Shiho Fujisaka

OBJECTIVE To characterize the phenotypic changes of adipose tissue macrophages (ATMs) under different conditions of insulin sensitivity. RESEARCH DESIGN AND METHODS The number and the expressions of marker genes for M1 and M2 macrophages from mouse epididymal fat tissue were analyzed using flow cytometry after the mice had been subjected to a high-fat diet (HFD) and pioglitazone treatment. RESULTS Most of the CD11c-positive M1 macrophages and the CD206-positive M2 macrophages in the epididymal fat tissue were clearly separated using flow cytometry. The M1 and M2 macrophages exhibited completely different gene expression patterns. Not only the numbers of M1 ATMs and the expression of M1 marker genes, such as tumor necrosis factor-α and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by subsequent pioglitazone treatment, suggesting the correlation with whole-body insulin sensitivity. We also found that the increased number of M2 ATMs after an HFD was associated with the upregulated expression of interleukin (IL)-10, an anti-inflammatory Th2 cytokine, in the adipocyte fraction as well as in adipose tissue. The systemic overexpression of IL-10 by an adenovirus vector increased the expression of M2 markers in adipose tissue. CONCLUSIONS M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.

Obesity, diabetes, and metabolic syndrome result from complex interactions between genetic and environmental factors, including the gut microbiota. To dissect these interactions, we utilized three commonly used inbred strains of mice-obesity/diabetes-prone C57Bl/6J mice, obesity/diabetes-resistant 129S1/SvImJ from Jackson Laboratory, and obesity-prone but diabetes-resistant 129S6/SvEvTac from Taconic-plus three derivative lines generated by breeding these strains in a new, common environment. Analysis of metabolic parameters and gut microbiota in all strains and their environmentally normalized derivatives revealed strong interactions between microbiota, diet, breeding site, and metabolic phenotype. Strain-dependent and strain-independent correlations were found between specific microbiota and phenotypes, some of which could be transferred to germ-free recipient animals by fecal transplantation. Environmental reprogramming of microbiota resulted in 129S6/SvEvTac becoming obesity resistant. Thus, development of obesity/metabolic syndrome is the result of interactions between gut microbiota, host genetics, and diet. In permissive genetic backgrounds, environmental reprograming of microbiota can ameliorate development of metabolic syndrome.

Overconsumption of high-fat diet (HFD) and sugar-sweetened beverages are risk factors for developing obesity, insulin resistance, and fatty liver disease. Here we have dissected mechanisms underlying this association using mice fed either chow or HFD with or without fructose- or glucose-supplemented water. In chow-fed mice, there was no major physiological difference between fructose and glucose supplementation. On the other hand, mice on HFD supplemented with fructose developed more pronounced obesity, glucose intolerance, and hepatomegaly as compared to glucose-supplemented HFD mice, despite similar caloric intake. Fructose and glucose supplementation also had distinct effects on expression of the lipogenic transcription factors ChREBP and SREBP1c. While both sugars increased ChREBP-β, fructose supplementation uniquely increased SREBP1c and downstream fatty acid synthesis genes, resulting in reduced liver insulin signaling. In contrast, glucose enhanced total ChREBP expression and triglyceride synthesis but was associated with improved hepatic insulin signaling. Metabolomic and RNA sequence analysis confirmed dichotomous effects of fructose and glucose supplementation on liver metabolism in spite of inducing similar hepatic lipid accumulation. Ketohexokinase, the first enzyme of fructose metabolism, was increased in fructose-fed mice and in obese humans with steatohepatitis. Knockdown of ketohexokinase in liver improved hepatic steatosis and glucose tolerance in fructose-supplemented mice. Thus, fructose is a component of dietary sugar that is distinctively associated with poor metabolic outcomes, whereas increased glucose intake may be protective.

As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs.The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages.Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1β. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1β and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia.Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.

Abstract Adipose tissue resident macrophages have important roles in the maintenance of tissue homeostasis and regulate insulin sensitivity for example by secreting pro-inflammatory or anti-inflammatory cytokines. Here, we show that M2-like macrophages in adipose tissue regulate systemic glucose homeostasis by inhibiting adipocyte progenitor proliferation via the CD206/TGFβ signaling pathway. We show that adipose tissue CD206 + cells are primarily M2-like macrophages, and ablation of CD206 + M2-like macrophages improves systemic insulin sensitivity, which was associated with an increased number of smaller adipocytes. Mice genetically engineered to have reduced numbers of CD206 + M2-like macrophages show a down-regulation of TGFβ signaling in adipose tissue, together with up-regulated proliferation and differentiation of adipocyte progenitors. Our findings indicate that CD206 + M2-like macrophages in adipose tissues create a microenvironment that inhibits growth and differentiation of adipocyte progenitors and, thereby, control adiposity and systemic insulin sensitivity.

<h2>Summary</h2> Diet, genetics, and the gut microbiome are determinants of metabolic status, in part through production of metabolites by the gut microbiota. To understand the mechanisms linking these factors, we performed LC-MS-based metabolomic analysis of cecal contents and plasma from C57BL/6J, 129S1/SvImJ, and 129S6/SvEvTac mice on chow or a high-fat diet (HFD) and HFD-treated with vancomycin or metronidazole. Prediction of the functional metagenome of gut bacteria by PICRUSt analysis of 16S sequences revealed dramatic differences in microbial metabolism. Cecal and plasma metabolites showed multifold differences reflecting the combined and integrated effects of diet, antibiotics, host background, and the gut microbiome. Eighteen plasma metabolites correlated positively or negatively with host insulin resistance across strains and diets. Over 1,000 still-unidentified metabolite peaks were also highly regulated by diet, antibiotics, and genetic background. Thus, diet, host genetics, and the gut microbiota interact to create distinct responses in plasma metabolites, which can contribute to regulation of metabolism and insulin resistance.

Obesity and diabetes in humans are associated with increased rates of anxiety and depression. To understand the role of the gut microbiome and brain insulin resistance in these disorders, we evaluated behaviors and insulin action in brain of mice with diet-induced obesity (DIO) with and without antibiotic treatment. We find that DIO mice have behaviors reflective of increased anxiety and depression. This is associated with decreased insulin signaling and increased inflammation in in the nucleus accumbens and amygdala. Treatment with oral metronidazole or vancomycin decreases inflammation, improves insulin signaling in the brain and reduces signs of anxiety and depression. These effects are associated with changes in the levels of tryptophan, GABA, BDNF, amino acids, and multiple acylcarnitines, and are transferable to germ-free mice by fecal transplant. Thus, changes in gut microbiota can control brain insulin signaling and metabolite levels, and this leads to altered neurobehaviors.

Dietary sugars, fructose and glucose, promote hepatic de novo lipogenesis and modify the effects of a high-fat diet (HFD) on the development of insulin resistance. Here, we show that fructose and glucose supplementation of an HFD exert divergent effects on hepatic mitochondrial function and fatty acid oxidation. This is mediated via three different nodes of regulation, including differential effects on malonyl-CoA levels, effects on mitochondrial size/protein abundance, and acetylation of mitochondrial proteins. HFD- and HFD plus fructose-fed mice have decreased CTP1a activity, the rate-limiting enzyme of fatty acid oxidation, whereas knockdown of fructose metabolism increases CPT1a and its acylcarnitine products. Furthermore, fructose-supplemented HFD leads to increased acetylation of ACADL and CPT1a, which is associated with decreased fat metabolism. In summary, dietary fructose, but not glucose, supplementation of HFD impairs mitochondrial size, function, and protein acetylation, resulting in decreased fatty acid oxidation and development of metabolic dysregulation.

The human body is inhabited by numerous bacteria, fungi, and viruses, and each part has a unique microbial community structure. The gastrointestinal tract harbors approximately 100 trillion strains comprising more than 1000 bacterial species that maintain symbiotic relationships with the host. The gut microbiota consists mainly of the phyla Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Of these, Firmicutes and Bacteroidetes constitute 70-90% of the total abundance. Gut microbiota utilize nutrients ingested by the host, interact with other bacterial species, and help maintain healthy homeostasis in the host. In recent years, it has become increasingly clear that a breakdown of the microbial structure and its functions, known as dysbiosis, is associated with the development of allergies, autoimmune diseases, cancers, and arteriosclerosis, among others. Metabolic diseases, such as obesity and diabetes, also have a causal relationship with dysbiosis. The present review provides a brief overview of the general roles of the gut microbiota and their relationship with metabolic disorders.

Nonalcoholic fatty liver disease (NAFLD) is an abnormal liver metabolism often observed with insulin resistance and metabolic syndrome. Calorie restriction is a useful treatment for NAFLD and reportedly prolongs the life spans of several species in which sirtuin plays an important role. In this study, we examined whether the activation of SIRT1, a mammalian ortholog of sirtuin, may ameliorate the development of NAFLD. Monosodium glutamate (MSG) mice, which exhibited obesity and insulin resistance, were treated with SRT1720, a specific SIRT1 activator from the age of 6-16 wk. Sixteen-week-old MSG mice exhibited increased liver triglyceride content and elevated levels of aminotransferase. SRT1720 treatment significantly reduced these levels without affecting body weight or food intake. These results suggested that the administration of SRT1720 ameliorated the development of NAFLD in MSG mice. The expressions of lipogenic genes, such as sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, and the serum lipid profiles, including free fatty acids, were elevated in MSG mice and were reduced by SRT1720 treatment. SRT1720 treatment also reduced the expressions of lipogenic genes in cultured HepG2 cells. Furthermore, SRT1720 treatment decreased the expressions of marker genes for oxidative stress and inflammatory cytokines in the liver of MSG mice. Taken together, SRT1720 treatment may reduce liver lipid accumulation, at least in part, by directly reducing the expressions of lipogenic genes. The reduction of oxidative stress and inflammation may also be involved in the amelioration of NAFLD.

Diabetes, obesity, and the metabolic syndrome are multifactorial diseases dependent on a complex interaction of host genetics, diet, and other environmental factors. Increasing evidence places gut microbiota as important modulators of the crosstalk between diet and development of obesity and metabolic dysfunction. In addition, host genetics can have important impact on the composition and function of gut microbiota. Indeed, depending on the genetic background of the host, diet and other environmental factors may produce different changes in gut microbiota, have different impacts on host metabolism, and create different interactions between the microbiome and the host.In this review, we highlight how appropriate animal models can help dissect the complex interaction of host genetics with the gut microbiome and how diet can lead to different degrees of weight gain, levels of insulin resistance, and metabolic outcomes, such as diabetes, in different individuals. We also discuss the challenges of identifying specific disease-associated microbiota and the limitations of simple metrics, such as phylogenetic diversity or the ratio of Firmicutes to Bacteroidetes.Understanding these complex interactions will help in the development of novel treatments for microbiome-related metabolic diseases. This article is part of a special issue on microbiota.

Interactions of diet, gut microbiota, and host genetics play important roles in the development of obesity and insulin resistance. Here, we have investigated the molecular links between gut microbiota, insulin resistance, and glucose metabolism in 3 inbred mouse strains with differing susceptibilities to metabolic syndrome using diet and antibiotic treatment. Antibiotic treatment altered intestinal microbiota, decreased tissue inflammation, improved insulin signaling in basal and stimulated states, and improved glucose metabolism in obesity- and diabetes-prone C57BL/6J mice on a high-fat diet (HFD). Many of these changes were reproduced by the transfer of gut microbiota from antibiotic-treated donors to germ-free or germ-depleted mice. These physiological changes closely correlated with changes in serum bile acids and levels of the antiinflammatory bile acid receptor Takeda G protein-coupled receptor 5 (TGR5) and were partially recapitulated by treatment with a TGR5 agonist. In contrast, antibiotic treatment of HFD-fed, obesity-resistant 129S1 and obesity-prone 129S6 mice did not improve metabolism, despite changes in microbiota and bile acids. These mice also failed to show a reduction in inflammatory gene expression in response to the TGR5 agonist. Thus, changes in bile acid and inflammatory signaling, insulin resistance, and glucose metabolism driven by an HFD can be modified by antibiotic-induced changes in gut microbiota; however, these effects depend on important interactions with the host's genetic background and inflammatory potential.

Diet-induced obesity is reported to induce a phenotypic switch in adipose tissue macrophages from an antiinflammatory M2 state to a proinflammatory M1 state. Telmisartan, an angiotensin II type 1 receptor blocker and a peroxisome proliferator-activated receptor-γ agonist, reportedly has more beneficial effects on insulin sensitivity than other angiotensin II type 1 receptor blockers. In this study, we studied the effects of telmisartan on the adipose tissue macrophage phenotype in high-fat-fed mice. Telmisartan was administered for 5 wk to high-fat-fed C57BL/6 mice. Insulin sensitivity, macrophage infiltration, and the gene expressions of M1 and M2 markers in visceral adipose tissues were then examined. An insulin- or a glucose-tolerance test showed that telmisartan treatment improved insulin resistance, decreasing the body weight gain, visceral fat weight, and adipocyte size without affecting the amount of energy intake. Telmisartan reduced the mRNA expression of CD11c and TNF-α, M1 macrophage markers, and significantly increased the expressions of M2 markers, such as CD163, CD209, and macrophage galactose N-acetyl-galactosamine specific lectin (Mgl2), in a quantitative RT-PCR analysis. A flow cytometry analysis showed that telmisartan decreased the number of M1 macrophages in visceral adipose tissues. In conclusion, telmisartan improves insulin sensitivity and modulates adipose tissue macrophage polarization to an antiinflammatory M2 state in high-fat-fed mice.

Insulin and IGF1 signaling are important for adipose tissue development and function; however, their role in mature adipocytes is unclear. Mice with a tamoxifen-inducible knockout of insulin and/or IGF1 receptors (IR/IGF1R) demonstrate a rapid loss of white and brown fat due to increased lipolysis and adipocyte apoptosis. This results in insulin resistance, glucose intolerance, hepatosteatosis, islet hyperplasia with hyperinsulinemia, and cold intolerance. This phenotype, however, resolves over 10–30 days due to a proliferation of preadipocytes and rapid regeneration of both brown and white adipocytes as identified by mTmG lineage tracing. This cycle can be repeated with a second round of receptor inactivation. Leptin administration prior to tamoxifen treatment blocks development of the metabolic syndrome without affecting adipocyte loss or regeneration. Thus, IR is critical in adipocyte maintenance, and this loss of adipose tissue stimulates regeneration of brown/white fat and reversal of metabolic syndrome associated with fat loss.

Ectopic lipid accumulation in the liver is an almost universal feature of human and rodent models of generalized lipodystrophy and is also a common feature of type 2 diabetes, obesity, and metabolic syndrome. Here we explore the progression of fatty liver disease using a mouse model of lipodystrophy created by a fat-specific knockout of the insulin receptor (F-IRKO) or both IR and insulin-like growth factor 1 receptor (F-IR/IGFRKO). These mice develop severe lipodystrophy, diabetes, hyperlipidemia, and fatty liver disease within the first weeks of life. By 12 weeks of age, liver demonstrated increased reactive oxygen species, lipid peroxidation, histological evidence of balloon degeneration, and elevated serum alanine aminotransferase and aspartate aminotransferase levels. In these lipodystrophic mice, stored liver lipids can be used for energy production, as indicated by a marked decrease in liver weight with fasting and increased liver fibroblast growth factor 21 expression and intact ketogenesis. By 52 weeks of age, liver accounted for 25% of body weight and showed continued balloon degeneration in addition to inflammation, fibrosis, and highly dysplastic liver nodules. Progression of liver disease was associated with improvement in blood glucose levels, with evidence of altered expression of gluconeogenic and glycolytic enzymes. However, these mice were able to mobilize stored glycogen in response to glucagon. Feeding F-IRKO and F-IR/IGFRKO mice a high-fat diet for 12 weeks accelerated the liver injury and normalization of blood glucose levels. Thus, severe fatty liver disease develops early in lipodystrophic mice and progresses to advanced nonalcoholic steatohepatitis with highly dysplastic liver nodules. The liver injury is propagated by lipotoxicity and is associated with improved blood glucose levels.

Abstract Background Skeletal muscle is mainly responsible for insulin‐stimulated glucose disposal. Dysfunction in skeletal muscle metabolism especially during obesity contributes to the insulin resistance. Astaxanthin (AX), a natural antioxidant, has been shown to ameliorate hepatic insulin resistance in obese mice. However, its effects in skeletal muscle are poorly understood. The current study aimed to investigate the molecular target of AX in ameliorating skeletal muscle insulin resistance. Methods We fed 6‐week‐old male C57BL/6J mice with normal chow (NC) or NC supplemented with AX (NC+AX) and high‐fat‐diet (HFD) or HFD supplemented with AX for 24 weeks. We determined the effect of AX on various parameters including insulin sensitivity, glucose uptake, inflammation, kinase signaling, gene expression, and mitochondrial function in muscle. We also determined energy metabolism in intact C2C12 cells treated with AX using the Seahorse XFe96 Extracellular Flux Analyzer and assessed the effect of AX on mitochondrial oxidative phosphorylation and mitochondrial biogenesis. Results AX‐treated HFD mice showed improved metabolic status with significant reduction in blood glucose, serum total triglycerides, and cholesterol ( p &lt; 0.05). AX‐treated HFD mice also showed improved glucose metabolism by enhancing glucose incorporation into peripheral target tissues, such as the skeletal muscle, rather than by suppressing gluconeogenesis in the liver as shown by hyperinsulinemic–euglycemic clamp study. AX activated AMPK in the skeletal muscle of the HFD mice and upregulated the expressions of transcriptional factors and coactivator, thereby inducing mitochondrial remodeling, including increased mitochondrial oxidative phosphorylation component and free fatty acid metabolism. We also assessed the effects of AX on mitochondrial biogenesis in the siRNA‐mediated AMPK‐depleted C2C12 cells and showed that the effect of AX was lost in the genetically AMPK‐depleted C2C12 cells. Collectively, AX treatment (i) significantly ameliorated insulin resistance and glucose intolerance through regulation of AMPK activation in the muscle, (ii) stimulated mitochondrial biogenesis in the muscle, (iii) enhanced exercise tolerance and exercise‐induced fatty acid metabolism, and (iv) exerted antiinflammatory effects via its antioxidant activity in adipose tissue. Conclusions We concluded that AX treatment stimulated mitochondrial biogenesis and significantly ameliorated insulin resistance through activation of AMPK pathway in the skeletal muscle.

Adipose tissue hypoxia is an important feature of pathological adipose tissue expansion. Hypoxia-inducible factor-1α (HIF-1α) in adipocytes reportedly induces oxidative stress and fibrosis, rather than neoangiogenesis via vascular endothelial growth factor (VEGF)-A. We previously reported that macrophages in crown-like structures (CLSs) are both hypoxic and inflammatory. In the current study, we examined how macrophage HIF-1α is involved in high-fat diet (HFD)-induced inflammation, neovascularization, hypoxia, and insulin resistance using mice with myeloid cell-specific HIF-1α deletion that were fed an HFD. Myeloid cell-specific HIF-1α gene deletion protected against HFD-induced inflammation, CLS formation, poor vasculature development in the adipose tissue, and systemic insulin resistance. Despite a reduced expression of Vegfa in epididymal white adipose tissue (eWAT), the preadipocytes and endothelial cells of HIF-1α-deficient mice expressed higher levels of angiogenic factors, including Vegfa, Angpt1, Fgf1, and Fgf10 in accordance with preferable eWAT remodeling. Our in vitro study revealed that lipopolysaccharide-treated bone marrow-derived macrophages directly inhibited the expression of angiogenic factors in 3T3-L1 preadipocytes. Thus, macrophage HIF-1α is involved not only in the formation of CLSs, further enhancing the inflammatory responses, but also in the inhibition of neoangiogenesis in preadipocytes. We concluded that these two pathways contribute to the obesity-related physiology of pathological adipose tissue expansion, thus causing systemic insulin resistance.

Abstract Obesity and insulin resistance are associated with dysbiosis of the gut microbiota and impaired intestinal barrier function. Herein, we report that Bofutsushosan (BFT), a Japanese herbal medicine, Kampo, which has been clinically used for constipation in Asian countries, ameliorates glucose metabolism in mice with diet–induced obesity. A 16S rRNA sequence analysis of fecal samples showed that BFT dramatically increased the relative abundance of Verrucomicrobia, which was mainly associated with a bloom of Akkermansia muciniphila (AKK). BFT decreased the gut permeability as assessed by FITC-dextran gavage assay, associated with increased expression of tight-junction related protein, claudin-1, in the colon. The BFT treatment group also showed significant decreases of the plasma endotoxin level and expression of the hepatic lipopolysaccharide-binding protein. Antibiotic treatment abrogated the metabolic effects of BFT. Moreover, many of these changes could be reproduced when the cecal contents of BFT-treated donors were transferred to antibiotic-pretreated high fat diet-fed mice. These data demonstrate that BFT modifies the gut microbiota with an increase in AKK, which may contribute to improving gut barrier function and preventing metabolic endotoxemia, leading to attenuation of diet-induced inflammation and glucose intolerance. Understanding the interaction between a medicine and the gut microbiota may provide insights into new pharmacological targets to improve glucose metabolism.

Because oxidative stress promotes insulin resistance in obesity and type 2 diabetes, it is crucial to find effective antioxidant for the purpose of decreasing this threat. In this study, we explored the effect of astaxanthin, a carotenoid antioxidant, on insulin signaling and investigated whether astaxanthin improves cytokine- and free fatty acid-induced insulin resistance in vitro. We examined the effect of astaxanthin on insulin-stimulated glucose transporter 4 (GLUT4) translocation, glucose uptake, and insulin signaling in cultured rat L6 muscle cells using plasma membrane lawn assay, 2-deoxyglucose uptake, and Western blot analysis. Next, we examined the effect of astaxanthin on TNFα- and palmitate-induced insulin resistance. The amount of reactive oxygen species generated by TNFα or palmitate with or without astaxanthin was evaluated by dichlorofluorescein staining. We also compared the effect of astaxanthin on insulin signaling with that of other antioxidants, α-lipoic acid and α-tocopherol. We observed astaxanthin enhanced insulin-stimulated GLUT4 translocation and glucose uptake, which was associated with an increase in insulin receptor substrate-1 tyrosine and Akt phosphorylation and a decrease in c-Jun N-terminal kinase (JNK) and insulin receptor substrate-1 serine 307 phosphorylation. Furthermore, astaxanthin restored TNFα- and palmitate-induced decreases in insulin-stimulated GLUT4 translocation or glucose uptake with a concomitant decrease in reactive oxygen species generation. α-Lipoic acid enhanced Akt phosphorylation and decreased ERK and JNK phosphorylation, whereas α-tocopherol enhanced ERK and JNK phosphorylation but had little effect on Akt phosphorylation. Collectively these findings indicate astaxanthin is a very effective antioxidant for ameliorating insulin resistance by protecting cells from oxidative stress generated by various stimuli including TNFα and palmitate.

Although phenotypically polarized macrophages are now generally classified into two major subtypes termed proinflammatory M1 and anti-inflammatory M2 macrophages, a contributory role of lung M2 macrophages in the pathophysiological features of acute lung injury is not fully understood. Herein, we show in an endotoxemic murine model that M2 macrophages serve as key anti-inflammatory cells that play a regulatory role in the severity of lung injury. To study whether M2 macrophages can modify inflammation, we depleted M2 macrophages from lungs of CD206-diphtheria toxin (DT) receptor transgenic (Tg) mice during challenge with lipopolysaccharide. The i.p. administration of DT depleted CD206-positive cells in bronchoalveolar lavage fluid. The use of M2 macrophage markers Ym1 and arginase-1 identified pulmonary CD206-positive cells as M2 macrophages. A striking increase in neutrophils in bronchoalveolar lavage fluid cell contents was found in DT-treated CD206-DT receptor Tg mice. In CD206-DT receptor Tg mice given DT, endotoxin challenge exaggerated lung inflammation, including up-regulation of proinflammatory cytokines and increased histological lung damage, but the endotoxemia-induced increase in NF-κB activity was significantly reduced, suggesting that M2 phenotype-dependent counteraction of inflammatory insult cannot be attributed to the inhibition of the NF-κB pathway. Our results indicate a critical role of CD206-positive pulmonary macrophages in triggering inflammatory cascade during endotoxemic lung inflammation. Although phenotypically polarized macrophages are now generally classified into two major subtypes termed proinflammatory M1 and anti-inflammatory M2 macrophages, a contributory role of lung M2 macrophages in the pathophysiological features of acute lung injury is not fully understood. Herein, we show in an endotoxemic murine model that M2 macrophages serve as key anti-inflammatory cells that play a regulatory role in the severity of lung injury. To study whether M2 macrophages can modify inflammation, we depleted M2 macrophages from lungs of CD206-diphtheria toxin (DT) receptor transgenic (Tg) mice during challenge with lipopolysaccharide. The i.p. administration of DT depleted CD206-positive cells in bronchoalveolar lavage fluid. The use of M2 macrophage markers Ym1 and arginase-1 identified pulmonary CD206-positive cells as M2 macrophages. A striking increase in neutrophils in bronchoalveolar lavage fluid cell contents was found in DT-treated CD206-DT receptor Tg mice. In CD206-DT receptor Tg mice given DT, endotoxin challenge exaggerated lung inflammation, including up-regulation of proinflammatory cytokines and increased histological lung damage, but the endotoxemia-induced increase in NF-κB activity was significantly reduced, suggesting that M2 phenotype-dependent counteraction of inflammatory insult cannot be attributed to the inhibition of the NF-κB pathway. Our results indicate a critical role of CD206-positive pulmonary macrophages in triggering inflammatory cascade during endotoxemic lung inflammation. Macrophages are critical regulators of many organ systems, including innate and adaptive immunity, systemic metabolism, hematopoiesis, vasculogenesis, malignancy, and reproduction.1Tugal D. Liao X. Jain M.K. 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Macrophage polarization in health and disease.ScientificWorldJournal. 2011; 11: 2391-2402Crossref PubMed Scopus (215) Google Scholar Acute lung injury (ALI) is a common and highly morbid clinical disease marked by respiratory failure attributable to increased pulmonary endothelial and epithelial permeability, leading to alveolar flooding, and neutrophil accumulation in alveolar spaces and in adjacent capillaries.13Ware L.B. Matthay M.A. The acute respiratory distress syndrome.N Engl J Med. 2000; 342: 1334-1349Crossref PubMed Scopus (4477) Google Scholar, 14Manicone A. Role of the pulmonary epithelium and inflammatory signals in acute lung injury.Expert Rev Clin Immunol. 2009; 5: 63-75Crossref PubMed Scopus (93) Google Scholar Although neutrophil influx and activation within the lung play a crucial role in the pathogenesis of ALI,15Grommes J. Soehnlein O. Contribution of neutrophils to acute lung injury.Mol Med. 2011; 17: 293-307Crossref PubMed Scopus (918) Google Scholar growing evidence indicates that macrophages also contribute to the modulation of lung inflammation and the resultant lung injury.16Lomas-Neira J. Chung C.S. Perl M. Gregory S. Biffl W. Ayala A. Role of alveolar macrophage and migrating neutrophils in hemorrhage-induced priming for ALI subsequent to septic challenge.Am J Physiol Lung Cell Mol Physiol. 2006; 290: L51-L58Crossref PubMed Scopus (125) Google Scholar, 17Kooguchi K. Hashimoto S. Kobayashi A. Kitamura Y. Kudoh I. Wiener-Kronish J. Sawa T. Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia.Infect Immun. 1998; 66: 3164-3169Crossref PubMed Google Scholar, 18Marriott H.M. Dockrell D.H. The role of the macrophage in lung disease mediated by bacteria.Exp Lung Res. 2007; 33: 493-505Crossref PubMed Scopus (77) Google Scholar Several studies have found that macrophage depletion with clodronate-lisosomes results in attenuation of lung injury after endotoxin administration, ischemia-reperfusion, and mechanical ventilation.19Koay M.A. Gao X. Washington M.K. Parman K.S. Sadikot R.T. Blackwell T.S. Christman J.W. Macrophages are necessary for maximal nuclear factor-kappa B activation in response to endotoxin.Am J Respir Cell Mol Biol. 2002; 26: 572-578Crossref PubMed Scopus (115) Google Scholar, 20Naidu B.V. Krishnadasan B. Farivar A.S. Woolley S.M. Thomas R. Van Rooijen N. Verrier E.D. Mulligan M.S. Early activation of the alveolar macrophage is critical to the development of lung ischemia-reperfusion injury.J Thorac Cardiovasc Surg. 2003; 126: 200-207Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Zhao M. Fernandez L.G. Doctor A. Sharma A.K. Zarbock A. Tribble C.G. Kron I.L. Laubach V.E. Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury.Am J Physiol Lung Cell Mol Physiol. 2006; 291: L1018-L1026Crossref PubMed Scopus (152) Google Scholar, 22Frank J.A. Wray C.M. McAuley D.F. Schwendener R. Matthay M.A. Alveolar macrophages contribute to alveolar barrier dysfunction in ventilator-induced lung injury.Am J Physiol Lung Cell Mol Physiol. 2006; 291: L1191-L1198Crossref PubMed Scopus (149) Google Scholar Conversely, other studies suggest that the recruitment of macrophages to the lung is associated with attenuated lung injury.23Knapp S. Leemans J.C. Florquin S. Branger J. Maris N.A. Pater J. van Rooijen N. van der Poll T. Alveolar macrophages have a protective anti-inflammatory role during murine pneumococcal pneumonia.Am J Respir Crit Care Med. 2003; 167: 171-179Crossref PubMed Scopus (247) Google Scholar, 24Amano H. Morimoto K. Senba M. Wang H. Ishida Y. Kumatori A. Yoshimine H. Oishi K. Mukaida N. Nagatake T. Essential contribution of monocyte chemoattractant protein1/C-C chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia.J Immunol. 2004; 172: 398-409Crossref PubMed Scopus (92) Google Scholar One possible explanation for such disparate roles of macrophages in ALI may be related to different depletion or recruitment of heterogeneous populations of proinflammatory and anti-inflammatory macrophages in the lung. However, how M2 macrophages have a protective anti-inflammatory role under the pathological condition of ALI is not fully understood. Herein, we decided to deplete M2 macrophages in mice during exposure to lipopolysaccharide (LPS). We generated a line of transgenic (Tg) mice expressing human diphtheria toxin (DT) receptor under the control of the Mrc1 gene promoter. Murine cells are naturally resistant to DT because they lack functional cell surface receptors for the toxin.25Pappenheimer A.M. Harper A.A. Moynihan M. Brockers J.P. Diphtheria toxin and related proteins: effect of route of injection on toxicity and the determination of cytotoxicity for various cultured cells.J Inf Dis. 1982; 145: 94-102Crossref PubMed Scopus (67) Google Scholar, 26Naglich J.G. Metherall J.E. Russell D.M. Eidels L. Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.Cell. 1992; 69: 1051-1061Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 27Mitamura T. Higashiyama S. Taniguchi N. Klagsbrun M. Mekada E. Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity.J Biol Chem. 1995; 270: 1015-1019Crossref PubMed Scopus (261) Google Scholar Thus, the technique with human DT receptor transduction allows us to achieve efficient inducible ablation of a specific type of murine cells by DT administration in vivo. Because Mrc1 (alias CD206) is a cell surface receptor as an M2 phenotypic marker, systemic administration of DT was able to lead to a sharp decline in lung M2 macrophages in CD206-DT receptor Tg mice. Herein, we identify lung M2 macrophages as key anti-inflammatory cells that play a regulatory role in the severity of endotoxin-induced lung injury. We obtained the mouse BAC clone RP 24-377 D19 carrying a 152-kbp insert containing the exon coding translational start Met and upstream 133-kbp sequence of CD206 gene from the BACPAC Resources Center CHORI (Oakland, CA). The plasmid pTRECK6 that includes noncoding exon and intron from rabbit β-globulin gene, human HB-EGF (DT receptor) cDNA, and rabbit β-globulin and simian virus 40 polyadenylation (pA) signals was kindly provided by Dr. Kenji Kohno (Nara Institute of Science and Technology, Nara, Japan).28Saito M. Iwawaki T. Taya C. Yonekawa H. Noda M. Inui Y. Mekada E. Kimata Y. Tsuru A. Kohno K. Diphtheria toxin receptor-mediated conditional and targeted cell ablation in transgenic mice.Nat Biotechnol. 2001; 19: 746-750Crossref PubMed Scopus (360) Google Scholar, 29Furukawa N. Saito M. Kimata Y. Kohno K. A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity.J Biochem. 2006; 140: 831-841Crossref PubMed Scopus (24) Google Scholar By using a Counterselection BAC modification kit (Gene Bridges, Dresden, Germany), we inserted the noncoding exon and intron from rabbit β-globulin gene, human DT receptor cDNA, and rabbit β-globulin and simian virus 40 pA signals at the initiation site of translation in the CD206 gene to yield the pTg-CD206-DT receptor (Figure 1A). The purified pTg-CD206-DT receptor BAC DNA was microinjected into pronuclei of fertilized one-cell embryos from C57BL/6 mice. Founder mice were crossed with C57BL/6 mice to produce +/CD206DT receptor mice. The wild-type (WT; +/+) and heterozygous CD206-DT receptor (+/CD206DTR) littermates were used for analysis. All mice were housed in a specific pathogen-free facility. Experiments were performed according to institutional guidelines. For the genotyping of the Tg mouse lines by Southern blot analysis, genomic DNA prepared from tail biopsy specimens was digested with BamHI, separated by electrophoresis on a 0.6% agarose gel, and transferred to a nylon membrane (Hybond-N+; GE Healthcare Japan, Tokyo, Japan). Hybridization was conducted with a 900-bp 32P-labeled DNA fragment derived from the exon 1 and intron 1 (Figure 1B). The detected band sizes of endogenous and Tg genes were 8.3 and 3.8 kb, respectively. In a first series of experiments to examine the effects of M2 macrophage depletion under normal noninflammatory conditions, CD206-DT receptor Tg mice and WT littermates received an i.p. injection of 15 μg/kg DT. Mice were administered DT for 3 consecutive days and sacrificed 24 hours after the last injection (Figure 2A). Blood samples were collected, and various tissues and organs were removed. Some mice were given phosphate-buffered saline (PBS) instead of DT. In another series of experiments to address the role of M2 macrophages in endotoxin-induced ALI, mice received an i.p. injection of 15 μg/kg DT for 3 consecutive days and were subsequently challenged with i.v. LPS (0.1 mg/kg; List Biological Laboratories, Campbell, CA) 24 hours after the last injection. Mice were sacrificed 2 hours later, and blood collection and lung tissue isolation were performed (Figure 3A). Total RNA was isolated from tissues with an RNeasy Mini Kit (Qiagen, Tokyo, Japan). RNA was reverse transcribed to cDNA, and real-time PCR analyses were performed as described previously30Oishi H. Takano K. Tomita K. Takebe M. Yokoo H. Yamazaki M. Hattori Y. Olprinone and colforsin daropate alleviate septic lung inflammation and apoptosis through CREB-independent activation of the Akt pathway.Am J Physiol Lung Cell Mol Physiol. 2012; 303: L130-L140Crossref PubMed Scopus (40) Google Scholar using a Takara RNA PCR kit (Takara Bio, Ohtsu, Japan). Thermal cycler parameters were as follows: 1 cycle of 50°C for 2 minutes, 50 cycles of denaturation (95°C for 15 seconds), and combined annealing/extension (60°C for 30 seconds). Gene expression changes were calculated by the comparative CT method, and 18S ribosomal RNA was used as the reference gene for normalization. Blood levels of IL-1β, TNF-α, monocyte chemotactic protein (MCP)-1, and IL-6 were measured by the use of a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. The plate was read on a microplate reader (Nippon-InterMed, Tokyo, Japan). Assays were performed in duplicate. For routine histological examination, inflation-fixed lungs were harvested, fixed, dehydrated, paraffin embedded, and sliced into sections (4 μm thick).30Oishi H. Takano K. Tomita K. Takebe M. Yokoo H. Yamazaki M. Hattori Y. Olprinone and colforsin daropate alleviate septic lung inflammation and apoptosis through CREB-independent activation of the Akt pathway.Am J Physiol Lung Cell Mol Physiol. 2012; 303: L130-L140Crossref PubMed Scopus (40) Google Scholar, 31Matsuda N. Nishihira J. Takahashi Y. Kemmotsu O. Hattori Y. Role of macrophage migration inhibitory factor in acute lung injury in mice with acute pancreatitis complicated by endotoxemia.Am J Respir Cell Mol Biol. 2006; 35: 198-205Crossref PubMed Scopus (54) Google Scholar After deparaffinization, slides were stained with hematoxylin and eosin (H&E) using standard methods. A semiquantitative morphometric analysis of lung injury was performed by scoring from 0 to 4: 0 indicates none; 1 indicates light; 2 indicates moderate; 3 indicates severe; and 4 indicates very severe. The scores are for the following categories: neutrophil infiltration, pulmonary edema, and disorganization of lung parenchyma and hemorrhage.30Oishi H. Takano K. Tomita K. Takebe M. Yokoo H. Yamazaki M. Hattori Y. Olprinone and colforsin daropate alleviate septic lung inflammation and apoptosis through CREB-independent activation of the Akt pathway.Am J Physiol Lung Cell Mol Physiol. 2012; 303: L130-L140Crossref PubMed Scopus (40) Google Scholar A total lung injury score was calculated by adding the individual scores in every animal and averaging the total values in each group. All of the histological studies were performed in a blinded manner (K.K.). Frozen lung sections (5 mm thick) were fixed in acetone for 10 minutes at −20°C and air dried. Bronchoalveolar lavage (BAL) was performed with five aliquots of 5 mL of PBS instilled into the lungs and gently withdrawn. Cells obtained from the total BAL fluid (BALF) were spun at 113 × g for 10 minutes onto glass slides by use of a Cytospin 4 (Thermo Fisher Scientific, Yokohama, Japan). Then, they were fixed with 4% buffered formalin solution for 10 minutes at room temperature. Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide for 15 minutes. After incubation overnight at 4°C with anti-CD206 antibody (AbD Serotec, Raleigh, NC) or anti–arginase-1 antibody (Sigma-Aldrich, St. Louis, MO), the samples were exposed to anti-rat or anti-rabbit IgG conjugated with horseradish peroxidase. Bound antibody was visualized by a light microscopy with diaminobenzidine. Omission of primary antibody and staining with nonimmune IgG served as negative control. Cells obtained from the total BALF were adhered to a glass-bottom dish, fixed with 4% paraformaldehyde, and permeabilized in Perm Buffer (BD Biosciences, San Diego, CA). Cells were exposed to the fluorescent antibody Alexa 546–conjugated anti-rat IgG and Alexa 488–conjugated anti-rabbit IgG (Life Technologies, Carlsbad, CA) after overnight incubation with the primary antibody, anti-CD206 antibody and anti–arginase-1 antibody. The nucleus was counterstained with DAPI. Immunofluorescence images were observed under an Olympus (Tokyo, Japan) BX-51 fluorescence microscope and processed using Adobe Photoshop CS3 software (Adobe, San Jose, CA). The alterations in cells in BALF were examined as described previously.31Matsuda N. Nishihira J. Takahashi Y. Kemmotsu O. Hattori Y. Role of macrophage migration inhibitory factor in acute lung injury in mice with acute pancreatitis complicated by endotoxemia.Am J Respir Cell Mol Biol. 2006; 35: 198-205Crossref PubMed Scopus (54) Google Scholar Cells in BALF obtained as mentioned above were counted with a hemacytometer. Slides were stained with Wright stain, and cell differentiation was tabulated using light microscopy. Nuclear protein extracts from freshly isolated lungs were obtained with a commercially available nuclear extraction kit (Sigma-Aldrich), as described in the manufacturer's manual. TransAM NF-κB and TransAM AP-1 kits (Active Motif, Carlsbad, CA) were used to quantify the binding of p65 to the NF-κB site and c-Jun and c-Fos to the activator protein (AP)-1 site, according to the manufacturer's instructions. TransAM kits are DNA-binding ELISAs that quantify transcription factor activation in tissues. NF-κB and AP-1 DNA binding reactions were performed according to the manufacturer's protocol using the Odyssey Infrared electrophoretic mobility shift assay kit (Li-Cor Biosciences, Lincoln, NE) and nuclei extracts from lung tissues. Double-stranded IRDye 700 infrared dye-labeled oligonucleotides with consensus sequences of NF-κB (forward, 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and reverse, 5′-GCCTGGGAAAHTCCCCTCAACT-3′) and AP-1 (forward, 5′-CGCTTGATGACTCAGCCGGAA-3′ and reverse, 5′-TTCCGGCTGAGTCATCAAGCG-3′) were used. Data were analyzed by the use of Prism software version 6 (GraphPad Software, San Diego, CA). Statistical analysis was performed by Student's unpaired t-test or one-way analysis of variance, followed by Tukey's multiple comparison test. Differences were considered significant at P ≤ 0.05. Data are presented as means ± SEM. CD206 mRNAs were transcribed in a variety of tissues, including lung, liver, spleen, and kidney, of normal C57BL/6 mice, but they were most abundant in lung tissues (Figure 1C). CD206 mRNA expression levels were evidently higher in BALF than in lung tissues when normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 1D). Likewise, high levels of mRNA expression of the classic M2 marker Ym1 were detected in BALF (Figure 1E). Immunohistochemical (IHC) studies showed that CD206-positive cells were present in the alveoli (Figure 1G) and constituted a great portion of the whole cells in BALF (Figure 1H). Arginase-1, a marker for the M2 subset, behaved in the same way as CD206 in terms of the alveolar localization (Figure 1F). Moreover, merging of CD206 and arginase-1 showed that CD206-positive cells in BALF were labeled with arginase-1 (Figure 1I). DT treatment resulted in a sharp reduction in CD206 mRNA levels in lungs of CD206-DT receptor Tg mice without any change in those of WT mice (Figure 2B). Similarly, Ym1 mRNA expression in lungs was reduced when DT was given to CD206-DT receptor Tg mice (Figure 2C). Consistent with the effect on CD206 mRNA in lung tissues, DT treatment caused marked decreases in the CD206 and Ym1 mRNA expression levels (Figure 2, D and E) and the CD206-positive cell number (Figure 2, F and G) in BALF from the CD206-DT receptor Tg animal compared with WT. Furthermore, an immunofluorescence labeling study showed that no cells labeled with CD206 or arginase-1 were detectable in BALF from CD206-DT receptor Tg mice (data not shown). DT treatment did not substantially affect mRNA levels of proinflammatory cytokines, IL-1β, TNF-α, MCP-1, and IL-6, in lungs of WT mice. On the other hand, CD206-DT receptor Tg mice exhibited up-regulation of these proinflammatory cytokine mRNAs in response to DT treatment (Figure 2H). However, the effects of DT treatment on inflammatory gene expression were much less pronounced when compared with those of further challenge with LPS in DT-treated CD206-DT receptor Tg mice (Supplemental Figure S1). When blood levels of proinflammatory cytokines were measured via an ELISA, the WT animals had low levels of the cytokines examined herein regardless of whether DT was given. In contrast, IL-1β, MCP-1, and IL-6 showed an evident increase in CD206-DT receptor Tg mice when treated with DT, except that TNF-α was undetectable even though DT was administered (Figure 2I). DT treatment was without effect on lung and BALF CD206 mRNA levels in WT mice. When a low dose (0.1 mg/kg) of LPS was i.v. injected to DT-treated WT mice, CD206 mRNA levels were substantially unchanged in both lungs and BALF (Figure 3, B and C). Interestingly, challenge with the low dose of LPS did not alter the total number of BALF cells in WT mice (Supplemental Figure S2). This is due to the BALF cells being mainly composed of CD206-positive macrophages. However, a notable increase in neutrophils in BALF cell contents was found in DT-treated CD206-DT receptor Tg mice (Supplemental Figure S2). LPS challenge led to up-regulation of IL-1β, TNF-α, MCP-1, and IL-6 mRNA levels in lungs of WT mice. This up-regulation was strikingly enhanced in DT-treated CD206-DT receptor Tg mice (Figure 3D). The ability of LPS at 0.1 mg/kg to up-regulate blood levels of TNF-α, MCP-1, and IL-6 was less pronounced in WT mice regardless of treatment with DT, although IL-1β responded relatively well to LPS. However, LPS caused a significant and substantial impact on all proinflammatory cytokines examined herein in DT-treated CD206-DT receptor Tg mice (Figure 3E). LPS also led to an increase in the mRNA level of the anti-inflammatory cytokine IL-10 (Supplemental Figure S3). DT treatment enhanced the LPS-induced increase in IL-10 mRNA in CD206-DT receptor Tg mice. Histological examination of H&E-stained sections of the lungs revealed that WT mice had alveolar septae that were normal in appearance, with no intra-alveolar inflammation (Figure 4A). Lung histopathological analysis of WT mice given LPS showed modest inflammatory cell infiltrate (Figure 4B). DT-treated CD206-DT receptor Tg mice that received LPS had massive cell infiltration, hemorrhage resulting from ruptured capillary vessels, and diffuse septal edema (Figure 4C). Semiquantitative assessment using the lung injury score demonstrated that the score was significantly higher in CD206-DT receptor Tg mice treated with DT than in untreated Tg animals (Figure 4D). When DT-treated CD206-DT receptor Tg mice were challenged with LPS, the score increased strikingly. To examine the activation of the proinflammatory transcription factor NF-κB in lungs after LPS challenge, NF-κB DNA binding activity in nuclear protein extracts from lungs was quantitatively measured with its ELISA. The DNA binding activity of NF-κB was greatly increased in WT mice after LPS injection. This increase was significantly attenuated in DT-treated CD206-DT receptor Tg mice (Figure 5A). Another proinflammatory transcription factor, AP-1, consists of Jun and Fos family proteins. Thus, c-Jun and c-Fos proto-oncogenes encode proteins that form a complex that regulates transcription from promoters containing AP-1 activation elements.32Halazonetis T.D. Georgopoulos K. Greenberg M.E. Leder P. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities.Cell. 1988; 55: 917-924Abstract Full Text PDF PubMed Scopus (768) Google Scholar Both c-Jun and c-Fos binding activities were significantly increased when WT mice were challenged with LPS. Further increases in these binding activities in response to LPS were observed in DT-treated CD206-DT receptor Tg mice (Figure 5A). These findings obtained using the ELISA method were confirmed by gel mobility shift assay. The LPS-induced great increase in the NF-κB binding was blunted when DT was given to CD206-DT receptor Tg mice (Figure 5B). In contrast, DT treatment markedly heightened the LPS-induced increase in the AP-1 binding in CD20

Isoliquiritigenin (ILG) has been reported to attenuate adipose tissue inflammation and metabolic disorder; however, the underlying mechanisms remain to be elucidated. The aim of this study is to elucidate whether ILG shows the anti-inflammatory and antimetabolic syndrome effects through gut microbiota modification.Mice are fed a high-fat diet (HFD) with or without ILG for up to 12 weeks. The effect of ILG on body weight, blood glucose level, adipose tissue inflammation, gut barrier function, and gut microbiota composition are investigated. ILG supplementation alleviates HFD-induced obesity, glucose tolerance, and insulin resistance and suppresses inflammatory gene expression in epididymal white adipose tissue (eWAT). Moreover, ILG supplementation modifies gut bacterial composition by increasing the abundance of antimetabolic disease-associated species (e.g., Parabacteroides goldsteinii and Akkemansia muciniphila) and up-regulated genes associated with gut barrier function. Fecal microbiome transplantation (FMT) from ILG-fed donors counteract HFD-induced body and eWAT weight changes, inflammation-related gene expression, glucose tolerance, and insulin resistance, thereby suggesting that ILG-responsive gut bacteria exerts anti-inflammatory and antimetabolic syndrome effects.Alterations in gut bacteria underly the beneficial effects of ILG against adipose tissue inflammation and metabolic disorders. ILG may be a promising prebiotic for the prevention and treatment of metabolic syndrome.

Muscle regeneration requires the coordination of muscle stem cells, mesenchymal fibro-adipogenic progenitors (FAPs), and macrophages. How macrophages regulate the paracrine secretion of FAPs during the recovery process remains elusive. Herein, we systemically investigated the communication between CD206+ M2-like macrophages and FAPs during the recovery process using a transgenic mouse model. Depletion of CD206+ M2-like macrophages or deletion of CD206+ M2-like macrophages-specific TGF-β1 gene induces myogenesis and muscle regeneration. We show that depletion of CD206+ M2-like macrophages activates FAPs and activated FAPs secrete follistatin, a promyogenic factor, thereby boosting the recovery process. Conversely, deletion of the FAP-specific follistatin gene results in impaired muscle stem cell function, enhanced fibrosis, and delayed muscle regeneration. Mechanistically, CD206+ M2-like macrophages inhibit the secretion of FAP-derived follistatin via TGF-β signaling. Here we show that CD206+ M2-like macrophages constitute a microenvironment for FAPs and may regulate the myogenic potential of muscle stem/satellite cells.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform leads to unresolved endoplasmic reticulum (ER) stress when coupled with a HFD intake. Conversely, a liver-specific knockdown of KHK in mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in mice with genetically induced obesity or metabolic dysfunction, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

Chronic inflammation in adipose tissue contributes to obesity-related insulin resistance. The 3-phosphoinositide-dependent protein kinase 1 (Pdk1)/forkhead transcription factor (Foxo1) pathway is important in regulating glucose and energy homeostasis, but little is known about this pathway in adipose tissue macrophages (ATMs). To investigate this, we generated transgenic mice that carried macrophage/granulocyte-specific mutations, including a Pdk1 knockout (LysMPdk1(-/-)), a Pdk1 knockout with transactivation-defective Foxo1 (Δ256LysMPdk1(-/-)), a constitutively active nuclear (CN) Foxo1 (CNFoxo1(LysM)), or a transactivation-defective Foxo1 (Δ256Foxo1(LysM)). We analyzed glucose metabolism and gene expression in ATM populations isolated with fluorescence-activated cell sorting. The LysMPdk1(-/-) mice exhibited elevated M1 macrophages in adipose tissue and insulin resistance. Overexpression of transactivation-defective Foxo1 rescued these phenotypes. CNFoxo1(LysM) promoted transcription of the C-C motif chemokine receptor 2 (Ccr2) in ATMs and increased M1 macrophages in adipose tissue. On a high-fat diet, CNFoxo1(LysM) mice exhibited insulin resistance. Pdk1 deletion or Foxo1 activation in bone marrow-derived macrophages abolished insulin and interleukin-4 induction of genes involved in alternative macrophage activation. Thus, Pdk1 regulated macrophage infiltration by inhibiting Foxo1-induced Ccr2 expression. This shows that the macrophage Pdk1/Foxo1 pathway is important in regulating insulin sensitivity in vivo.

Obesity and insulin resistance are closely associated with a state of low-grade inflammation in the body, and adipose tissue macrophages (ATMs) play central roles in this inflammation. ATMs are known to exhibit marked functional heterogeneity. M1 ATMs produce inflammatory cytokines and induce insulin resistance. On the other hand, the majority of ATMs in lean individuals are M2 ATMs, which have anti-inflammatory potential. We found that M1 and M2 ATMs can be clearly distinguished using CD11c and CD206 as markers, and that both the number of the M1 and M2 ATMs and the M1/M2 ratio are correlated with the degree of insulin resistance. M1/M2 polarity in the adipose tissue is influenced not only by the level of secretion of various polarizing adipokines and chemokines, but also by factors in the local microenvironment, such as hypoxia. M1 ATMs acquire their polarity via activation of hypoxia-inducible factor-1α (HIF-1α) by local hypoxia, and absence of HIF-1α in the myeloid cells appears to enhance insulin sensitivity by promoting angiogenesis in adipose tissue. On the other hand, the resident M2 ATMs interact with adipose tissue progenitors to control adiposity. Thus, beyond their role as immunoregulatory cells, the M1/M2 ATMs also regulate the microenvironment in the adipose tissue and control insulin sensitivity. Recently, we have shown that interventions in the gut microbiota may be effective in controlling obesity-induced chronic inflammation. Control of M1/M2 ATM polarity is a potential therapeutic target for the treatment of insulin resistance associated with obesity.

Serine phosphorylation of insulin receptor substrate (IRS)-1 and the induction of suppressor of cytokine signaling 3 (SOCS3) is recently well documented as the mechanisms for the insulin resistance. However, the relationship between these two mechanisms is not fully understood. In this study, we investigated the involvement of SOCS3 and IRS-1 serine phosphorylation in TNFα-induced insulin resistance in 3T3-L1 adipocytes. TNFα transiently stimulated serine phosphorylation of IRS-1 from 10 min to 1 h, whereas insulin-stimulated IRS-1 tyrosine phosphorylation was inhibited only after TNFα treatment longer than 4 h. These results suggest that serine phosphorylation of IRS-1 alone is not the major mechanism for the inhibited insulin signaling by TNFα. TNFα stimulation longer than 4 h enhanced the expression of SOCS3 and signal transducer and activator of transcription-3 phosphorylation, concomitantly with the production of IL-6. Anti-IL-6 neutralizing antibody ameliorated suppressed insulin signaling by 24 h TNFα treatment, when it partially decreased SOCS3 induction and signal transducer and activator of transcription-3 phosphorylation. These results suggest that SOCS3 induction is involved in inhibited insulin signaling by TNFα. However, low-level expression of SOCS3 by IL-6 or adenovirus vector did not affect insulin-stimulated IRS-1 tyrosine phosphorylation. Interestingly, when IRS-1 serine phosphorylation was enhanced by TNFα or anisomycin in the presence of low-level SOCS3, IRS-1 degradation was remarkably enhanced. Taken together, both IRS-1 serine phosphorylation and SOCS3 induction are necessary, but one of the pair is not sufficient for the inhibited insulin signaling. Chronic TNFα may inhibit insulin signaling effectively because it causes both IRS-1 serine phosphorylation and the following SOCS3 induction in 3T3-L1 adipocytes.

Telmisartan, a new angiotensin II type 1 receptor blocker (ARB), was recently reported to stimulate PPARγ, and stronger effects of Telmisartan on insulin sensitivity has been expected than the class effect of ARB. In the present study, we examined the effects of Telmisartan on insulin sensitivity and adipokine levels in hypertensive and type 2 diabetic patients. Outpatients with both hypertension and type 2 diabetes mellitus (n = 36; male 23, female 13), received 20–40 mg Telmisartan orally once daily for 6 months. Physical examinations and blood or urine tests were performed before and 3 or 6 months after starting Telmisartan treatment. Results were statistically compared using Wilcoxon analysis. Telmisartan treatment for 3 or 6 months reduced systolic and diastolic blood pressure and urinary albumin excretion. Fasting plasma glucose, HbA1c, total and HDL-cholesterol, triglyceride, body weight, BMI and waist length were not changed. Fasting IRI and HOMA-IR were significantly decreased after Telmisartan treatment, suggesting the improved insulin sensitivity. Total and high molecular adiponectin were not changed. Interestingly, serum leptin was significantly increased by 3 months Telmisartan treatment, suggesting a possible involvement of leptin in improved insulin sensitivity. In conclusion, Telmisartan improved insulin resistance with increased serum leptin level in hypertensive and type 2 diabetic patients.

Pioglitazone is widely used for the treatment of diabetic patients with insulin resistance. The mechanism of pioglitazone to improve insulin sensitivity is not fully understood. Recent studies have shown that the induction of suppressor of cytokine signaling 3 (SOCS3) is related to the development of insulin resistance. Here, we examined whether the insulin-sensitizing effect of pioglitazone affects the SOCS induction. In db/db mice and high-fat-fed mice, expression of SOCS3 mRNA in fat tissue was increased compared with that in lean control mice, and pioglitazone suppressed SOCS3 levels. In 3T3-L1 adipocytes, mediators of insulin resistance such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6, growth hormone, and insulin increased SOCS3 expression, which was partially inhibited by pioglitazone. The ability of pioglitazone to suppress SOCS3 induction by TNF-alpha was greatly augmented by peroxisome proliferator-activated receptor gamma overexpression. SOCS3 overexpression and tyrphostin AG490, a Janus kinase 2 inhibitor, or dominant-negative STAT3 expression partially inhibited adiponectin secretion and was accompanied by decreased STAT3 phosphorylation. Conversely, pioglitazone increased adiponectin secretion and STAT3 phosphorylation in fat tissue of db/db mice and in 3T3-L1 adipocytes. These results suggest that pioglitazone exerts its effect to improve whole-body insulin sensitivity in part through the suppression of SOCS3, which is associated with the increase in STAT3 phosphorylation and adiponectin production in fat tissue.

Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-α (TNF-α) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-α. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-α induces their apoptosis in vitro.

Recent accumulating evidence suggests that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. Here, we show that a Toll-like receptor (TLR) protein, radioprotective 105 (RP105)/myeloid differentiation protein (MD)-1 complex, contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance. An HFD dramatically increased RP105 mRNA and protein expression in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. RP105 mRNA expression also was significantly increased in the visceral adipose tissue of obese human subjects relative to nonobese subjects. The RP105/MD-1 complex was expressed by most adipose tissue macrophages (ATMs). An HFD increased RP105/MD-1 expression on the M1 subset of ATMs that accumulate in eWAT. Macrophages also acquired this characteristic in coculture with 3T3-L1 adipocytes. RP105 knockout (KO) and MD-1 KO mice had less HFD-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance compared with wild-type (WT) and TLR4 KO mice. Finally, the saturated fatty acids, palmitic and stearic acids, are endogenous ligands for TLR4, but they did not activate RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling and may represent a novel therapeutic target for obesity-associated metabolic disorders.

Insulin receptors (IRs) and IGF-I receptors (IGF-IR) are major regulators of metabolism and cell growth throughout the body; however, their roles in the intestine remain controversial. Here we show that genetic ablation of the IR or IGF-IR in intestinal epithelial cells of mice does not impair intestinal growth or development or the composition of the gut microbiome. However, the loss of IRs alters intestinal epithelial gene expression, especially in pathways related to glucose uptake and metabolism. More importantly, the loss of IRs reduces intestinal glucose uptake. As a result, mice lacking the IR in intestinal epithelium retain normal glucose tolerance during aging compared with controls, which show an age-dependent decline in glucose tolerance. Loss of the IR also results in a reduction of glucose-dependent insulinotropic polypeptide (GIP) expression from enteroendocrine K-cells and decreased GIP release in vivo after glucose ingestion but has no effect on glucagon-like peptide 1 expression or secretion. Thus, the IR in the intestinal epithelium plays important roles in intestinal gene expression, glucose uptake, and GIP production, which may contribute to pathophysiological changes in individuals with diabetes, metabolic syndrome, and other insulin-resistant states.

Beige adipocytes are an inducible form of thermogenic adipocytes that become interspersed within white adipose tissue (WAT) depots in response to cold exposure. Previous studies have shown that type 2 cytokines and M2 macrophages induce cold-induced browning in inguinal WAT (ingWAT) by producing catecholamines. Exactly how the conditional and partial depletion of CD206+ M2-like macrophages regulates the cold-induced browning of ingWAT, however, remains unknown. We examined the role of CD206+ M2-like macrophages in the cold-induced browning of WAT using genetically engineered CD206DTR mice, in which CD206+ M2-like macrophages were conditionally depleted. The partial depletion of CD206+ M2-like enhanced UCP1 expression in ingWAT, as shown by immunostaining, and also upregulated the expression of Ucp1 and other browning-related marker genes in ingWAT after cold exposure. A flow cytometry analysis showed that the partial depletion of CD206+ M2-like macrophages caused an increase in the number of beige progenitors in ingWAT in response to cold. Thus, we concluded that CD206+ M2-like macrophages inhibit the proliferation of beige progenitors and that the partial depletion of CD206+ M2-like macrophages releases this inhibition, thereby enhancing browning and insulin sensitivity.

Growing evidence indicates an important link between gut microbiota, obesity, and metabolic syndrome. Alterations in exocrine pancreatic function are also widely present in patients with diabetes and obesity. To examine this interaction, C57BL/6J mice were fed a chow diet, a high-fat diet (HFD), or an HFD plus oral vancomycin or metronidazole to modify the gut microbiome. HFD alone leads to a 40% increase in pancreas weight, decreased glucagon-like peptide 1 and peptide YY levels, and increased glucose-dependent insulinotropic peptide in the plasma. Quantitative proteomics identified 138 host proteins in fecal samples of these mice, of which 32 were significantly changed by the HFD. The most significant of these were the pancreatic enzymes. These changes in amylase and elastase were reversed by antibiotic treatment. These alterations could be reproduced by transferring gut microbiota from donor C57BL/6J mice to germ-free mice. By contrast, antibiotics had no effect on pancreatic size or exocrine function in C57BL/6J mice fed the chow diet. Further, 1 week vancomycin administration significantly increased amylase and elastase levels in obese men with prediabetes. Thus, the alterations in gut microbiota in obesity can alter pancreatic growth, exocrine function, and gut endocrine function and may contribute to the alterations observed in patients with obesity and diabetes.

Polyphenols have health-promoting effects, such as improving insulin resistance. Isoxanthohumol (IX), a prenylated flavonoid found in beer hops, has been suggested to reduce obesity and insulin resistance; however, the mechanism remains unknown.High-fat diet-fed mice were administered IX. We analyzed glucose metabolism, gene expression profiles and histology of liver, epididymal adipose tissue and colon. Lipase activity, fecal lipid profiles and plasma metabolomic analysis were assessed. Fecal 16s rRNA sequencing was obtained and selected bacterial species were used for in vitro studies. Fecal microbiota transplantation and monocolonization were conducted to antibiotic-treated or germ-free (GF) mice.The administration of IX lowered weight gain, decreased steatohepatitis and improved glucose metabolism. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption by decreasing the expression of the fatty acid transporter CD36 in the small intestine, which was confirmed by increased lipid excretion in feces. IX administration increased markers of intestinal barrier function, including thickening the mucin layer and increasing caludin-1, a tight-junction related protein in the colon. In contrast, the effects of IX were nullified by antibiotics. As revealed using 16S rRNA sequencing, the microbial community structure changed with a significant increase in the abundance of Akkermansia muciniphila in the IX-treated group. An anaerobic chamber study showed that IX selectively promoted the growth of A. muciniphila while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct effect of A. muciniphila on lipid and glucose metabolism, we monocolonized either A. muciniphila or Bacteroides thetaiotaomicron to GF mice. A. muciniphila monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism, with decreased levels of multiple classes of fatty acids determined using plasma metabolomic analysis.Our study demonstrated that IX prevents obesity and enhances glucose metabolism by inhibiting dietary fat absorption. This mechanism is linked to suppressing pancreatic lipase activity and shifts in microbial composition, notably an increase in A. muciniphila. These highlight new treatment strategies for preventing metabolic syndrome by boosting the gut microbiota with food components.

Adipose tissue not only functions as the major energy-storing tissue, but also functions as an endocrine organ that regulates systemic metabolism by releasing various hormones called adipokines. Macrophages play a critical role in maintaining adipocyte health in a lean state and in remodeling during the progression of obesity. Large numbers of classically activated (M1) macrophages accumulate in adipose tissue as adipocytes become larger because of excessive energy conditions, and they adversely affect insulin resistance by triggering local and systemic inflammation. In contrast, alternatively activated (M2) macrophages seem to maintain the health of adipose tissues in a lean state. In addition, they play a role in adapting to excess energy states, because M2 macrophage dysfunction caused by genetic disruption of the M2 gene results in metabolic disorders under high-fat-fed conditions that are probably attributable to their anti-inflammatory functions. Nonetheless, how M2 macrophages contribute to maintaining the health of adipose tissue and therefore to insulin sensitivity is largely unknown. In this article, we review the literature on the role of M1 and M2 macrophages in metabolism, with a special focus on the role of M2 macrophages in adipose tissue. Likewise, we raise topics of M2 macrophages in non-adipose tissues to expand our understanding of macrophage heterogeneity.

The potential roles of the gut microbiota in the pathogenesis of non-alcoholic fatty liver disease, including non-alcoholic steatohepatitis (NASH), have attracted increased interest. We have investigated the links between gut microbiota and NASH development in Tsumura-Suzuki non-obese mice fed a high-fat/cholesterol/cholate-based (iHFC) diet that exhibit advanced liver fibrosis using antibiotic treatments. The administration of vancomycin, which targets Gram-positive organisms, exacerbated the progression of liver damage, steatohepatitis, and fibrosis in iHFC-fed mice, but not in mice fed a normal diet. F4/80+-recruited macrophages were more abundant in the liver of vancomycin-treated iHFC-fed mice. The infiltration of CD11c+-recruited macrophages into the liver, forming hepatic crown-like structures, was enhanced by vancomycin treatment. The co-localization of this macrophage subset with collagen was greatly augmented in the liver of vancomycin-treated iHFC-fed mice. These changes were rarely seen with the administration of metronidazole, which targets anaerobic organisms, in iHFC-fed mice. Finally, the vancomycin treatment dramatically modulated the level and composition of bile acid in iHFC-fed mice. Thus, our data demonstrate that changes in inflammation and fibrosis in the liver by the iHFC diet can be modified by antibiotic-induced changes in gut microbiota and shed light on their roles in the pathogenesis of advanced liver fibrosis.

The activation of platelet-derived growth factor receptor-β (PDGFR-β) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-β signalling during the development of diabetic nephropathy. We recently generated pancreatic beta cell-specific Ca2+/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-β (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-β gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. The plasma glucose concentrations and HbA1c levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-β gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-β gene deletion. The activation of PDGFR-β signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Sirt1 plays an important role in regulating glucose and lipid metabolism in obese animal models. Impaired adipose tissue angiogenesis in the obese state decreases adipogenesis and thereby contributes to glucose intolerance and lipid metabolism. However, the mechanism by which Sirt1 activation affects obesity-associated impairments in angiogenesis in the adipose tissue is not fully understood. Here, we show that SRT1720 treatment induces angiogenic genes in cultured 3T3-L1 preadipocytes and ex vivo preadipocytes. siRNA-mediated knockdown of Sirt1 in 3T3-L1 preadipocytes downregulated angiogenic genes in the preadipocytes. SRT1720 treatment upregulated metabolically favorable genes and reduced inflammatory gene expressions in the adipose tissue of diet-induced obese (DIO) mice. Collectively, these findings suggest a novel role of SRT1720-induced Sirt1 activation in the induction of angiogenic genes in preadipocytes, thereby reducing inflammation and fibrosis in white adipose tissue (WAT) and promoting insulin sensitivity.

Proinflammatory cytokines are well-known to inhibit insulin signaling to result in insulin resistance. IL-1α is also one of the proinflammatory cytokines, but the mechanism of how IL-1α induces insulin resistance remains unclear. We have now examined the effects of IL-1α on insulin signaling in 3T3-L1 adipocytes. Prolonged IL-1α treatment for 12 to 24 hours partially decreased the protein levels as well as the insulin-stimulated tyrosine phosphorylation of IRS-1 and Akt phosphorylation. mRNA for SOCS3, an endogenous inhibitor of insulin signaling, was dramatically augmented 4 hours after IL-1α treatment. Concomitantly, the level of IL-6 in the medium and STAT3 phosphorylation were increased by the prolonged IL-1α treatment. Addition of anti-IL-6 neutralizing antibody to the medium or overexpression of dominant-negative STAT3 decreased the IL-1α-stimulated STAT3 activation and SOCS3 induction, and ameliorated insulin signaling. These results suggest that the IL-1α-mediated deterioration of insulin signaling is largely due to the IL-6 production and SOCS3 induction in 3T3-L1 adipocytes.

Expansion of adipose tissue during obesity through the recruitment of newly generated adipocytes (hyperplasia) is metabolically healthy, whereas that through the enlargement of pre-existing adipocytes (hypertrophy) leads to metabolic complications. Accumulating evidence from genetic fate mapping studies suggests that in animal models receiving a high-fat diet (HFD), only adipocyte progenitors (APs) in gonadal white adipose tissue (gWAT) have proliferative potential. However, the proliferative potential and differentiating capacity of APs in the inguinal WAT (iWAT) of male mice remains controversial. The objective of this study was to investigate the proliferative and adipogenic potential of APs in the iWAT of HFD-fed male mice.We generated PDGFRα-GFP-Cre-ERT2/tdTomato (KI/td) mice and traced PDGFRα-positive APs in male mice fed HFD for 8 weeks. We performed a comprehensive phenotypic analysis, including the histology, immunohistochemistry, flow cytometry, and gene expression analysis, of KI/td mice fed HFD.Contrary to the findings of others, we found an increased number of newly generated tdTomato+ adipocytes in the iWAT of male mice, which was smaller than that observed in the gWAT. We found that in male mice, the iWAT has more proliferating tdTomato+ APs than the gWAT. We also found that tdTomato+ APs showed a higher expression of Dpp4 and Pi16 than tdTomato- APs, and the expression of these genes was significantly higher in the iWAT than in the gWAT of mice fed HFD for 8 weeks. Collectively, our results reveal that HFD feeding induces the proliferation of tdTomato+ APs in the iWAT of male mice.In male mice, compared with gWAT, iWAT undergoes hyperplasia in response to 8 weeks of HFD feeding through the recruitment of newly generated adipocytes due to an abundance of APs with a high potential for proliferation and differentiation.

Monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) in adipose tissue are thought to induce systemic insulin resistance in rodents; but the precise mechanism is not fully clarified. We examined the mechanism of Ang II-induced and/or tumor necrosis factor-α (TNF-α)-induced MCP-1 production from 3T3-L1 preadipocytes. The MCP-1 protein and MCP-1 mRNA expression in 3T3-L1 preadipocytes were increased significantly by stimulation with TNF-α. We found no significant increase in MCP-1 concentrations by Ang II alone; but it enhanced the TNF-α-induced MCP-1 mRNA expression in a dose-dependent manner. Then, we examined the effect of Ang II and/or TNF-α on phosphorylation of extracellular signal-regulated kinase (ERK), p38MAPK, and IκB-α. Ang II and TNF-α clearly enhanced ERK and p38MAPK phosphorylation. IκB-α phosphorylation was enhanced by TNF-α, but not by Ang II. The MCP-1 mRNA expression induced by TNF-α and co-stimulation with Ang II was inhibited by either ERK inhibitor, p38MAPK inhibitor or NF-κB inhibitor. Moreover, Ang II enhanced the activation of AP-1 (c-fos) induced by TNF-α. Our results suggest that Ang II may serve as an additional stimulus on the TNF-α-induced MCP-1 production through the ERK-and p38MAPK-dependent pathways probably due to AP-1 activation.

We examined whether oral administration of a hop-derived prenylflavonoid isoxanthohumol (IX) would show anti-obesity activity and the underlying mechanism of the potential activity using a high-fat diet (HFD)-induced obese mouse model. Oral administration of 180 mg/kg IX for 8 weeks suppressed HFD-induced accumulation of visceral fat and body weight gain in mice. Simultaneously, IX changed the composition of the microbiome, as determined by a significant increase in the relative abundances of Akkermansia muciniphila, Blautia, and Escherichia coli. A. muciniphila accounted for 23% and 24% of the total microbiome in the HFD+60 mg/kg and 180 mg/kg IX groups, respectively, while it was undetectable in the normal diet (ND) and HFD groups. Similarly, Blautia accounted for 8% and 10% of the total microbiome in the HFD+60 mg/kg and 180 mg/kg IX groups, respectively, while it accounted for less than 1% in the ND and HFD groups. In contrast, a significant decrease in the relative abundance of Oscillospira was observed in the HFD+60 mg/kg and 180 mg/kg IX groups compared with the HFD group. We further examined the anti-obesity effect of IX using a germ-free (GF) mouse model to clarify the relationship between the microbiome and the effect of IX. IX showed no significant anti-obesity effect on fat accumulation and weight gain in GF mice. These results suggest that the anti-obesity effect of IX may involve microbial changes.

A single-arm prospective study was conducted among Japanese patients with type 2 diabetes having preserved ejection fraction. The aim was to investigate (1) whether liraglutide therapy could improve B-type natriuretic peptide (BNP) levels and diastolic cardiac function assessed by the E-wave to E' ratio (E/E') using transthoracic echocardiography (TTE), and (2) whether E/E' contributed to BNP improvement independent of bodyweight reduction (UMIN000005565).Patients with type 2 diabetes and left ventricular ejection fraction (LVEF) ≥ 40% without heart failure symptoms were enrolled, and daily injection with liraglutide (0.9 mg) was introduced. Cardiac functions were assessed by TTE before and after 26 weeks of liraglutide treatment. Diastolic cardiac function was defined as septal E/E' ≥ 13.0.Thirty-one patients were analyzed. BNP and E/E' improved, with BNP levels declining from 36.8 ± 30.5 pg/mL to 26.3 ± 25.9 pg/mL (p = 0.0014) and E/E' dropping from 12.7 ± 4.7 to 11.0 ± 3.3 (p = 0.0376). The LVEF showed no significant changes. E/E' improved only in patients with E/E' ≥ 13.0. Favorable changes in E/E' were canceled when adjusted for body mass index (BMI). Multivariate linear regression analysis revealed that the left ventricular diastolic diameter and ∆E/E'/∆BMI contributed to ∆BNP/baseline BNP (p = 0.0075, R2 = 0.49264).Liraglutide had favorable effects on BNP and E/E' but not on LVEF. E/E' improvement was only seen in patients with diastolic cardiac function. Body weight reduction affected the change of E/E'. The BMI-adjusted E/E' significantly contributed to the relative change of BNP. GLP-1 analog treatment could be considered a therapeutic option against diabetic diastolic cardiac dysfunction regardless of body weight. This trial is registered with the University Hospital Medical Information Network in Japan, with clinical trial registration number: UMIN000005565.

Recently, obesity-induced insulin resistance, type 2 diabetes, and cardiovascular disease have become major social problems. We have previously shown that Astaxanthin (AX), which is a natural antioxidant, significantly ameliorates obesity-induced glucose intolerance and insulin resistance. It is well known that AX is a strong lipophilic antioxidant and has been shown to be beneficial for acute inflammation. However, the actual effects of AX on chronic inflammation in adipose tissue (AT) remain unclear. To observe the effects of AX on AT functions in obese mice, we fed six-week-old male C57BL/6J on high-fat-diet (HFD) supplemented with or without 0.02% of AX for 24 weeks. We determined the effect of AX at 10 and 24 weeks of HFD with or without AX on various parameters including insulin sensitivity, glucose tolerance, inflammation, and mitochondrial function in AT. We found that AX significantly reduced oxidative stress and macrophage infiltration into AT, as well as maintaining healthy AT function. Furthermore, AX prevented pathological AT remodeling probably caused by hypoxia in AT. Collectively, AX treatment exerted anti-inflammatory effects via its antioxidant activity in AT, maintained the vascular structure of AT and preserved the stem cells and progenitor's niche, and enhanced anti-inflammatory hypoxia induction factor-2α-dominant hypoxic response. Through these mechanisms of action, it prevented the pathological remodeling of AT and maintained its integrity.

We report a sporadic case of maturity-onset diabetes of the young type 5 (MODY5) with a whole-gene deletion of the hepatocyte nuclear factor-1beta (HNF1B) gene. A 44-year-old Japanese man who had been diagnosed with early-onset non-autoimmune diabetes mellitus at the age of 23 was examined. He showed multi-systemic symptoms, including a solitary congenital kidney, pancreatic hypoplasia, pancreatic exocrine dysfunction, elevation of the serum levels of liver enzymes, hypomagnesemia, and hyperuricemia. These clinical characteristics, in spite of the absence of a family history of diabetes, prompted us to make the diagnosis of maturity-onset diabetes of the young 5 (MODY 5). One allele deletion of the entire HNF1B gene revealed by multiplex ligation-dependent probe amplification (MLPA) led us to the diagnoses of 17q12 microdeletion syndrome even though there were negative chromosomal analyses with array comparative genomic hybridization (CGH). 17q12 microdeletion syndrome, which is not rare especially in sporadic cases since 17q12 is a typical hot spot for chromosomal deletion, could have complicated the clinical heterogeneity of MODY5.

The gut microbiota metabolizes the nutrients to produce various metabolites that play crucial roles in host metabolism. However, the links between the microbiota established by different nutrients and the microbiota-influenced changes in the plasma lipids remain unclear. Diets rich in cornstarch, fructose, branched chain amino acids, soybean oil (SO), or lard established a unique microbiota and had influence on glucose metabolism, which was partially reproduced by transferring the microbiota. Comparison of plasma lipidomic analysis between germ-free and colonized mice revealed significant impacts of the microbiota on various lipid classes, and of note, the microbiota established by the SO diet, which was associated with the greatest degree of glucose intolerance, caused the maximum alteration of the plasma lipid profile. Thus, the gut microbiota composed of dietary nutrients was associated with dynamic changes in the lipids potentially having differential effects on glucose metabolism.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

Mitochondrial DNA m.3243A > G mutation causes mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and its associated multi-organ disorders, including diabetes. To clarify associations between m.3243A > G organ heteroplasmy and clinical phenotypes, including the age at death, we combined genetic and pathological examinations from seven unreported and 36 literature cases of autopsied subjects. Clinical characteristics of subjects were as follows: male, 13; female, 28; unknown, 2; the age at death, 36.9 ± 20.2 [4-82] years; BMI, 16.0 ± 2.9 [13.0-22.3]; diabetes, N = 21 (49%), diabetes onset age 38.6 ± 14.2 years; deafness, N = 27 (63%); stroke-like episodes (StLEp), N = 25 (58%); congestive heart failure (CHF), N = 15 (35%); CHF onset age, 51.3 ± 14.5 years. Causes of death (N = 32) were as follows: cardiac, N = 13 (41%); infection, N = 8 (25%); StLEp, N = 4 (13%); gastrointestinal, N = 4 (13%); renal, N = 2 (6%); hepatic, N = 1 (2%). High and low heteroplasmies were confirmed in non-regenerative and regenerative organs, respectively. Heteroplasmy of the liver, spleen, leukocytes, and kidney for all subjects was significantly associated with the age at death. Furthermore, the age at death was related to juvenile-onset (any m.3243A > G-related symptoms appeared before 20) and stroke-like episodes. Multiple linear regression analysis with the age at death as an objective variable showed the significant contribution of liver heteroplasty and juvenile-onset to the age at death. m.3243A > G organ heteroplasmy levels, particularly hepatic heteroplasmy, are significantly associated with the age at death in deceased cases.

Among the three adiponectin isoforms, a lower ratio of high molecular weight (HMW) adiponectin to total adiponectin (TA) is well known to cause insulin resistance and type 2 diabetes (T2D). However, how the levels of other adiponectin isoforms, such as the middle molecular weight (MMW) and low molecular weight (LMW) isoforms, and their relative ratio to TA change in T2D subjects has not been determined. Therefore, we investigated the association of these adiponectin-related parameters with T2D.We examined the associations between adiponectin-related parameters and diabetes in a group of 394 T2D subjects and 374 controls (1st group) randomly selected from among the participants in our previous study. The associations between these parameters and the HOMA-IR in a 2nd group, consisting of the subjects remaining in the 1st group after the exclusion of subjects receiving diabetic medication, were also examined.In the 1st group, after adjusting for confounding factor, the levels of all the adiponectin isoforms and the HMW/TA ratio were significantly lower among the diabetic subjects than among the controls (all P values < 0.01). On the contrary, the LMW/TA ratio was significantly higher among the diabetic subjects (P < 0.01) and was positively associated with T2D (odds ratio = 8.64, P < 0.01). In the 2nd group, the HMW/TA ratio was inversely associated with the HOMA-IR; however, the LMW/TA ratio was positively associated with the HOMA-IR (β for LMW/TA ratio = 0.89, SE = 0.24, P < 0.001), similar to the association with T2D. The MMW/TA ratio was not associated with T2D or the HOMA-IR.The current investigation demonstrated that, unlike the reduction in the levels of all the adiponectin isoforms and the HMW/TA ratio, an increased LMW/TA ratio was associated with T2D through its relation to insulin resistance.

(Cell Metabolism 22, 516–530; September 1, 2015) In preparing the final version of Figure 5, two errors occurred as the format was switched from horizontal to vertical, which resulted in incorrect labeling. In Figure 5A, the column next to the heatmap that lists the OTU (bacterial taxonomy) was inverted, such that top label (2014/bacteroides) should be labeling the bottom row of the heatmap, and so forth. In Figure 5B, the labeling over the fourth and sixth columns (B6J/Jos and 129t/Jos) were also transposed. Both of these panels are based on data presented in Table S4. The actual data in that table are unchanged and correct. The changes in Figure 5, which are now published online, make it consistent with the data and the statements in the text, which were written using a correct version of the figure. The authors apologize for any confusion that this error may have caused.Figure 5Representation of Bacterial Taxa in the Fecal Microbiota (original)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Interactions between Gut Microbiota, Host Genetics and Diet Modulate the Predisposition to Obesity and Metabolic SyndromeUssar et al.Cell MetabolismAugust 20, 2015In BriefIn a longitudinal analysis of host genetics, diet, and gut microbiota interations, Ussar et al. demonstrate how interactions between the gut microbiota, host genetics, and diet influence the development of metabolic syndrome. The authors find that specific bacterial taxa appear to be linked to specific phenotypes. Changing the environment in early life not only changes the microbiota but also changes development of metabolic syndrome. Full-Text PDF Open Archive

Chronic inflammation is a pathophysiology of insulin resistance in metabolic diseases, such as obesity and type 2 diabetes. Adipose tissue macrophages (ATMs) play important roles in this inflammatory process. SIRT1 is implicated in the regulation of glucose metabolism in some metabolic tissues, such as liver or skeletal muscle. This study was performed to investigate whether SIRT1 in macrophages played any roles in the regulation of inflammation and glucose metabolism. Myeloid cell-specific SIRT1-knockout mice were originally generated and analyzed under chow-fed and high-fat-fed conditions. Myeloid cell-specific SIRT1 deletion impaired insulin sensitivity and glucose tolerance assessed by the glucose- or insulin-tolerance test, which was associated with the enhanced expression of inflammation-related genes in epididymal adipose tissue of high-fat-fed mice. Interestingly, the M1 ATMs from the SIRT1-knockout mice showed more hypoxic and inflammatory phenotypes than those from control mice. The expressions of some inflammatory genes, such as Il1b and Nos2, which were induced by in vitro hypoxia treatment, were further enhanced by SIRT1 deletion along with the increased acetylation of HIF-1α in cultured macrophages. These results suggest that deletion of SIRT1 in myeloid cells impairs glucose metabolism by enhancing the hypoxia and inflammatory responses in ATMs, thereby possibly representing a novel therapeutic target for metabolic diseases, such as type 2 diabetes.

Meflin ( Islr ) expression has gained attention as a marker for mesenchymal stem cells, but its function remains largely unexplored. Here, we report the generation of Meflin - CreER T2 mice with CreER T2 inserted under the Meflin gene promoter to label Meflin -expressing cells genetically, thereby enabling their lineages to be traced. We found that in adult mice, Meflin -expressing lineage cells were present in adipose tissue stroma and had differentiated into mature adipocytes. These cells constituted Crown-like structures in the adipose tissue of mice after high-fat diet loading. Cold stimulation led to the differentiation of Meflin -expressing lineage cells into beige adipocytes. Thus, the Meflin-CreER T2 mouse line is a useful new tool for visualizing and tracking the lineage of Meflin -expressing cells.

The aim of this study was to verify the effect of treatment with isoxanthohumol (IX) on the metabolomics profile of mouse feces to explore the host-intestinal bacterial interactions at the molecular level.The fecal contents of several amino acids in the high-fat diet (HFD) + 0.1% IX group (treated with IX mixed in diets for 12 weeks) were significantly lower than in the HFD group.The fecal contents of the secondary bile acid deoxycholic acid (DCA) in the HFD + 180 mg/kg IX group (orally treated with IX for 8 weeks) were significantly lower than in the HFD group; the values in the HFD group were higher than those in the normal diet (ND) group.Administration of IX changed the fecal metabolomics profile.For some metabolites, IX normalized HFD-induced fluctuations.

Chronic inflammation of the liver is often observed with obesity or type 2 diabetes. In these pathological conditions, the immunological cells, such as macrophages, play important roles in the development or growth of liver cancer. Recently, it was reported that hypoxia-inducible factor-1α (HIF-1α) is a key molecule for the acquisition of inflammatory M1 polarity of macrophages. In the present study, we examined the effects of altered macrophage polarity on obesity- and diabetes-associated liver cancer using macrophage-specific HIF-1α knockout (KO) mice.To induce liver cancer in the mice, diethylnitrosamine, a chemical carcinogen, was used. Both KO mice and wild-type littermates were fed either a high-fat diet (HFD) or normal chow. They were mainly analyzed 6 months after HFD feeding.Development of liver cancer after HFD feeding was 45% less in KO mice than in wild-type littermates mice. Phosphorylation of extracellular signal-regulated kinase 2 was also lower in the liver of KO mice. Those effects of HIF-1α deletion in macrophages were not observed in normal chow-fed mice. Furthermore, the size of liver tumors did not differ between KO and wild-type littermates mice, even those on a HFD. These results suggest that the activation of macrophage HIF-1α by HFD is involved not in the growth, but in the development of liver cancer with the enhanced oncogenic extracellular signal-regulated kinase 2 signaling in hepatocytes.The activation of macrophage HIF-1α might play important roles in the development of liver cancer associated with diet-induced obesity and diabetes.

Abstract Aims/Introduction Fragmented QRS (fQRS) on electrocardiography is a marker of myocardial fibrosis and myocardial scar formation. This study aimed to clarify the relationship of fQRS with diabetes mellitus and metabolic syndrome (MetS) in Japanese patients. Materials and Methods Approximately 702 individuals who had a routine health checkup at the Hokuriku Health Service Association (Toyama, Japan) in October 2014 were enrolled and categorized into one of the following four groups based on MetS and diabetes mellitus status: with diabetes mellitus (+) MetS+ (164 participants); diabetes mellitus+ without MetS (Mets−; 103 participants); diabetes mellitus− MetS+ (133 participants); and diabetes mellitus− MetS− (302 participants). fQRS was assessed using the results of electrocardiography. Results The prevalence of fQRS was statistically higher in patients with diabetes mellitus+ MetS+ (37%) and diabetes mellitus+ MetS− (35%), than those with diabetes mellitus− MetS+ (14%) or diabetes mellitus− MetS− (10%; P &lt; 0.0001). Significant differences were observed between the fQRS(+) and fQRS(–) groups for age, sex, waist circumference, heart rate, hypertension, hemoglobin A1c, total cholesterol, MetS and diabetes mellitus. The area under the receiver operating characteristic curve for traditional risk factors and diabetes mellitus was 0.72 ( P = 0.0007, 95% confidence interval 0.67–0.76), and for traditional risk factors and MetS it was 0.67 ( P = 0.28, 95% confidence interval 0.62–0.72). Patients with diabetes mellitus had more than threefold higher likelihood of showing fQRS (odds ratio 3.41; 95% confidence interval 2.25–5.22; P &lt; 0.0001) compared with the reference group without diabetes mellitus, after adjusting for age, sex, dyslipidemia, hypertension and waist circumference. Conclusions fQRS was observed more frequently in diabetes mellitus patients than in MetS and control individuals. Diabetes mellitus was the most significant determinant for fQRS among MetS and other traditional metabolic risk factors.

Bofutsushosan (BFT), an oriental herbal medicine, has been clinically used for obese patients. To explore the impact of BFT on glucose metabolism, male C57BL6 mice were fed on high-fat diet (HFD) for 12 weeks and administered either BFT (25 mg /day) or saline for the last 8 weeks. Oral glucose tolerance test and insulin tolerance test revealed improved glucose metabolism with improved insulin sensitivity in BFT treated mice, which was associated with decreased inflammatory gene expressions and improved insulin signaling in the adipose tissue. Whereas the weight of liver and adipose tissue was not changed, cecum weight was significantly increased by BFT. 16S rRNA sequence analysis of fecal samples showed that microbial composition was markedly changed. BFT reduced the relative abundance of Bacteroidetes from 52% to 34%, whereas it increased Verrucomicrobia from 3.4% to 24%. An increase in Verrucomicrobia was mainly associated with Akkermansia mucuniphila (Akk). The bloom of Akk was observed at one week of BFT treatment. Consistent with the previous reports that Akk improves gut barrier function and prevents from metabolic endotoxemia in obese subjects, BFT decreased the gut permeability as assessed by FITC-dextran gavage assay, which was associated with increased claudin-1 protein in the colon. Furthermore, plasma endotoxin level and hepatic lipopolysaccharide binding protein expression were significantly decreased in BFT group. Antibiotic treatment canceled the metabolic effect of BFT. Moreover, when the gut microbiota of BFT-treated mice were transferred to HFD-fed mice, glucose metabolism was significantly improved with decreased diet -induced inflammation with AKK bloom. These data demonstrate that BFT increases Akk in the gut, which may contribute to improving gut barrier function and preventing metabolic endotoxemia, leading to improved diet-induced inflammation, thereby controlling glucose metabolism. Disclosure S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. I. Usui: None. A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. Y. Igarashi: None. T. Kado: None. K. Okabe: None. K. Yagi: None. T. Nakagawa: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura &amp; Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co.

Diastolic cardiac dysfunction in type 2 diabetes (DD2D) is a critical risk of heart failure with preserved ejection fraction. However, there is no established biomarker to detect DD2D. We aimed to investigate the predictive impact of fragmented QRS (fQRS) on electrocardiography on the existence of DD2D.We included in-hospital patients with type 2 diabetes without heart failure symptoms who were admitted to our institution for glycemic management between November 2017 and April 2021. An fQRS was defined as an additional R' wave or notching/splitting of the S wave in two contiguous electrocardiography leads. DD2D was diagnosed according to the latest guidelines of the American Society of Echocardiography.Of 320 participants, 122 patients (38.1%) had fQRS. DD2D was diagnosed in 82 (25.6%). An fQRS was significantly associated with the existence of DD2D (odds ratio 4.37, 95% confidence interval 2.33-8.20; p < 0.0001) adjusted for seven potential confounders. The correlation between DD2D and diabetic microvascular disease was significant only among those with fQRS. Classification and regression tree analysis showed that fQRS was the most relevant optimum split for DD2D.An fQRS might be a simple and promising predictor of the existence of DD2D. The findings should be validated in a larger-scale cohort.

An immune-related adverse event is a new disease entity that occurs after the introduction and extensive use of anticancer immune agents, such as anti-programmed cell death protein 1 (PD-1) or anti-programmed death ligand 1 (PD-L1) antibodies. Anti-PD-L1-related type 1 diabetes is the immune-related adverse event that typically presents as fulminant type 1 diabetes, characterized by an absence of anti-islet autoantibodies. We are presenting a case of anti-PD-L1 therapy-related type 1 diabetes with highly positive anti-GAD and anti-islet antigen 2 antibodies. The present patient was a 66-year-old Japanese men with type 2 diabetes diagnosed at the age of 57 years. Since then, he had been treated with nateglinide, teneligliptin and biphasic insulin aspart 30. He was diagnosed with squamous cell lung carcinoma in the right lower lobe at the age of 63 years. His lung cancer was initially treated with concurrent chemotherapy and radiation therapy. After a recurrence, the anti-PD-L1 antibody, atezolizumab, was introduced at the age of 64 years. Although his biopsied lung cancer tissue specimens showed negative PD-L1 expression, the atezolizumab had been effective. The diameter of the primary nodule changed from 43 to 34 mm. Two months after starting atezolizumab, his glycated hemoglobin level rapidly increased from 7.8 to 10.0%. The serum C-peptide level, which was 4.3 ng/mL 1 year earlier, became undetectable. However, no obvious ketosis and no hyperglycemic symptoms were observed during the whole clinical course, and fulminant type 1 diabetes was less likely in this case. Interestingly, the patient showed multiple islet-related autoantibodies: anti-GAD autoantibody, with a titer of 44.8 U/mL (normal range <5.0 U/mL), and anti-islet antigen 2 autoantibody, with a titer >30.0 U/mL (normal range <0.6 U/mL). The results were negative regarding anti-zinc transporter 8, anti-thyroid peroxidase and anti-thyroglobulin autoantibodies. Conforming to the provisions of the Declaration of Helsinki, written informed consent was obtained before examining the patient's human leukocyte antigen (HLA) types. HLA deoxyribonucleic acid typing detected by polymerase chain reaction sequenced-based typing methods were as follows: DRB1 01:01:01 and 09:01:02, and DQB1 05:01:01 and 03:03:02. As the limitation of the present study, islet autoantibodies were not checked before, and there still exists the possibility of undiagnosed slowly progressive type 1 diabetes. However, clinical features strongly suggested the patient had type 2 diabetes with middle-aged onset, obesity and family history of type 2 diabetes. The rapid progression of type 1 diabetes from the introduction of atezolizumab should also be discussed. Usui et al.1 reviewed and reported that eight out of 13 patients presented with newly onset type 1 diabetes within 10 weeks from the introduction of anti-PD-1/PD-L1 antibodies. In summary, we considered that the present patient developed newly onset type 1 diabetes, during type 2 diabetes, as a result of anti-PD-L1 therapy. Baden et al.2 explored 22 cases of anti-PD-1 therapy-related type 1 diabetes, and only one example showed the single islet-related autoantibody, anti-GAD-antibody. Out of all the cases we have examined, this is the first case of type 1 diabetes with HLA-DR9 related to immune checkpoint blockade therapy, presenting multiple islet-related autoantibodies. Although Clotman et al.3 reported five cases of anti-PD-L1-related type 1 diabetes showing multiple autoantibodies, no case showed HLA-DR9. As HLA-DR9 is unique to Asian individuals, immunological backgrounds were different from the present patient. The present patient had phenotypically acute-onset autoimmune type 1 diabetes. Interestingly, Tsutsumi et al.4 reported that patients with HLA-DRB1 09:01 and DQB1 03:03 are susceptible to fulminant type 1 diabetes, and those with DRB1 01:01 are resistant to it, but might develop classical type 1 diabetes with positive anti-islet autoantibodies. Although it is scientifically meaningless to discuss the association of clinical phenotypes in an individual case with the HLA haplotype, clinical findings on this case suggest the possible reflection of the mixture of class II HLA genotypes and the contribution of medications affecting chronic inflammations, such as dipeptidyl peptidase-4 inhibitors. The authors declare no conflict of interest.

Sensor-augmented insulin pump therapy with a predictive low glucose suspend (SAP-PLGS) feature is a remarkably progressed modality for the glycemic management of patients with type 1 diabetes. This technology avoids nocturnal hypoglycemia and severe hypoglycemia. A Brazilian woman developed type 1 diabetes at age 11 and was treated with multiple daily insulin injections. At age 20, she was admitted to our internal medicine department for her first pregnancy. Her HbA1c was 7.9% in the 6 weeks of gestation. Although the combination of continuous subcutaneous insulin infusion and a sensor-augmented pump was introduced, she had a miscarriage in the next week. After 6 months, she became pregnant again. Despite an HbA1c of 7.2%, she had another miscarriage. Thereafter, she returned to multiple daily insulin injections and began using intermittently scanned continuous glycemic monitoring. At age 22, she had her third pregnancy. Her HbA1c was 7.3%. SAP-PLGS was then introduced, which reduced her frequent hypoglycemic events and blood glucose fluctuations. She gave birth to a 4137 g boy at 39 weeks without significant complications. Successful delivery can be obtained in women with type 1 diabetes following repeated miscarriages after introducing SAP-PLGS. We hypothesize that the modality might contributed to our patient's miscarriage avoidance by reducing her glycemic fluctuations.

Endogenous insulin secretion could be recovered by improving hyperglycemia in patients with type 2 diabetes (T2D). However, no reports have showed the associations between short-term insulin secretion recovery and future glycemic control. In this study, we investigated whether short-term C-peptide index (CPI) recovery during hospitalization could predict future glycemic control. In this study, 117 patients with T2D were included. The recovery of fasting and postprandial CPI were defined as (fasting CPI at discharge - fasting CPI at admission [index-A]) and (postprandial CPI at discharge - postprandial CPI at admission [index-B]). We compared the clinical background between the low-recovery and high-recovery groups of each index and assessed the association between the indices and metabolic parameters after discharge. Using index-A, age was significantly younger (P = 0.017) and body mass index (BMI) was significantly higher (P = 0.004) in the high-recovery group than that in low-recovery group. Using index-B, the age was significantly younger (P = 0.019) and BMI was significantly higher (P = 0.004) in the high-recovery group. Additionally, visceral fat area (P = 0.043), serum TG (P &amp;lt; 0.001), and γ-GTP levels (P = 0.009) were significantly higher in the high-recovery group. Regarding the association between CPI recovery and future glycemic control, GA levels were significantly lower in the high-recovery group than that in low-recovery group at 6 months (16.7 vs 21.1%, P = 0.045) and 12 months (16.8 vs 21.7%, P = 0.033) after discharge in the analysis using index-B. Furthermore, postprandial CPI (cut-off; 3.79 [AUC 0.698, P = 0.007]) at discharge and index-B (cut-off; 1.66 [AUC 0.657, P = 0.018]) were identified as indicators to predict HbA1c ≤ 7.0% at 6 months after discharge. In conclusion, short-term postprandial CPI recovery during hospitalization may predict future glycemic control. Disclosure A.Enkaku: None. D.Chujo: None. M.Kamigishi: None. S.Inagawa: None. W.Sakai: None. S.Matsukoshi: None. A.Takikawa: None. S.Fujisaka: None. K.Tobe: Other Relationship; MSD K.K., Novo Nordisk Pharma Ltd., Takeda Pharmaceutical Company Limited, Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Suntory Global Corporation, Ltd.,, Taiho Pharmaceutical Co. Ltd., Japan Diabetes Foundation, Japan Association for Diabetes Education and Care.

Gut microbiota is an important driver of metabolic status. Recent studies have demonstrated that dysbiosis precedes the development of metabolic disorders. However, the bacterial species associated with metabolic syndrome and their physiological role in obesity and glucose metabolism have not been fully elucidated. In this study, we performed 16S rRNA sequencing analysis on fecal samples from 49 metabolically healthy volunteers, a detailed lifestyle questionnaire survey including diet, and an analysis on metabolic parameters. We also examined the metabolic effects of some microbes by transplantation into HFD-fed mice. The average age, BMI and HbA1c were 43.5±9.0, 23.9±3.9 and 5.5±0.3, respectively. A clear correlation was observed between lifestyle and metabolic parameters. Analysis of fecal microbial structure divided the participants into three enterotypes according to their predominant phylogenic pattern; 1) Prevotella copri group, 2) Bacteroides group, and 3) others. Enterotype 1 had higher random blood glucose (BG), and lower HDL-C, while enterotype 2 had lower random BG, BMI, and waist circumference. Eleven bacterial species were identified as being positively or negatively correlated with metabolic parameters. Relative abundances of the Family Fusobacteriaceae and Prevotella stercorea were positively correlated with BMI, fasting BG, and visceral fat mass. In contrast, the Family Rikenellaceae and Bacteroides uniformis (Bu) negatively correlated with metabolic parameters. Transplantation of Alistipes indistinctus (Ali), a major bacterium of the Family Rikenellaceae, or Bu into HFD-fed C57BL/6J mice suppressed body weight gain, improved glucose tolerance and histological findings without altering food intake. In summary, our results suggest that microbial community structure can be altered by lifestyle even in good health. Moreover, maintaining microbial species such as Ali and Bu may contribute to the prevention of metabolic disorders in Japanese adults. Disclosure H. Honoki: None. S. Fujisaka: None. Y. Watanabe: None. M. Oku: None. Y. Kondo: None. A. Nishimura: None. T. Kado: None. M. Bilal: None. A. Enkaku: None. A. Takikawa: None. D. Chujo: None. Y. Morinaga: None. K. Tobe: Other Relationship; MSD K.K., Novo Nordisk Pharma Ltd., Takeda Pharmaceutical Company Limited, Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Suntory Global Corporation, Ltd.,, Taiho Pharmaceutical Co. Ltd., Japan Diabetes Foundation, Japan Association for Diabetes Education and Care. Funding Japan Society for the Promotion of Science (20K10318); Japan Association for Diabetes Education and Care

Dysbiosis is an important factor that leads to metabolic disorders via disruption of the gut barrier function. A decrease in Akkermansia muciniphila (AM) is a phenotype of obesity-induced disruption of the gut barrier. Although interventions to increase AM have been reported to improve glucose metabolism, the underlying mechanism has not been fully understood. The administration of IX suppressed weight gain and steatohepatitis and improved glucose metabolism in diet-induced obese mice. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption via decreased expression of the lipid absorption transporter, CD36, in the jejunum. Moreover, IX administration improved the gut barrier function and reduced metabolic endotoxemia. In contrast, the observed effects of IX were cancelled by antibiotics and reproduced by fecal microbiota transplantation to GF mice. 16S rRNA sequencing revealed that the microbial community structure changed with a significant increase in AM in the IX-administration group. An anaerobic chamber study showed that IX promoted the growth of AM, while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct impact of AM on lipid and glucose metabolism in monocolonized mice, we transplanted either AM or Bacteroides thetaiotaomicron to GF mice. AM monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism with decreased multiple classes of fatty acids in plasma metabolomics analysis. In summary, IX contributes to metabolic improvement by decreasing CD36 and lipid absorption in the small intestine which is associated with a bloom of AM, suggesting an important interaction between food ingredients and AM abundance in controlling metabolism. Disclosure Y.Watanabe: None. S.Fujisaka: None. S.Watanabe: None. A.Nishimura: None. T.Kado: None. M.Aslam: None. M.Bilal: None. Y.Morinaga: None. K.Tobe: Other Relationship; MSD K.K., Novo Nordisk Pharma Ltd., Takeda Pharmaceutical Company Limited, Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Suntory Global Corporation, Ltd.,, Taiho Pharmaceutical Co. Ltd., Japan Diabetes Foundation, Japan Association for Diabetes Education and Care. Funding Japan Society for the Promotion of Science (21K20896, 22K16424); Lotte Foundation; Yakult Bio-Science Foundation

Abstract Aims Dysbiosis is an important factor that leads to metabolic disorders by disrupting energy balance and insulin sensitivity. A decrease in Akkermansia muciniphila is a phenotype of obesity-induced dysbiosis. Although interventions to increase A. muciniphila are expected to improve glucose metabolism, the underlying mechanism has not been fully understood. Methods Isoxanthohumol (IX), a prenylated flavonoid found in beer hops was administered to high fat diet-fed mice. We analyzed glucose metabolism, gene expression profiles and histology of liver, epididymal adipose tissue and colon. Lipase activity, fecal lipid profiles and plasma metabolomic analysis were assessed. Fecal 16s rRNA sequencing was obtained and selected bacterial species were used for in vitro studies. Fecal microbiota transplantation and monocolonization were conducted to antibiotic-treated or germ-free (GF) mice. Results The administration of IX lowered weight gain, decreased steatohepatitis and improved glucose metabolism. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption by decreasing the expression of the fatty acid transporter CD36 in the small intestine, which was confirmed by increased lipid excretion in feces. IX administration improved the gut barrier function and reduced metabolic endotoxemia. In contrast, the effects of IX were nullified by antibiotics. As revealed using 16S rRNA sequencing, the microbial community structure changed with a significant increase in the abundance of A. muciniphila in the IX-treated group. An anaerobic chamber study showed that IX selectively promoted the growth of A. muciniphila while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct effect of A. muciniphila on lipid and glucose metabolism, we monocolonized either A. muciniphila or Bacteroides thetaiotaomicron to GF mice. A. muciniphila monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism, with decreased levels of multiple classes of fatty acids determined using plasma metabolomic analysis. Conclusion Our study confirmed a direct role of A. muciniphila in energy metabolism, which was induced by microbial actions of IX. These highlight new treatment strategies for preventing metabolic syndrome by boosting the gut microbiota with food components.

Abstract Aims/Introduction Endogenous insulin secretion could be recovered by improving hyperglycemia in patients with type 2 diabetes. This study aimed to investigate the association between short‐term recovery of insulin secretion during hospitalization and clinical background or future glycemic control in patients with type 2 diabetes. Materials and Methods A total of 127 patients with type 2 diabetes were included. The recovery of endogenous insulin secretion was determined using the following indices: index A: fasting C‐peptide index (CPI) at discharge – fasting CPI on admission; index B: postprandial CPI at discharge – postprandial CPI on admission; and index C: Δ C‐peptide immunoreactivity (CPR) (postprandial CPR − fasting CPR) at discharge – ΔCPR on admission. We examined the associations of each index with clinical background and future glycemic control measured by glycosylated hemoglobin and continuous glucose monitoring. Results Using index A and B, the age was significantly younger, whereas BMI and visceral fat area were significantly higher in the high‐recovery group than in the low‐recovery group. Changes in glycosylated hemoglobin levels were significantly greater at 6 and 12 months in the high‐recovery group in the analysis of index C. The receiver operating characteristic curve analysis identified the index B and index C as indicators to predict glycosylated hemoglobin &lt;7.0% at 6 months after discharge. Furthermore, index C was positively correlated with the time in the target glucose range, and inversely correlated with the standard deviation of glucose at 3 and 12 months after discharge. Conclusions Short‐term recovery of insulin secretion in response to a meal during hospitalization, evaluated with the index‐C, might predict future glycemic control.

Type 2 diabetes and insulin resistance are one of the major consequences of obesity as a result of inflammation in adipose tissues. Adipose tissue-resident macrophages (ATMs) have a primary role in tissue remodeling and maintenance of homeostasis within adipose tissue. Two different types of macrophages are present in adipose tissue i.e. M1 and M2-type. Obesity is associated phenotypic transformation of macrophages, from anti-inflammatory M2 to pro-inflammatory M1 macrophages. M1-type macrophages increases in obesity and contribute to the development of type 2 diabetes and insulin resistacne. In contrast, M2 ATMs secretes anti-inflammatory cytokines and are involved in maintaining insulin sensitivity. However, little is known about the role of M2 macrophages in adipose tissue. Literature related to the role of M1 and M2 macrophages in metabolism have been reviewed in this article with emphasis on the macrophages associated with adipose tissues. Role of M2 macrophages in adipose tissues have also been highlighted in this article to enhance our knowledge and understanding of macrophages homogeneity.

Nonalcoholic fatty liver disease (NAFLD) is an emerging worldwide health concern. The disease may involve immune cells including T cells, but little is known about the role(s) of the innate-like T cells in the liver. Furthermore, the most abundant innate-like T cells in the human liver are mucosal-associated invariant T (MAIT) cells, but the involvement of MAIT cells in NAFLD remains largely unexplored because of their paucity in mice. In this study, we used a novel mouse line, Vα19, in which the number of MAIT cells is equivalent to or greater than that in humans. Compared with the control mice, Vα19 mice fed a high-fat diet (HFD) exhibited a reduction in lipid accumulation, NAFLD activity score, and transcripts relevant to lipogenesis. In addition, serum triglyceride and non-esterified fatty acids were lower in Vα19 mice fed normal chow or HFD. In contrast, the Vα19 mice showed little or no change in glucose tolerance, insulin sensitivity, inflammation in adipose tissues, or intestinal permeability compared with the controls, irrespective of diet. These results suggest that the presence of MAIT cells is associated with reduced lipogenesis and lipid accumulation in the liver; however, further studies are needed to clarify the role of MAIT cells in hepatic lipid metabolism.

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Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Growing evidence indicates an important link between gut microbiota, obesity, and metabolic syndrome. Alterations in exocrine pancreatic function are also widely present in patients with diabetes and obesity. To examine this interaction, C57BL/6J mice were fed either a chow diet, high-fat diet (HFD) or HFD plus oral vancomycin or metronidazole to modify the gut microbiome. HFD alone leads to a 40% increase in pancreas weight, decreased glucagon-like peptide-1 and peptide YY levels, and increased glucose-dependent insulinotropic peptide in the plasma. Quantitative proteomics identified 138 host proteins in fecal samples of these mice, of which 32 were significantly changed by HFD. The most significant of these were the pancreatic enzymes. These changes in amylase and elastase were reversed by antibiotic treatment. These alterations could be reproduced by transferring gut microbiota from donor C57BL/6J mice to germ-free. By contrast, antibiotics had no effect on pancreatic size or exocrine function in C57BL/6J mice fed a chow diet&lt;a&gt;.&lt;/a&gt; Further, one week vancomycin administration significantly increased amylase and elastase levels in obese prediabetic men. Thus, the alterations in gut microbiota in obesity can alter pancreatic growth, exocrine function and gut endocrine function, and may contribute to the alterations observed in patients with obesity and diabetes.

Growing evidence indicates an important link between gut microbiota, obesity, and metabolic syndrome. Alterations in exocrine pancreatic function are also widely present in patients with diabetes and obesity. To examine this interaction, C57BL/6J mice were fed either a chow diet, high-fat diet (HFD) or HFD plus oral vancomycin or metronidazole to modify the gut microbiome. HFD alone leads to a 40% increase in pancreas weight, decreased glucagon-like peptide-1 and peptide YY levels, and increased glucose-dependent insulinotropic peptide in the plasma. Quantitative proteomics identified 138 host proteins in fecal samples of these mice, of which 32 were significantly changed by HFD. The most significant of these were the pancreatic enzymes. These changes in amylase and elastase were reversed by antibiotic treatment. These alterations could be reproduced by transferring gut microbiota from donor C57BL/6J mice to germ-free. By contrast, antibiotics had no effect on pancreatic size or exocrine function in C57BL/6J mice fed a chow diet&lt;a&gt;.&lt;/a&gt; Further, one week vancomycin administration significantly increased amylase and elastase levels in obese prediabetic men. Thus, the alterations in gut microbiota in obesity can alter pancreatic growth, exocrine function and gut endocrine function, and may contribute to the alterations observed in patients with obesity and diabetes.

Mol. Nutr. Food Res. 2022, 66, 202101119 DOI: 10.1002/mnfr.202101119 Isoliquiritigenin (ILG), a flavonoid with a chalcone structure derived from Glycyrrhiza uralensis, beneficially modified gut microbiota by increasing the abundance of Akkermansia muciniphila and Parabacteroides goldsteinii. The altered composition of the gut bacteria by ILG restored impaired gut barrier function and prevented metabolic disorder in high fat diet-fed mice. This is reported by Riko Ishibashi and co-workers in article number 2101119.

This study aimed to identify the clinical factors affecting postoperative residual pancreatic β-cell function, as assessed by the C-peptide index (CPI), and to investigate the association between perioperative CPI and the status of diabetes management after pancreatectomy.The associations between perioperative CPI and clinical background, including surgical procedures of pancreatectomy, were analyzed in 47 patients who underwent pancreatectomy, and were assessed for pre-and postoperative CPI. The association between perioperative CPI and glycemic control after pancreatectomy was investigated.The low postoperative CPI group (CPI <0.7) had longer duration of diabetes (17.5 ± 14.5 vs 5.5 ± 11.0 years, P = 0.004), a higher percentage of sulfonylurea users (41.7 vs 8.7%, P = 0.003) and a greater number of drug categories used for diabetes treatment (1.9 ± 1.1 vs 0.8 ± 0.8, P <0.001) than did the high postoperative CPI group. Postoperative CPI was higher (1.4 ± 1.2 vs 0.7 ± 0.6, P = 0.039) in patients with low glycosylated hemoglobin (<7.0%) at 6 months after pancreatectomy; preoperative (2.0 ± 1.5 vs 0.7 ± 0.5, P = 0.012) and postoperative CPI (2.5 ± 1.4 vs 1.4 ± 1.1, P = 0.020) were higher in non-insulin users than in insulin users at 6 months after surgery.The duration of diabetes and preoperative diabetes treatment were associated with residual pancreatic β-cell function after pancreatectomy. Furthermore, perioperative β-cell function as assessed by CPI was associated with diabetes management status after pancreatectomy.

This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.

Growing evidence indicates an important link between gut microbiota, obesity, and metabolic syndrome. Alterations in exocrine pancreatic function are also widely present in patients with diabetes and obesity. To examine this interaction, C57BL/6J mice were fed either a chow diet, high-fat diet (HFD) or HFD plus oral vancomycin or metronidazole to modify the gut microbiome. HFD alone leads to a 40% increase in pancreas weight, decreased glucagon-like peptide-1 and peptide YY levels, and increased glucose-dependent insulinotropic peptide in the plasma. Quantitative proteomics identified 138 host proteins in fecal samples of these mice, of which 32 were significantly changed by HFD. The most significant of these were the pancreatic enzymes. These changes in amylase and elastase were reversed by antibiotic treatment. These alterations could be reproduced by transferring gut microbiota from donor C57BL/6J mice to germ-free. By contrast, antibiotics had no effect on pancreatic size or exocrine function in C57BL/6J mice fed a chow diet&lt;a&gt;.&lt;/a&gt; Further, one week vancomycin administration significantly increased amylase and elastase levels in obese prediabetic men. Thus, the alterations in gut microbiota in obesity can alter pancreatic growth, exocrine function and gut endocrine function, and may contribute to the alterations observed in patients with obesity and diabetes.

During cell differentiation, intracellular energetic pathways are drastically reorganized. Although this alteration had been considered as a passive process to adapt to environmental change during differentiation, recent studies suggested that the metabolic alteration rather actively regulates the differentiation process. It is known that the dynamic metabolic reprogramming occurs during differentiation of preadipocytes, however its implication in adipogenesis is unknown. In this study, we employed mass spectrometry-based metabolomics and investigated the metabolic change during differentiation of 3T3-L1 preadipocytes. We found that the level of nicotinamide adenine dinucleotide (NAD), a cofactor mediating redox reaction and protein modification, was significantly increased during the differentiation. Consistently Nampt, a rate-limiting enzyme of NAD synthesis, was also upregulated in the early phase of adipogenesis. Further, we found that pharmacological or genetic inhibition of Nampt reduced NAD synthesis and repressed the metabolic reprogramming during adipogenesis. Notably, inhibition of Nampt blocked the gene expression of Pparg and impeded the preadipocyte differentiation. The effect of Nampt inhibition was cancelled by the supplementation of nicotinamide mononucleotide, a precursor of NAD. Our metabolomic analysis also revealed that TCA cycle intermediate, α-ketoglutarate (αKG) was upregulated during the differentiation and Nampt inhibition disturbed the rise of αKG. Interestingly, αKG is known as a cofactor of the demethylation of histones. The ChIP assay revealed that αKG mediated the demethylation of H3K9me3 on Pparg promoter region and promote the gene expression of Pparg during adipogenesis. This process was inhibited by Nampt inhibitor treatment, and was reversed by the supplementation of αKG. Altogether, our data indicated that Nampt-mediated NAD synthesis is necessary for differentiation of preadipocyte. Disclosure K. Okabe: None. I. Usui: None. A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. T. Kado: None. Y. Igarashi: None. K. Yagi: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura &amp; Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. T. Nakagawa: None.

We have previously shown that CD2M2-like macrophages constitute a microenvironment for adipocyte progenitors (APs), in a Tgfβ-dependent manner, to retain systemic insulin sensitivity by tuning the quiescence/proliferation balance of APs to adapt to changes in nutritional status. Previously we generated CD206DTR transgenic mice and CD206-CreERT2/ Tgfβ1flox/flox (Tgfβ1 KO) mice to determine the role of CD2M2-like macrophages a role of CD206+ cell specific Tgfβ1 respectively, in the APs proliferation. Previous reports have shown that M2-like macrophages play a key role in the browning of WAT via activation of type 2 cytokines production during cold exposure. These findings revealed the relationship between the occurrence of beige adipocytes and M2-like macrophage recruitment. In order to investigate the role of CD2M2-like macrophages in the induction of browning into the WAT, we extended our study to see the browning phenomenon by using CD206DTR and Tgfβ1 KO mice. Here, we show that depletion of CD2M2-like macrophages promotes the browning of WAT. We report that cold stimulation resulted in the generation of smaller adipocytes, upregulation of UCP1 and CD137, a marker for beige progenitors, in the WAT of CD206-ablated mice. Flow cytometry analysis further confirmed enhanced beige progenitors in the WAT. Since blocking of Tgfβ is known to promote browning of the WAT. Mechanistically, we assume that enhanced browning into the WAT of CD206DTR mice might be due to the deletion of Tgfβ1 from CD206-M2-like macrophages. Taken together, we concluded that CD2M2-like macrophages induce the proliferation of beige progenitors in the WAT, which may serve as effective therapeutic tools for the prevention and treatment of obesity. Disclosure A. Nawaz: Research Support; Spouse/Partner; Kobayashi Foundation, Kobayashi Foundation, Kobayashi Foundation. S. Fujisaka: Research Support; Self; Mochida Pharmaceutical Co., Ltd., Eli Lilly and Company, MSD K.K., Toyama First Bank. I. Usui: None. K. Yagi: None. T. Nakagawa: None. T. Kado: None. K. Tobe: Research Support; Self; Bristol-Myers Squibb Company, Takeda Pharmaceutical Company, Teijin Pharma Limited, Japan Diabetes Society, Eli Lilly and Company, MSD K.K., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Pfizer Inc., Astellas Parma Inc, Chugai Pharmaceutical Co., Ltd., Daiichi Sankyo Company, Limited, Novo Nordisk Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Tsumura &amp; Co., Ono Pharmaceutical Co., Ltd., Novartis Pharma K.K., Sumitomo Dainippon Pharma Co. Y. Igarashi: None. K. Okabe: None. K. Saeki: None.

Nampt is the rate-limiting enzyme in salvage pathway in which NAD is mainly synthesized in mammalian cells. NAD plays very important role in various biological processes, such as energy production, energy consumption, aging, cell death, and DNA damage repair as a coenzyme of redox reactions or as a substrate of Sirtuins or Poly (ADP-ribose) polymerases. Besides, Nampt is highly expressed in mature adipocyte and secreted into extracellular. The extracellular Nampt is known to regulate systemic metabolic state. In short, Nampt works as a metabolic sensor and NAD integrates systemic metabolism. Recently, metabolism is thought to be closely related to cell differentiation. However, how NAD metabolism affects to adipogenesis remains unknown. In this study, we generated the adipose tissue specific Nampt knock out mice, Adiponectin-Cre Nampt flox/flox (KO) and conducted targeted-metabolomic analysis to investigate the metabolic effect, employing liquid-chromatograph mass spectrometry and gas-chromatograph mass spectrometry. Interestingly, KO showed resistant against diet-induced obesity (DIO), fed high fat high sucrose diet. Body weight and both inguinal and epididymal fat mass were significantly reduced in KO compared to wild type control mice. On the other hand, we revealed that Nampt-mediated NAD synthesis was up-regulated during differentiation of 3T3-L1 preadipocytes and the inhibition of Nampt significantly suppressed the differentiation. Our metabolomic analysis and ChIP-qPCR assay showed NAD contributed to the differentiation through the metabolic reprogramming and the subsequent epigenetic alteration to activate the transcription of adipogenic genes. This result indicated Nampt-mediated NAD synthesis regulated differentiation of preadipocytes to mature adipocytes. Together, our data suggested that adipose tissue specific Nampt deletion resulted in the resistance against DIO and it could be partly attributed to the inhibited differentiation of preadipocytes. Disclosure K. Okabe: None. A. Nawaz: Research Support; Spouse/Partner; Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company. Research Support; Self; Japan Society for the Promotion of Science. Research Support; Spouse/Partner; Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Shionogi &amp; Co., Ltd., Takeda Pharmaceutical Company Limited. S. Fujisaka: None. K. Yagi: None. T. Kado: None. I. Usui: None. T. Nakagawa: None. K. Tobe: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Japan Diabetes Society. Research Support; Self; Bristol-Myers Squibb Company. Funding Japan Society for the Promotion of Science

Growing evidence suggests a link between alterations in gut microbiota, diabetes, and metabolic syndrome. We recently showed that treatment with antibiotics to alter gut microbiota could improve insulin signaling and glucose metabolism in obesity- and diabetes-prone C57BL/6J mice on high-fat diet (HFD). This occurred in part through changing metabolite composition in plasma and decreasing adipose tissue inflammation. We also showed that these changes could be induced in germ-free mice by transferring the gut microbiota. It is known that diabetes is associated with exocrine pancreatic dysfunction, and diseases of the exocrine pancreas can be associated with diabetes. To explore the role of gut microbiota on host pancreatic exocrine function and its crosstalk with the endocrine system, mice were given either normal chow or HFD, or HFD and treatment with vancomycin or metronidazole. As expected, HFD alone produced signs of metabolic syndrome but also increased total pancreas weight by ~20%, an amount that could not be explained by islet hyperplasia. Analysis of the fecal proteome of these mice identified 138 host proteins of which 32 changed with either HFD or antibiotic treatment. Notably, the most significantly changed proteins by diet and altered microbiome were pancreatic enzymes: amylase, lipase and elastase. Furthermore, the plasma levels of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) levels were decreased, while glucose-dependent insulinotropic peptide (GIP) was increased by the HFD. These alterations of pancreas growth, acinar cell hyperplasia, exocrine pancreas function, and hormone secretion were reversed by antibiotic treatment of the host. These changes could also be reproduced in germ free mice by transfer of gut microbiota. Together, these data indicate that gut microbiota can regulate pancreatic growth and both exocrine and endocrine function. Better understanding pancreas-gut microbiome crosstalk can help us develop probiotics to cure pancreas dysfunction in diabetes. Disclosure E. Altindis: None. M. Soto: Employee; Self; LNC Therapeutics. Other Relationship; Self; biomeostasis. L. Orliaguet: None. Q. Huang: None. C. Cederquist: None. E. Dirice: None. S. Fujisaka: None. J. Hu: None. R. Kulkarni: None. C. Kahn: Advisory Panel; Self; MedImmune. Board Member; Self; Kaleido Biosciences. Consultant; Self; AntriaBio, Inc., Cobalt Therapeutics, Flagship Pioneering. Research Support; Self; Alnylam. Funding National Institutes of Health

Several pieces of evidence indicate that Wnt/β-catenin pathway may regulate the mesenchymal stem cells fate proliferation in vivo. The Wnt signaling is also known to inhibit preadipocyte differentiation into adipocyte. Adipose tissue macrophages play important roles in the maintenance of tissue homeostasis by regulating insulin sensitivity via their anti-inflammatory actions. However, how CD206 M2-like macrophages linked to Wnt signaling in maintaining the preadipocyte differentiation, remains elusive. In the present study, we aimed to investigate the physiological role of CD206 linked to Wnt signaling in preadipocyte differentiation. We show that C1q, which can mediate Wnt signaling, is abundantly expressed in CD206 M2-like macrophages in epididymal adipose tissue. Then, we examined whether CD206 M2-macrophages depletion activate or inhibit Wnt signaling and preadipocyte differentiation. Here, we show that the expression of M2 marker genes was reduced with a reduced expression of Wnt signaling genes in adipose tissue of CD206-reduced mice. Western blot analysis confirmed reduced protein level of β-catenin in CD206-reduced mice that can contribute to regulate the preadipocyte/progenitor’s activity. We presumed that the reduction of C1q-induced Wnt signaling promoted adipogenesis. We also found that depletion of CD206 M2-like macrophages resulted in the generation of the smaller adipocyte, upregulated expression of metabolically favorable genes and enhanced insulin sensitivity in both chow and high-fat diet-fed CD206-reduced mice. Taken together, we show that partial depletion of CD206 M2-like macrophages inhibits Wnt/β-catenin pathway activity in mice and increases preadipocyte differentiation, thus ameliorating insulin resistance in lean and obese mice. Disclosure A. Nawaz: Research Support; Spouse/Partner; Astellas Pharma Inc., Astellas Pharma Inc., AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company. Research Support; Self; Japan Society for the Promotion of Science. Research Support; Spouse/Partner; Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Shionogi &amp; Co., Ltd., Takeda Pharmaceutical Company Limited. T. Nakagawa: None. K. Yagi: None. S. Fujisaka: None. K. Tobe: Advisory Panel; Self; Novo Nordisk A/S. Board Member; Self; Japan Diabetes Society. Research Support; Self; Bristol-Myers Squibb Company. K. Okabe: None. T. Kado: None. J. Liu: None. M. Bilal: None. Funding Japan Society for the Promotion of Science

Abstract Background Fragmented QRS (fQRS) on electrocardiography is a marker of myocardial fibrosis and myocardial scar formation. fQRS has been reported as a reliable predictor of adverse cardiac events in several populations. We investigated the relationship of fQRS with diabetes mellitus (DM) and metabolic syndrome (MetS) in Japanese patients. Material and methods Our study enrolled a total of 702 subjects (435 without DM and 267 with type 2 DM) who had a routine health checkup at the Hokuriku Health Service Association (Toyama, Japan) in October 2014. Based on MetS and DM status, participants were categorized into one of the following four groups: DM+ MetS + (157 subjects); DM+ MetS - (110 subjects); DM- MetS + (82 subjects); and DM- MetS- (353 subjects). fQRS was assessed using the results of electrocardiography. Results The prevalence of fQRS was statistically higher in patients with DM+MetS+ (36%) and DM+MetS- (36%), than those with DM-MetS+ (18%) or DM-MetS- (9%) (p &lt; 0.001). Significant differences were observed between the fQRS(+) and fQRS(-) groups for age, gender, waist circumference (WC), heart rate, hypertension, HbA1c, TC, MetS, and DM. The area under the receiver-operating curve for traditional risk factors and DM was 0.72 (p=0.0021, 95% confidence interval [CI]: 0.68-0.77), and for traditional risk factors and MetS it was 0.69 (p=0.1478, 95% CI: 0.64-0.73). Patients with DM had more than three-fold higher likelihood of showing fQRS (odds ratio, 3.41; 95% CI, 2.25-5.22; p&lt;0.0001) compared to the reference group without DM, after adjusting for age, gender, dyslipidemia, hypertension, and WC. Conclusion fQRS was observed more frequently in DM than in MetS and control subjects. DM was the most significant determinant for fQRS among MetS and other traditional metabolic risk factors in the general Japanese population.