence completely arrests steatohepatitis development and confers protection against diet-induced systemic insulin resistance and glucose intolerance.

Plasma C-reactive protein (CRP) concentration is increased in the metabolic syndrome, which consists of a cluster of cardiovascular disease risk factors, including insulin resistance. It is not known, however, whether CRP is merely a marker of accompanying inflammation or whether it contributes causally to insulin resistance. The objective of this study is to investigate the role that CRP may play in the development of insulin resistance. We examined the effect of single-dose intravenous administration of purified human (h)CRP on insulin sensitivity in Sprague-Dawley rats using the euglycemic, hyperinsulinemic clamp technique. hCRP was associated with impaired insulin suppression of endogenous glucose production with no reduction in peripheral tissue glucose uptake, suggesting that hCRP mediated insulin resistance in the liver but not extrahepatic tissues. We further assessed components of the insulin signaling pathway and mitogen-activated protein kinases (MAPKs) in the liver. Liver tissues derived from hCRP-treated rats showed reduced insulin-stimulated insulin receptor substrate (IRS) tyrosine phosphorylation, IRS/phosphatidylinositol 3-kinase (PI3K) association, and Akt phosphorylation, consistent with hCRP-induced impairment of hepatic insulin signaling. Furthermore, hCRP enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK as well as IRS-1 Ser(612) . Finally, we observed in primary cultured rat hepatocytes that U0126 (a selective inhibitor of MAPK/ERK kinase1/2) corrected hCRP-induced impairment of insulin signaling.hCRP plays an active role in inducing hepatic insulin resistance in the rat, at least in part by activating ERK1/2, with downstream impairment in the insulin signaling pathway.

Retrospective studies suggest that adolescents with craniopharyngioma and hypothalamic obesity have increased sleep-disordered breathing (SDB).The objectives of this study were to compare the prevalence of SDB in adolescents with craniopharyngioma-related obesity compared with body mass index (BMI)-matched controls and to explore possible relationships between SDB, insulin resistance, and adipocytokines.This was a cross-sectional study of obese craniopharyngioma and obese control adolescents.Subjects were evaluated in the clinical investigation unit at the Hospital for Sick Children, Toronto.Fifteen patients with craniopharyngioma-related obesity and 15 BMI-matched controls were recruited and tested.Each subject underwent fasting blood work, frequent sampled iv glucose tolerance test, polysomnography, and abdominal magnetic resonance imaging with calculation of visceral and sc adipose tissue.Main measures included insulin sensitivity, sleep efficiency, and fragmentation.Insulin sensitivity was lower in craniopharyngioma subjects compared with control subjects (0.96 +/- 0.34 vs. 1.67 +/- 0.7, P = 0.01). Sleep-onset latency (19.3 +/- 27.8 vs. 31.9 +/- 23.4, P = 0.03) and oxygen saturations (rapid eye movement sleep: 89.0 +/- 5.1 vs. 94.2 +/- 2.3, P < 0.001; non-rapid eye movement sleep: 88.4 +/- 5.6 vs. 94.3 +/- 1.5, P < 0.001) were lower in craniopharyngioma. Obstructive apnea-hypopnea index (OAHI) (7.5 +/- 9.0 vs. 1.5 +/- 1.5, P = 0.03) was higher in craniopharyngioma. Respiratory distress index and OAHI correlated negatively with adiponectin concentrations (r = -0.61, P = 0.03, r = -0.71, P = 0.006, respectively) in craniopharyngioma. On multiple regression, TNF-alpha and craniopharyngioma were independent positive predictors of sleep-onset latency and adiponectin and craniopharyngioma were significant predictors (negative and positive, respectively) of OAHI.SDB is increased in adolescents with craniopharyngioma-related obesity compared with BMI-matched controls. Routine polysomnography should be considered in obese patients with craniopharyngioma and appropriate treatment initiated.

Regulated cell metabolism involves acute and chronic regulation of gene expression by various nutritional and endocrine stimuli. To respond effectively to endogenous and exogenous signals, cells require rapid response mechanisms to modulate transcript expression and protein synthesis and cannot, in most cases, rely on control of transcriptional initiation that requires hours to take effect. Thus, co- and posttranslational mechanisms have been increasingly recognized as key modulators of metabolic function. This review highlights the critical role of mRNA translational control in modulation of global protein synthesis as well as specific protein factors that regulate metabolic function. First, the complex lifecycle of eukaryotic mRNAs will be reviewed, including our current understanding of translational control mechanisms, regulation by RNA binding proteins and microRNAs, and the role of RNA granules, including processing bodies and stress granules. Second, the current evidence linking regulation of mRNA translation with normal physiological and metabolic pathways and the associated disease states are reviewed. A growing body of evidence supports a key role of translational control in metabolic regulation and implicates translational mechanisms in the pathogenesis of metabolic disorders such as type 2 diabetes. The review also highlights translational control of apolipoprotein B (apoB) mRNA by insulin as a clear example of endocrine modulation of mRNA translation to bring about changes in specific metabolic pathways. Recent findings made on the role of 5'-untranslated regions (5'-UTR), 3'-UTR, RNA binding proteins, and RNA granules in mediating insulin regulation of apoB mRNA translation, apoB protein synthesis, and hepatic lipoprotein production are discussed.

To develop an accurate assay and establish the normal reference intervals for serum cortisol, corticosterone, 11-deoxycortisol, androstenedione, 21-hydroxyprogesterone, testosterone, 17-hydroxyprogesterone, and progesterone. These steroids are commonly used as biomarkers for the diagnosis and monitoring of endocrine diseases such as congenital adrenal hyperplasia. Appropriate age- and gender-stratified reference intervals are essential in accurate interpretation of steroid hormone levels. The samples analyzed in this study were collected from healthy, ethnically diverse children in the Greater Toronto Area as part of the CALIPER program. A total of 337 serum samples from children between the ages of 0 and 18 years were analyzed. The concentrations were measured by using an LC–MS/MS method. The data were analyzed for outliers and age- and gender-specific partitions were established prior to establishing the 2.5th and 97.5th percentiles for the reference intervals. Reference intervals for all hormones required significant age-dependent stratification while testosterone and progesterone required additional sex-dependent stratification. We report a sensitive, accurate and relatively fast LC–MS/MS method for the simultaneous measurement of eight steroid hormones. Detailed reference intervals partitioned based on both age and gender were also established for all eight steroid hormones.

Sphingolipids have emerged as important bioactive lipid species involved in the pathogenesis of type 2 diabetes and cardiovascular disease. However, little is known of the regulatory role of sphingolipids in dyslipidemia of insulin-resistant states. We employed hamster models of dyslipidemia and insulin resistance to investigate the role of sphingolipids in hepatic VLDL overproduction, induction of insulin resistance, and inflammation. Hamsters were fed either a control chow diet, a high fructose diet, or a diet high in fat, fructose and cholesterol (FFC diet). They were then treated for 2 weeks with vehicle or 0.3 mg/kg myriocin, a potent inhibitor of de novo sphingolipid synthesis. Both fructose and FFC feeding induced significant increases in hepatic sphinganine, which was normalized to chow-fed levels with myriocin (P < 0.05); myriocin also lowered hepatic ceramide content (P < 0.05). Plasma TG and cholesterol as well as VLDL-TG and -apoB100 were similarly reduced with myriocin treatment in all hamsters, regardless of diet. Myriocin treatment also led to improved insulin sensitivity and reduced hepatic SREBP-1c mRNA, though it did not appear to ameliorate the activation of hepatic inflammatory pathways. Importantly, direct treatment of primary hamster hepatocytes ex vivo with C2 ceramide or sphingosine led to an increased secretion of newly synthesized apoB100. Taken together, these data suggest that a) hepatic VLDL-apoB100 overproduction may be stimulated by ceramides and sphingosine and b) inhibition of sphingolipid synthesis can reduce circulating VLDL in hamsters and improve circulating lipids--an effect that is possibly due to improved insulin signaling and reduced lipogenesis but is independent of changes in inflammation.

A high anion gap in diabetic ketoacidosis (DKA) suggests that some unmeasured anions must contribute to the generation of the anion gap. We investigated the contribution of d-lactate to the anion gap in DKA. Diabetic patients with and without DKA and high anion gap were recruited. Plasma d-lactate was quantified by HPLC. Plasma methylglyoxal was assayed by liquid chromatography-tandem mass spectrometry. The plasma fasting glucose, β-hydroxybutyrate, and blood HbA1c levels were highly elevated in DKA. Plasma anion gap was significantly increased in DKA (20.59 ± 6.37) compared to either the diabetic (7.50 ± 1.88) or the control group (6.53 ± 1.75) (p < 0.001, respectively). Moreover, plasma d-lactate levels were markedly increased in DKA (3.82 ± 2.50 mmol/l) compared to the diabetic (0.47 ± 0.55 mmol/l) or the control group (0.25 ± 0.35 mmol/l) (p < 0.001, respectively). Regression analysis demonstrated that d-lactate was associated with acidosis and anion gap (r = 0.686, p < 0.001). Plasma d-lactate levels are highly elevated and associated with metabolic acidosis and the high anion gap in DKA. Laboratory monitoring of d-lactate will provide valuable information for assessment of patients with DKA.

Bronchial carcinoids are pulmonary neuroendocrine cell-derived tumors comprising typical (TC) and atypical (AC) malignant phenotypes. The 5-year survival rate in metastatic carcinoid, despite multiple current therapies, is 14-25%. Hence, we are testing novel therapies that can affect the proliferation and survival of bronchial carcinoids.In vitro studies were used for the dose-response (AlamarBlue) effects of acetazolamide (AZ) and sulforaphane (SFN) on clonogenicity, serotonin-induced growth effect and serotonin content (LC-MS) on H-727 (TC) and H-720 (AC) bronchial carcinoid cell lines and their derived NOD/SCID mice subcutaneous xenografts. Tumor ultra structure was studied by electron microscopy. Invasive fraction of the tumors was determined by matrigel invasion assay. Immunohistochemistry was conducted to study the effect of treatment(s) on proliferation (Ki67, phospho histone-H3) and neuroendocrine phenotype (chromogranin-A, tryptophan hydroxylase).Both compounds significantly reduced cell viability and colony formation in a dose-dependent manner (0-80 μM, 48 hours and 7 days) in H-727 and H-720 cell lines. Treatment of H-727 and H-720 subcutaneous xenografts in NOD/SCID mice with the combination of AZ + SFN for two weeks demonstrated highly significant growth inhibition and reduction of 5-HT content and reduced the invasive capacity of H-727 tumor cells. In terms of the tumor ultra structure, a marked reduction in secretory vesicles correlated with the decrease in 5-HT content.The combination of AZ and SFN was more effective than either single agent. Since the effective doses are well within clinical range and bioavailability, our results suggest a potential new therapeutic strategy for the treatment of bronchial carcinoids.

We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr−/−xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr−/−xLcat−/− mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr−/−xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr−/−xLcat−/− mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr−/−xLcat−/− mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr−/−xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr−/−xLcat−/− mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance. We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr−/−xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr−/−xLcat−/− mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr−/−xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr−/−xLcat−/− mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr−/−xLcat−/− mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr−/−xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr−/−xLcat−/− mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance.

To develop a simple and sensitive LC-MS/MS procedure for quantification of serum 25-OH-vitamin D₃ (25-OH-D₃), 25-OH-vitamin D₂ (25-OH-D₂), and their C3-epimers.Serum 25-OH-vitamin D metabolites were extracted with MTBE and quantified by LC-MS/MS. Commercially available calibrators and QC materials were employed. The ion-transition 401.2→365.2 was monitored for 25-OH-D₃ and C3-epi-25-OH-D₃, 407.2→371.3 for d6-25-OH-D₃, 413.2→331.2 for 25-OH-D₂ and C3-epi-25-OH-D₂ and 419.2→337.1 for, d6-25-OH-D₂. As a proof-of-principle, 25-OH-D₃ and C3-epi-25-OH-D₃ were quantified in 200 pediatric subjects (0-20 years of age). Cholecalciferol supplements were examined as a potential source of C3-epimer.The assay provided an LLOQ of ≤2.8 nmol/L for all 25-OH-D metabolites, with a linear response up to 400 nmol/L. The CV was <10% for 25-OH-D₂/₃ and <15% for C3-epi-25-OH-D₃. C3-epi-25-OH-D₃ was quantified in all subjects, with higher concentrations observed in infants ≤1 year of age (11.44 nmol/L vs. 4.4 nmol/L; p<0.001). Within the first year of life, 25-OH-D₃ concentrations increased linearly, while C3-epi-25-OH-D₃ concentrations remained constant. At 12 months of age, C3-epi-25-OH-D₃ concentration dropped by almost 50% (11.4 nmol/L in infants ≤1year of age vs. 5.4 nmol/L in infants 1-2years of age; p<0.001). Liquid vitamin D₃ supplements did not contain appreciable amounts of C3-epi-D₃.The proposed LC-MS/MS procedure is suitable for quantifying 25-OH-D₃ metabolites. Although the C3-epimer is present in all pediatric subjects, it is significantly elevated in individuals ≤1 year of age and drops at 12 months of age. Oral vitamin D supplements are unlikely to be a significant source of C3-vitamin D epimer.

Abstract Background Insulin resistance is strongly associated with the development of type 2 diabetes and cardiovascular disease. However, the underlying mechanisms linking insulin resistance and the development of atherosclerosis have not been fully elucidated. Moreover, the protective effect of antihyperglycemic agent, metformin, is not fully understood. This study investigated the protective effects and underlying mechanisms of metformin in balloon-injury induced stenosis in insulin resistant rats. Methods After 4 weeks high fructose diet, rats received balloon catheter injury on carotid arteries and were sacrificed at 1 and 4 weeks post injury. Biochemical, histological, and molecular changes were investigated. Results Plasma levels of glucose, insulin, total cholesterol, triglyceride, free fatty acids, and methylglyoxal were highly increased in fructose-induced insulin resistant rats and treatment with metformin significantly improved this metabolic profile. The neointimal formation of the carotid arteries was enhanced, and treatment with metformin markedly attenuated neointimal hyperplasia. A significant reduction in BrdU-positive cells in the neointima was observed in the metformin-treated group (P &lt; 0.01). Insulin signaling pathways were inhibited in insulin resistant rats while treatment with metformin enhanced the expression of insulin signaling pathways. Increased expression of JNK and NFKB was suppressed following metformin treatment. Vasoreactivity was impaired while treatment with metformin attenuated phenylephrine-induced vasoconstriction and enhanced methacholine-induced vasorelaxation of the balloon injured carotid arteries in insulin resistant rats. Conclusion The balloon-injury induced neointimal formation of the carotid arteries is enhanced by insulin resistance. Treatment with metformin significantly attenuates neointimal hyperplasia through inhibition of smooth muscle cell proliferation, migration, and inflammation as well as by improvement of the insulin signaling pathway.

Poor vitamin D status (i.e. low serum 25-hydroxyvitamin D (25(OH)D)) has been associated with adverse clinical outcomes during pregnancy and childhood. However, the interpretation of serum 25(OH)D levels may be complicated by the presence of the C3-epimer of 25(OH)D. We aimed to quantify C3-epi-25(OH)D3 in pregnant women and fetuses, to explore the relationship of the C3-epimer between maternal and cord samples, and to establish whether infant C3-epimer abundance is explained by prenatal formation.In a sub-study of a randomized trial of prenatal vitamin D3, 25(OH)D3 and C3-epi-25(OH)D3 were quantified by LC-MS/MS in 71 sets of mother-fetus-infant serum samples, including maternal delivery specimens, cord blood, and infant specimens acquired at 3-28 weeks of age.Without supplementation, median concentrations of C3-epi-25(OH)D₃ were higher in infants (6.80 nmol/L) than mothers (0.45 nmol/L) and cord blood (0 nmol/L). However, there was substantial variation such that C3-epi-25(OH)D₃ accounted for up to 11% (maternal), 14% (cord), and 25% (infant) of the total 25(OH)D₃. Supplemental vitamin D₃ significantly increased maternal-fetal C3-epi-25(OH)D₃, and was a preferential source of C3-epi-25(OH)D₃ compared to basal vitamin D, possibly due to C3-epi-cholecalciferol in the supplement. Multivariate regression did not suggest transplacental transfer of C3-epi-25(OH)D₃, but rather indicated its generation within the fetal-placental unit from maternally-derived 25(OH)D₃. Neither maternal nor fetal C3-epi-25(OH)D₃ is accounted for the relatively high concentrations of infant C3-epi-25(OH)D₃, suggesting rapid postnatal generation.C3-epi-25(OH)D₃ is present in some pregnant women and fetuses, but does not appear to be efficiently transferred transplacentally. High C3-epimer concentrations in infancy are probably due to postnatal formation rather than fetal stores.

With no effective therapies to attenuate cartilage degeneration in osteoarthritis (OA), the result is pain and disability. Activation of hedgehog (HH) signaling causes changes related to the progression of OA, with higher levels of Gli-mediated transcriptional activation associated with increased disease severity. To elucidate the mechanism through which this occurs, this study sought to identify genes regulated by HH signaling in human OA chondrocytes.Using human OA cartilage samples, microarray analyses were performed to detect changes in gene expression when the HH pathway was modulated. Results were analyzed for differentially expressed genes, grouped into functional networks, and validated in independent samples. To investigate the effects of chondrocyte-specific sterol accumulation, we generated mice lacking Insig1 and Insig2, which are major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements.HH signaling was found to regulate genes that govern cholesterol homeostasis, and this led to alterations in cholesterol accumulation in chondrocytes. A higher level of Gli-mediated transcription resulted in accumulation of intracellular cholesterol. In genetically modified mice, chondrocyte-specific cholesterol accumulation was associated with an OA phenotype. Reducing cholesterol accumulation attenuated the severity of OA in mice in vivo and decreased the expression of proteases in human OA cartilage in vitro.HH signaling regulates cholesterol homeostasis in chondrocytes, and intracellular cholesterol accumulation contributes to the severity of OA. Our findings have therapeutic implications, since reduction of HH signaling reversed cholesterol accumulation and statin treatment attenuated cartilage degeneration.

Ezetimibe is a cholesterol uptake inhibitor that targets the Niemann-Pick C1-like 1 cholesterol transporter. Ezetimibe treatment has been shown to cause significant decreases in plasma cholesterol levels in patients with hypercholesterolemia and familial hypercholesterolemia. A recent study in humans has shown that ezetimibe can decrease the release of atherogenic postprandial intestinal lipoproteins. In the present study, we evaluated the mechanisms by which ezetimibe treatment can lower postprandial apoB48-containing chylomicron particles, using a hyperlipidemic and insulin-resistant hamster model fed a diet rich in fructose and fat (the FF diet) and fructose, fat, and cholesterol (the FFC diet). Male Syrian Golden hamsters were fed either chow or the FF or FFC diet ± ezetimibe for 2 wk. After 2 wk, chylomicron production was assessed following intravenous triton infusion. Tissues were then collected and analyzed for protein and mRNA content. FFC-fed hamsters treated with ezetimibe showed improved glucose tolerance, decreased fasting insulin levels, and markedly reduced circulating levels of TG and cholesterol in both the LDL and VLDL fractions. Examination of triglyceride (TG)-rich lipoprotein (TRL) fractions showed that ezetimibe treatment reduced postprandial cholesterol content in TRL lipoproteins as well as reducing apoB48 content. Although ezetimibe did not decrease TRL-TG levels in FFC hamsters, ezetimibe treatment in FF hamsters resulted in decreases in TRL-TG. Jejunal apoB48 protein expression was lower in ezetimibe-treated hamsters. Reductions in jejunal protein levels of scavenger receptor type B-1 (SRB-1) and fatty acid transport protein 4 were also observed. In addition, ezetimibe-treated hamsters showed significantly lower jejunal mRNA expression of a number of genes involved in lipid synthesis and transport, including srebp-1c, sr-b1, ppar-γ, and abcg1. These data suggest that treatment with ezetimibe not only inhibits cholesterol uptake, but may also alter intestinal function to promote improved handling of dietary lipids and reduced chylomicron production. These, in turn, promote decreases in fasting and postprandial lipid levels and improvements in glucose homeostasis.

Individuals with metabolic syndrome and frank type 2 diabetes are at increased risk of atherosclerotic cardiovascular disease, partially due to the presence of lipid and lipoprotein abnormalities. In these conditions, the liver and intestine overproduce lipoprotein particles, exacerbating the hyperlipidemia of fasting and postprandial states. Incretin-based, antidiabetes therapies (i.e., glucagon-like peptide [GLP]-1 receptor agonists and dipeptidyl peptidase-4 inhibitors) have proven efficacy for the treatment of hyperglycemia. Evidence is accumulating that these agents also improve fasting and postprandial lipemia, the latter more significantly than the former. In contrast, the gut-derived peptide GLP-2, cosecreted from intestinal L cells with GLP-1, has recently been demonstrated to enhance intestinal lipoprotein release. Understanding the roles of these emerging regulators of intestinal lipoprotein secretion may offer new insights into the regulation of intestinal lipoprotein assembly and secretion and provide new opportunities for devising novel strategies to attenuate hyperlipidemia, with the potential for cardiovascular disease reduction.

Defining laboratory biomarker reference values in a healthy population and understanding the fluctuations in biomarker concentrations throughout life and between sexes are critical to clinical interpretation of laboratory test results in different disease states. The Canadian Health Measures Survey (CHMS) has collected blood samples and health information from the Canadian household population. In collaboration with the Canadian Laboratory Initiative on Pediatric Reference Intervals (CALIPER), the data have been analyzed to determine reference value distributions and reference intervals for several endocrine and special chemistry biomarkers in pediatric, adult, and geriatric age groups.CHMS collected data and blood samples from thousands of community participants aged 3 to 79 years. We used serum samples to measure 13 immunoassay-based special chemistry and endocrine markers. We assessed reference value distributions and, after excluding outliers, calculated age- and sex-specific reference intervals, along with corresponding 90% CIs, according to CLSI C28-A3 guidelines.We observed fluctuations in biomarker reference values across the pediatric, adult, and geriatric age range, with stratification required on the basis of age for all analytes. Additional sex partitions were required for apolipoprotein AI, homocysteine, ferritin, and high sensitivity C-reactive protein.The unique collaboration between CALIPER and CHMS has enabled, for the first time, a detailed examination of the changes in various immunochemical markers that occur in healthy individuals of different ages. The robust age- and sex-specific reference intervals established in this study provide insight into the complex biological changes that take place throughout development and aging and will contribute to improved clinical test interpretation.