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The current dogma is that obesity-associated hepatic inflammation is due to increased Kupffer cell (KC) activation. However, recruited hepatic macrophages (RHMs) were recently shown to represent a sizable liver macrophage population in the context of obesity. Therefore, we assessed whether KCs and RHMs, or both, represent the major liver inflammatory cell type in obesity. We used a combination of in vivo macrophage tracking methodologies and adoptive transfer techniques in which KCs and RHMs are differentially labeled with fluorescent markers. With these approaches, the inflammatory phenotype of these distinct macrophage populations was determined under lean and obese conditions. In vivo macrophage tracking revealed an approximately sixfold higher number of RHMs in obese mice than in lean mice, whereas the number of KCs was comparable. In addition, RHMs comprised smaller size and immature, monocyte-derived cells compared with KCs. Furthermore, RHMs from obese mice were more inflamed and expressed higher levels of tumor necrosis factor-α and interleukin-6 than RHMs from lean mice. A comparison of the MCP-1/C-C chemokine receptor type 2 (CCR2) chemokine system between the two cell types showed that the ligand (MCP-1) is more highly expressed in KCs than in RHMs, whereas CCR2 expression is approximately fivefold greater in RHMs. We conclude that KCs can participate in obesity-induced inflammation by causing the recruitment of RHMs, which are distinct from KCs and are not precursors to KCs. These RHMs then enhance the severity of obesity-induced inflammation and hepatic insulin resistance., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: Adipose tissue is the primary site for lipid deposition that protects the organisms in cases of nutrient excess during obesogenic diets. The histone deacetylase Sirtuin 1 (SIRT1) inhibits adipocyte differentiation by targeting the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ)., Methods: To assess the specific role of SIRT1 in adipocytes, we generated Sirt1 adipocyte-specific knockout mice (ATKO) driven by aP2 promoter onto C57BL/6 background. Sirt1 (flx/flx) aP2Cre (+) (ATKO) and Sirt1 (flx/flx) aP2Cre (-) (WT) mice were fed high-fat diet for 5 weeks (short-term) or 15 weeks (chronic-term). Metabolic studies were combined with gene expression analysis and phosphorylation/acetylation patterns in adipose tissue., Results: On standard chow, ATKO mice exhibit low-grade chronic inflammation in adipose tissue, along with glucose intolerance and insulin resistance compared with control fed mice. On short-term HFD, ATKO mice become more glucose intolerant, hyperinsulinemic, insulin resistant and display increased inflammation. During chronic HFD, WT mice developed a metabolic dysfunction, higher than ATKO mice, and thereby, knockout mice are more glucose tolerant, insulin sensitive and less inflamed relative to control mice. SIRT1 attenuates adipogenesis through PPARγ repressive acetylation and, in the ATKO mice adipocyte PPARγ was hyperacetylated. This high acetylation was associated with a decrease in Ser273-PPARγ phosphorylation. Dephosphorylated PPARγ is constitutively active and results in higher expression of genes associated with increased insulin sensitivity., Conclusion: Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.

Insulin resistance results from several pathophysiologic mechanisms, including chronic tissue inflammation and defective insulin signaling. We found that liver, muscle and adipose tissue exhibit higher levels of the chemotactic eicosanoid LTB4 in obese high-fat diet (HFD)-fed mice. Inhibition of the LTB4 receptor Ltb4r1, through either genetic or pharmacologic loss of function, led to an anti-inflammatory phenotype with protection from insulin resistance and hepatic steatosis. In vitro treatment with LTB4 directly enhanced macrophage chemotaxis, stimulated inflammatory pathways, reduced insulin-stimulated glucose uptake in L6 myocytes, and impaired insulin-mediated suppression of hepatic glucose output in primary mouse hepatocytes. This was accompanied by lower insulin-stimulated Akt phosphorylation and higher Irs-1/2 serine phosphorylation, and all of these events were dependent on Gαi and Jnk1, two downstream mediators of Ltb4r1 signaling. These observations elucidate a novel role of the LTB4-Ltb4r1 signaling pathway in hepatocyte and myocyte insulin resistance, and they show that in vivo inhibition of Ltb4r1 leads to robust insulin-sensitizing effects.

The systemic expression of the bile acid (BA) sensor farnesoid X receptor (FXR) has led to promising new therapies targeting cholesterol metabolism, triglyceride production, hepatic steatosis and biliary cholestasis. In contrast to systemic therapy, bile acid release during a meal selectively activates intestinal FXR. By mimicking this tissue-selective effect, the gut-restricted FXR agonist fexaramine (Fex) robustly induces enteric fibroblast growth factor 15 (FGF15), leading to alterations in BA composition, but does so without activating FXR target genes in the liver. However, unlike systemic agonism, we find that Fex reduces diet-induced weight gain, body-wide inflammation and hepatic glucose production, while enhancing thermogenesis and browning of white adipose tissue (WAT). These pronounced metabolic improvements suggest tissue-restricted FXR activation as a new approach in the treatment of obesity and metabolic syndrome.

Fibroblast growth factor 1 (FGF1) is an autocrine/paracrine regulator whose binding to heparan sulphate proteoglycans effectively precludes its circulation. Although FGF1 is known as a mitogenic factor, FGF1 knockout mice develop insulin resistance when stressed by a high-fat diet, suggesting a potential role in nutrient homeostasis. Here we show that parenteral delivery of a single dose of recombinant FGF1 (rFGF1) results in potent, insulin-dependent lowering of glucose levels in diabetic mice that is dose-dependent but does not lead to hypoglycaemia. Chronic pharmacological treatment with rFGF1 increases insulin-dependent glucose uptake in skeletal muscle and suppresses the hepatic production of glucose to achieve whole-body insulin sensitization. The sustained glucose lowering and insulin sensitization attributed to rFGF1 are not accompanied by the side effects of weight gain, liver steatosis and bone loss associated with current insulin-sensitizing therapies. We also show that the glucose-lowering activity of FGF1 can be dissociated from its mitogenic activity and is mediated predominantly via FGF receptor 1 signalling. Thus we have uncovered an unexpected, neomorphic insulin-sensitizing action for exogenous non-mitogenic human FGF1 with therapeutic potential for the treatment of insulin resistance and type 2 diabetes.

It is well known that the ω-3 fatty acids (ω-3-FAs; also known as n-3 fatty acids) can exert potent anti-inflammatory effects. Commonly consumed as fish products, dietary supplements and pharmaceuticals, ω-3-FAs have a number of health benefits ascribed to them, including reduced plasma triglyceride levels, amelioration of atherosclerosis and increased insulin sensitivity. We reported that Gpr120 is the functional receptor for these fatty acids and that ω-3-FAs produce robust anti-inflammatory, insulin-sensitizing effects, both in vivo and in vitro, in a Gpr120-dependent manner. Indeed, genetic variants that predispose to obesity and diabetes have been described in the gene encoding GPR120 in humans (FFAR4). However, the amount of fish oils that would have to be consumed to sustain chronic agonism of Gpr120 is too high to be practical, and, thus, a high-affinity small-molecule Gpr120 agonist would be of potential clinical benefit. Accordingly, Gpr120 is a widely studied drug discovery target within the pharmaceutical industry. Gpr40 is another lipid-sensing G protein-coupled receptor, and it has been difficult to identify compounds with a high degree of selectivity for Gpr120 over Gpr40 (ref. 11). Here we report that a selective high-affinity, orally available, small-molecule Gpr120 agonist (cpdA) exerts potent anti-inflammatory effects on macrophages in vitro and in obese mice in vivo. Gpr120 agonist treatment of high-fat diet-fed obese mice causes improved glucose tolerance, decreased hyperinsulinemia, increased insulin sensitivity and decreased hepatic steatosis. This suggests that Gpr120 agonists could become new insulin-sensitizing drugs for the treatment of type 2 diabetes and other human insulin-resistant states in the future.

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.

Chronic, low-grade adipose tissue inflammation is a key etiological mechanism linking the increasing incidence of type 2 diabetes (T2D) and obesity. It is well recognized that the immune system and metabolism are highly integrated, and macrophages, in particular, have been identified as critical effector cells in the initiation of inflammation and insulin resistance. Recent advances have been made in the understanding of macrophage recruitment and retention to adipose tissue and the participation of other immune cell populations in the regulation of this inflammatory process. Here we discuss the pathophysiological link between macrophages, obesity, and insulin resistance, highlighting the dynamic immune cell regulation of adipose tissue inflammation. We also describe the mechanisms by which inflammation causes insulin resistance and the new therapeutic targets that have emerged., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. Here, we report that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1α induction, setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1α can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Macrophage infiltration plays an important role in obesity-induced insulin resistance. CCAAT enhancer-binding protein-α (C/EBPα) is a transcription factor that is highly expressed in macrophages. To examine the roles of C/EBPα in regulating macrophage functions and energy homeostasis, macrophage-specific C/EBPα knockout (MαKO) mice were created. Chow-fed MαKO mice exhibited higher body fat mass and decreased energy expenditure despite no change in food intake. However, the obese phenotype disappeared after high-fat (HF) diet feeding. Although there was a transient decrease in insulin sensitivity of chow-fed young MαKO mice, systemic insulin sensitivity was protected during HF-feeding due to preserved insulin sensitivity in skeletal muscle. We also found that C/EBPα-deficient macrophages exhibited a blunted response of cytokine-induced expression of M1 and M2 macrophage markers, suggesting that C/EBPα controls both M1 and M2 polarization. Consistent with decreased exercise capacity, mitochondrial respiration rates and signal pathways for fatty acid oxidation were remarkably reduced in the skeletal muscle of chow-fed MαKO mice. Furthermore, expression levels of inflammatory cytokines were reduced in skeletal muscle of HF-fed MαKO mice. Together, these results imply that C/EBPα is required for macrophage activation, which plays an important role in maintaining skeletal muscle energy metabolism.

Objective: In obesity, immune cells infiltrate adipose tissue. Skeletal muscle is the major tissue of insulin-dependent glucose disposal, and indices of muscle inflammation arise during obesity, but whether and which immune cells increase in muscle remain unclear., Methods: Immune cell presence in quadriceps muscle of wild type mice fed high-fat diet (HFD) was studied for 3 days to 10 weeks, in CCL2-KO mice fed HFD for 1 week, and in human muscle. Leukocyte presence was assessed by gene expression of lineage markers, cyto/chemokines and receptors; immunohistochemistry; and flow cytometry., Results: After 1 week HFD, concomitantly with glucose intolerance, muscle gene expression of Ly6b, Emr1 (F4/80), Tnf, Ccl2, and Ccr2 rose, as did pro- and anti-inflammatory markers Itgax (CD11c) and Mgl2. CD11c+ proinflammatory macrophages in muscle increased by 76%. After 10 weeks HFD, macrophages in muscle increased by 47%. Quadriceps from CCL2-KO mice on HFD did not gain macrophages and maintained insulin sensitivity. Muscle of obese, glucose-intolerant humans showed elevated CD68 (macrophage marker) and ITGAX, correlating with poor glucose disposal and adiposity., Conclusion: Mouse and human skeletal muscles gain a distinct population of inflammatory macrophages upon HFD or obesity, linked to insulin resistance in humans and CCL2 availability in mice., (© 2013 The Obesity Society.)

Insulin resistance plays a major role in the development of type 2 diabetes and obesity and affects a number of biological processes such as mitochondrial biogenesis. Though mitochondrial dysfunction has been linked to the development of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism linking the two is not well understood. We used high fat diet (HFD)-induced obesity dependent diabetes mouse models to gain insight into the potential pathways altered with metabolic disease, and carried out quantitative proteomic analysis of liver mitochondria. As previously reported, proteins involved in fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of HFD fed mice compared with that of normal diet. Further, our studies revealed that retinol metabolism is distinctly down-regulated and the mitochondrial structural proteins-components of mitochondrial inter-membrane space bridging (MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins-essential for protein import, are significantly up-regulated in HFD fed mice. Structural and functional studies on HFD and normal diet liver mitochondria revealed remodeling of HFD mitochondria to a more condensed form with increased respiratory capacity and higher ATP levels compared with normal diet mitochondria. Thus, it is likely that the structural remodeling is essential to accommodate the increased protein content in presence of HFD: the mechanism could be through the MIB complex promoting contact site and crista junction formation and in turn facilitating the lipid and protein uptake.

Macrophage-mediated inflammation is a major contributor to obesity-associated insulin resistance. The corepressor NCoR interacts with inflammatory pathway genes in macrophages, suggesting that its removal would result in increased activity of inflammatory responses. Surprisingly, we find that macrophage-specific deletion of NCoR instead results in an anti-inflammatory phenotype along with robust systemic insulin sensitization in obese mice. We present evidence that derepression of LXRs contributes to this paradoxical anti-inflammatory phenotype by causing increased expression of genes that direct biosynthesis of palmitoleic acid and ω3 fatty acids. Remarkably, the increased ω3 fatty acid levels primarily inhibit NF-κB-dependent inflammatory responses by uncoupling NF-κB binding and enhancer/promoter histone acetylation from subsequent steps required for proinflammatory gene activation. This provides a mechanism for the in vivo anti-inflammatory insulin-sensitive phenotype observed in mice with macrophage-specific deletion of NCoR. Therapeutic methods to harness this mechanism could lead to a new approach to insulin-sensitizing therapies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Female obesity is associated with insulin resistance, hyperandrogenemia, and reproductive dysfunction. We hypothesized that elevated free fatty acids (FFAs) might directly modulate pituitary gonadotropin production. FFAs caused a time- and dose-dependent increase in phosphorylation of the MAPKs p38MAPK, c-Jun N-terminal kinase (JNK)-1/2, and ERK1/2 in LβT2 gonadotrope cells. Furthermore, FFAs up-regulated Lhb mRNA expression acutely, an effect that was blocked by JNK inhibition, but suppressed Fshb mRNA expression, an effect that was independent of MAPK signaling. FFAs enhanced the activation of the MAPKs in the presence of GnRH, although the cotreatment did not alter Lhb induction but did eliminate the GnRH induction of Fshb. FFAs also suppressed activin-induced Fshb expression. Knockdown experiments showed that the FFA effect on the inflammatory kinases p38MAPK and JNK and on Lhb, but not Fshb, mRNA expression is mediated via toll-like receptor-2 and toll-like receptor-4 and was mimicked by lipopolysaccharide stimulation. In vivo, male C57BL/6 mice on a high-fat diet showed reduced FSH levels consistent with the suppression of Fshb seen in vitro. Histological analysis of the testes showed an increased number of abnormal seminiferous tubules. Female mice on a high-fat diet lacked the expected proestrus LH and FSH surge and exhibited an increase in the number of days at estrus and a reduced number of days at proestrus, and ovaries had significantly fewer corpora lutea. Taken together, our findings suggest that lipid excess can lead to reproductive defects in both male and female mice.

Sirt1 is a NAD(+)-dependent class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. To assess this idea, we generated Sirt1 neuron-specific knockout (SINKO) mice. On both standard chow and HFD, SINKO mice were more insulin sensitive than Sirt1(f/f) mice. Thus, SINKO mice had lower fasting insulin levels, improved glucose tolerance and insulin tolerance, and enhanced systemic insulin sensitivity during hyperinsulinemic euglycemic clamp studies. Hypothalamic insulin sensitivity of SINKO mice was also increased over controls, as assessed by hypothalamic activation of PI3K, phosphorylation of Akt and FoxO1 following systemic insulin injection. Intracerebroventricular injection of insulin led to a greater systemic effect to improve glucose tolerance and insulin sensitivity in SINKO mice compared with controls. In line with the in vivo results, insulin-induced AKT and FoxO1 phosphorylation were potentiated by inhibition of Sirt1 in a cultured hypothalamic cell line. Mechanistically, this effect was traced to a reduced effect of Sirt1 to directly deacetylate and repress IRS-1 function. The enhanced central insulin signaling in SINKO mice was accompanied by increased insulin receptor signal transduction in liver, muscle, and adipose tissue. In summary, we conclude that neuronal Sirt1 negatively regulates hypothalamic insulin signaling, leading to systemic insulin resistance. Interventions that reduce neuronal Sirt1 activity have the potential to improve systemic insulin action and limit weight gain on an obesigenic diet.

Gastrointestinal bypass surgeries that result in rerouting and subsequent exclusion of nutrients from the duodenum appear to rapidly alleviate hyperglycemia and hyperinsulinemia independent of weight loss. While the mechanism(s) responsible for normalization of glucose homeostasis remains to be fully elucidated, this rapid normalization coupled with the well-known effects of vagal inputs into glucose homeostasis suggests a neurohormonally mediated mechanism. Our results show that duodenal bypass surgery on obese, insulin-resistant Zucker fa/fa rats restored insulin sensitivity in both liver and peripheral tissues independent of body weight. Restoration of normoglycemia was attributable to an enhancement in key insulin-signaling molecules, including insulin receptor substrate-2, and substrate metabolism through a multifaceted mechanism involving activation of AMP-activated protein kinase and downregulation of key regulatory genes involved in both lipid and glucose metabolism. Importantly, while central nervous system-derived vagal nerves were not essential for restoration of insulin sensitivity, rapid normalization in hepatic gluconeogenic capacity and basal hepatic glucose production required intact vagal innervation. Lastly, duodenal bypass surgery selectively altered the tissue concentration of intestinally derived glucoregulatory hormone peptides in a segment-specific manner. The present data highlight and support the significance of vagal inputs and intestinal hormone peptides toward normalization of glucose and lipid homeostasis after duodenal bypass surgery.

Purpose: Recognition of the complex, multidimensional relationship between excess adiposity and cancer control outcomes has motivated the scientific community to seek new research models and paradigms., Methods: The National Cancer Institute developed an innovative concept to establish a center grant mechanism in nutrition, energetics, and physical activity, referred to as the Transdisciplinary Research on Energetics and Cancer (TREC) Initiative. This paper gives an overview of the 2011-2016 TREC Collaborative Network and the 15 research projects being conducted at the centers., Results: Four academic institutions were awarded TREC center grants in 2011: Harvard University, University of California San Diego, University of Pennsylvania, and Washington University in St. Louis. The Fred Hutchinson Cancer Research Center is the Coordination Center. The TREC research portfolio includes three animal studies, three cohort studies, four randomized clinical trials, one cross-sectional study, and two modeling studies. Disciplines represented by TREC investigators include basic science, endocrinology, epidemiology, biostatistics, behavior, medicine, nutrition, physical activity, genetics, engineering, health economics, and computer science. Approximately 41,000 participants will be involved in these studies, including children, healthy adults, and breast and prostate cancer survivors. Outcomes include biomarkers of cancer risk, changes in weight and physical activity, persistent adverse treatment effects (e.g., lymphedema, urinary and sexual function), and breast and prostate cancer mortality., Conclusion: The NIH Science of Team Science group will evaluate the value added by this collaborative science. However, the most important outcome will be whether this transdisciplinary initiative improves the health of Americans at risk of cancer as well as cancer survivors.

Obesity is associated with insulin resistance, a major risk factor for type 2 diabetes and cardiovascular disease. However, not all obese individuals are insulin resistant, which confounds our understanding of the mechanistic link between these conditions. We conducted transcriptome analyses on 835 obese subjects with mean BMI of 48.8, on which we have previously reported genetic associations of gene expression. Here, we selected ~320 nondiabetic (HbA(1c) <7.0) subjects and further stratified the cohort into insulin-resistant versus insulin-sensitive subgroups based on homeostasis model assessment-insulin resistance. An unsupervised informatics analysis revealed that immune response and inflammation-related genes were significantly downregulated in the omental adipose tissue of obese individuals with extreme insulin sensitivity and, to a much lesser extent, in subcutaneous adipose tissue. In contrast, genes related to β-oxidation and the citric acid cycle were relatively overexpressed in adipose of insulin-sensitive patients. These observations were verified by querying an independent cohort of our published dataset of 37 subjects whose subcutaneous adipose tissue was sampled before and after treatment with thiazolidinediones. Whereas the immune response and inflammation pathway genes were downregulated by thiazolidinedione treatment, β-oxidation and citric acid cycle genes were upregulated. This work highlights the critical role that omental adipose inflammatory pathways might play in the pathophysiology of insulin resistance, independent of body weight.

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKK-ɛ and TANK-binding kinase 1 (TBK1) are induced in liver and fat by NF-κB activation upon high-fat diet feeding and in turn initiate a program of counterinflammation that preserves energy storage. Here we report that amlexanox, an approved small-molecule therapeutic presently used in the clinic to treat aphthous ulcers and asthma, is an inhibitor of these kinases. Treatment of obese mice with amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis. Because of its record of safety in patients, amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.

Obesity-induced insulin resistance is the major determinant of metabolic syndrome, which precedes the development of type 2 diabetes mellitus and is thus the driving force behind the emerging diabetes epidemic. The precise causes of insulin resistance are varied, and the relative importance of each is a matter of ongoing research. Here, we offer a Perspective on the heterogeneous etiology of insulin resistance, focusing in particular on the role of inflammation, lipid metabolism, and the gastrointestinal microbiota., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Chronic low-grade adipose tissue and liver inflammation is a major cause of systemic insulin resistance and is a key component of the low degree of insulin sensitivity that exists in obesity and type 2 diabetes. Immune cells, such as macrophages, T cells, B cells, mast cells and eosinophils, have all been implicated as having a role in this process. Neutrophils are typically the first immune cells to respond to inflammation and can exacerbate the chronic inflammatory state by helping to recruit macrophages and by interacting with antigen-presenting cells. Neutrophils secrete several proteases, one of which is neutrophil elastase, which can promote inflammatory responses in several disease models. Here we show that treatment of hepatocytes with neutrophil elastase causes cellular insulin resistance and that deletion of neutrophil elastase in high-fat-diet–induced obese (DIO) mice leads to less tissue inflammation that is associated with lower adipose tissue neutrophil and macrophage content. These changes are accompanied by improved glucose tolerance and increased insulin sensitivity. Taken together, we show that neutrophils can be added to the extensive repertoire of immune cells that participate in inflammation-induced metabolic disease.

GPR105, a G protein-coupled receptor for UDP-glucose, is highly expressed in several human tissues and participates in the innate immune response. Because inflammation has been implicated as a key initial trigger for type 2 diabetes, we hypothesized that GPR105 (official gene name: P2RY14) might play a role in the initiation of inflammation and insulin resistance in obesity. To this end, we investigated glucose metabolism in GPR105 knockout (KO) and wild-type (WT) mice fed a high-fat diet (HFD). We also examined whether GPR105 regulates macrophage recruitment to liver or adipose tissues by in vivo monocyte tracking and in vitro chemotaxis experiments, followed by transplantation of bone marrow from either KO or WT donors to WT recipients. Our data show that genetic deletion of GPR105 confers protection against HFD-induced insulin resistance, with reduced macrophage infiltration and inflammation in liver, and increased insulin-stimulated Akt phosphorylation in liver, muscle, and adipose tissue. By tracking monocytes from either KO or WT donors, we found that fewer KO monocytes were recruited to the liver of WT recipients. Furthermore, we observed that uridine 5-diphosphoglucose enhanced the in vitro migration of bone marrow-derived macrophages from WT but not KO mice, and that plasma uridine 5-diphosphoglucose levels were significantly higher in obese versus lean mice. Finally, we confirmed that insulin sensitivity improved in HFD mice with a myeloid cell-specific deletion of GPR105. These studies indicate that GPR105 ablation mitigates HFD-induced insulin resistance by inhibiting macrophage recruitment and tissue inflammation. Hence GPR105 provides a novel link between innate immunity and metabolism.

Obesity-induced inflammation is a key component of systemic insulin resistance, which is a hallmark of type 2 diabetes. A major driver of this inflammation/insulin resistance syndrome is the accumulation of proinflammatory macrophages in adipose tissue and liver. We found that the orphan GPCR Gpr21 was highly expressed in the hypothalamus and macrophages of mice and that whole-body KO of this receptor led to a robust improvement in glucose tolerance and systemic insulin sensitivity and a modest lean phenotype. The improvement in insulin sensitivity in the high-fat diet-fed (HFD-fed) Gpr21 KO mouse was traced to a marked reduction in tissue inflammation caused by decreased chemotaxis of Gpr21 KO macrophages into adipose tissue and liver. Furthermore, mice lacking macrophage expression of Gpr21 were protected from HFD-induced inflammation and displayed improved insulin sensitivity. Results of in vitro chemotaxis studies in human monocytes suggested that the defect in chemotaxis observed ex vivo and in vivo in mice is also translatable to humans. Cumulatively, our data indicate that GPR21 has a critical function in coordinating macrophage proinflammatory activity in the context of obesity-induced insulin resistance.

Obesity-associated hepatic steatosis is a manifestation of selective insulin resistance whereby lipogenesis remains sensitive to insulin but the ability of insulin to suppress glucose production is impaired. We created a mouse model of liver-specific knockdown of p70 S6 kinase (S6K) (L-S6K-KD) by systemic delivery of an adeno-associated virus carrying a shRNA for S6K and examined the effects on steatosis and insulin resistance. High fat diet (HFD) fed L-S6K-KD mice showed improved glucose tolerance and systemic insulin sensitivity compared with controls, with no changes in food intake or body weight. The induction of lipogenic gene expression was attenuated in the L-S6K-KD mice with decreased sterol regulatory element-binding protein (SREBP)-1c expression and mature SREBP-1c protein, as well as decreased steatosis on HFD. Our results demonstrate the importance of S6K: 1) as a modulator of the hepatic response to fasting/refeeding, 2) in the development of steatosis, and 3) as a key node in selective hepatic insulin resistance in obese mice.

Although feast and famine cycles illustrate that remodelling of adipose tissue in response to fluctuations in nutrient availability is essential for maintaining metabolic homeostasis, the underlying mechanisms remain poorly understood. Here we identify fibroblast growth factor 1 (FGF1) as a critical transducer in this process in mice, and link its regulation to the nuclear receptor PPARγ (peroxisome proliferator activated receptor γ), which is the adipocyte master regulator and the target of the thiazolidinedione class of insulin sensitizing drugs. FGF1 is the prototype of the 22-member FGF family of proteins and has been implicated in a range of physiological processes, including development, wound healing and cardiovascular changes. Surprisingly, FGF1 knockout mice display no significant phenotype under standard laboratory conditions. We show that FGF1 is highly induced in adipose tissue in response to a high-fat diet and that mice lacking FGF1 develop an aggressive diabetic phenotype coupled to aberrant adipose expansion when challenged with a high-fat diet. Further analysis of adipose depots in FGF1-deficient mice revealed multiple histopathologies in the vasculature network, an accentuated inflammatory response, aberrant adipocyte size distribution and ectopic expression of pancreatic lipases. On withdrawal of the high-fat diet, this inflamed adipose tissue fails to properly resolve, resulting in extensive fat necrosis. In terms of mechanisms, we show that adipose induction of FGF1 in the fed state is regulated by PPARγ acting through an evolutionarily conserved promoter proximal PPAR response element within the FGF1 gene. The discovery of a phenotype for the FGF1 knockout mouse establishes the PPARγ–FGF1 axis as critical for maintaining metabolic homeostasis and insulin sensitization.

Inflammation and lipid signaling are intertwined modulators of homeostasis and immunity. In addition to the extensively studied eicosanoids and inositol phospholipids, emerging studies indicate that many other lipid species act to positively and negatively regulate inflammatory responses. Conversely, inflammatory signaling can significantly alter lipid metabolism in the liver, adipose tissue, skeletal muscle, and macrophage in the context of infection, diabetes, and atherosclerosis. Here, we review recent findings related to this interconnected network from the perspective of immunity and metabolic disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Human loss-of-function gene variants in GPR120 have recently been identified that confer increased risk for obesity and metabolic syndrome. In addition, GPR120 KO mice develop obesity, increased inflammation, and insulin resistance, consistent with a role for GPR120 signaling in the metabolic syndrome and diabetes mellitus., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75(NTR)) is a regulator of glucose uptake and insulin resistance. p75(NTR) knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75(NTR) gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75(NTR) in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75(NTR) in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75(NTR) forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75(NTR) primarily via helix 4 of the p75(NTR) death domain. Adipocytes from p75(NTR) knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75(NTR) knockout adipocytes. Our results identify p75(NTR) as a unique player in glucose metabolism and suggest that signaling from p75(NTR) to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes.

It is now recognized that obesity is driving the type 2 diabetes epidemic in Western countries. Obesity-associated chronic tissue inflammation is a key contributing factor to type 2 diabetes and cardiovascular disease, and a number of studies have clearly demonstrated that the immune system and metabolism are highly integrated. Recent advances in deciphering the various cellular and signaling networks that participate in linking the immune and metabolic systems together have contributed to understanding of the pathogenesis of metabolic diseases and may also inform new therapeutic strategies based on immunomodulation. Here we discuss how these various networks underlie the etiology of the inflammatory component of insulin resistance, with a particular focus on the central roles of macrophages in adipose tissue and liver.

Macrophage-mediated inflammation is a key component of insulin resistance; however, the initial events of monocyte migration to become tissue macrophages remain poorly understood. We report a new method to quantitate in vivo macrophage tracking (i.e., blood monocytes from donor mice) labeled ex vivo with fluorescent PKH26 dye and injected into recipient mice. Labeled monocytes appear as adipose, liver, and splenic macrophages, peaking in 1-2 days. When CCR2 KO monocytes are injected into wild-type (WT) recipients, or WT monocytes given to MCP-1 KO recipients, adipose tissue macrophage (ATM) accumulation is reduced by ~40%, whereas hepatic macrophage content is decreased by ~80%. Using WT donor cells, ATM accumulation is several-fold greater in obese recipient mice compared with lean mice, regardless of the source of donor monocytes. After their appearance in adipose tissue, ATMs progressively polarize from the M2- to the M1-like state in obesity. In summary, the CCR2/MCP-1 system is a contributory factor to monocyte migration into adipose tissue and is the dominant signal controlling the appearance of recruited macrophages in the liver. Monocytes from obese mice are not programmed to become inflammatory ATMs but rather the increased proinflammatory ATM accumulation in obesity is in response to tissue signals.

Objective: Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation., Research Design and Methods: We used antisense oligonucleotides to reduce SirT1 to levels similar to those seen during overnutrition and studied SirT1-overexpressing transgenic mice and fat-specific SirT1 knockout animals. Finally, we analyzed subcutaneous adipose tissue biopsies from two independent cohorts of human subjects., Results: We found that inducible or genetic reduction of SirT1 in vivo causes macrophage recruitment to adipose tissue, whereas overexpression of SirT1 prevents adipose tissue macrophage accumulation caused by chronic high-fat feeding. We also found that SirT1 expression in human subcutaneous fat is inversely related to adipose tissue macrophage infiltration., Conclusions: Reduction of adipose tissue SirT1 expression, which leads to histone hyperacetylation and ectopic inflammatory gene expression, is identified as a key regulatory component of macrophage influx into adipose tissue during overnutrition in rodents and humans. Our results suggest that SirT1 regulates adipose tissue inflammation by controlling the gain of proinflammatory transcription in response to inducers such as fatty acids, hypoxia, and endoplasmic reticulum stress.

Insulin resistance, tissue inflammation, and adipose tissue dysfunction are features of obesity and Type 2 diabetes. We generated adipocyte-specific Nuclear Receptor Corepressor (NCoR) knockout (AKO) mice to investigate the function of NCoR in adipocyte biology, glucose and insulin homeostasis. Despite increased obesity, glucose tolerance was improved in AKO mice, and clamp studies demonstrated enhanced insulin sensitivity in liver, muscle, and fat. Adipose tissue macrophage infiltration and inflammation were also decreased. PPARγ response genes were upregulated in adipose tissue from AKO mice and CDK5-mediated PPARγ ser-273 phosphorylation was reduced, creating a constitutively active PPARγ state. This identifies NCoR as an adaptor protein that enhances the ability of CDK5 to associate with and phosphorylate PPARγ. The dominant function of adipocyte NCoR is to transrepress PPARγ and promote PPARγ ser-273 phosphorylation, such that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity, phenocopying the TZD-treated state., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Skeletal muscle insulin resistance is a key component of the etiology of type 2 diabetes. Caloric restriction (CR) enhances the sensitivity of skeletal muscle to insulin. However, the molecular signals within skeletal muscle linking CR to improved insulin action remain largely unknown. Recently, the mammalian ortholog of Sir2, sirtuin 1 (Sirt1), has been identified as a potential transducer of perturbations in cellular energy flux into subsequent metabolic adaptations, including modulation of skeletal muscle insulin action. Here, we have demonstrated that CR increases Sirt1 deacetylase activity in skeletal muscle in mice, in parallel with enhanced insulin-stimulated phosphoinositide 3-kinase (PI3K) signaling and glucose uptake. These adaptations in skeletal muscle insulin action were completely abrogated in mice lacking Sirt1 deacetylase activity. Mechanistically, Sirt1 was found to be required for the deacetylation and inactivation of the transcription factor Stat3 during CR, which resulted in decreased gene and protein expression of the p55α/p50α subunits of PI3K, thereby promoting more efficient PI3K signaling during insulin stimulation. Thus, these data demonstrate that Sirt1 is an integral signaling node in skeletal muscle linking CR to improved insulin action, primarily via modulation of PI3K signaling.

The past decade has seen great progress in the understanding of the molecular pharmacology, physiological function and therapeutic potential of G-protein-coupled receptors (GPCRs). Free fatty acids (FFAs) have been demonstrated to act as ligands of several GPCRs including GPR40, GPR43, GPR84, GPR119 and GPR120. We have recently shown that GPR120 acts as a physiological receptor of ω3 fatty acids in macrophages and adipocytes, which mediate potent anti-inflammatory and insulin sensitizing effects. The important role GPR120 plays in the control of inflammation raises the possibility that targeting this receptor could have therapeutic potential in many inflammatory diseases including obesity and type 2 diabetes. In this review paper, we discuss lipid-sensing GPCRs and highlight potential outcomes of targeting such receptors in ameliorating disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A connection between diet, obesity and diabetes exists in multiple species and is the basis of an escalating human health problem. The factors responsible provoke both insulin resistance and pancreatic beta cell dysfunction but remain to be fully identified. We report a combination of molecular events in human and mouse pancreatic beta cells, induced by elevated levels of free fatty acids or by administration of a high-fat diet with associated obesity, that comprise a pathogenic pathway to diabetes. Elevated concentrations of free fatty acids caused nuclear exclusion and reduced expression of the transcription factors FOXA2 and HNF1A in beta cells. This resulted in a deficit of GnT-4a glycosyltransferase expression in beta cells that produced signs of metabolic disease, including hyperglycemia, impaired glucose tolerance, hyperinsulinemia, hepatic steatosis and diminished insulin action in muscle and adipose tissues. Protection from disease was conferred by enforced beta cell-specific GnT-4a protein glycosylation and involved the maintenance of glucose transporter expression and the preservation of glucose transport. We observed that this pathogenic process was active in human islet cells obtained from donors with type 2 diabetes; thus, illuminating a pathway to disease implicated in the diet- and obesity-associated component of type 2 diabetes mellitus.

Resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenol found in red wine, has multiple beneficial activities that are similar to caloric restriction. In this study, we analyzed the effect of resveratrol on the gonadotropin genes, follicle-stimulating hormone (FSHβ) and luteinizing hormone (LHβ) in LβT2 immortalized mouse gonadotrope cells. Resveratrol specifically inhibited activin-induced FSHβ mRNA and protein expression, and reduced activin-stimulated Smad2/3 phosphorylation. Knockdown of SirT1 gene expression or SirT1 inhibition did not block repression of FSHβ expression or suppression of Smad2/3 phosphorylation, but did increase p53 acetylation. Taken together, our results suggest that resveratrol down-regulates Smad2/3 phosphorylation and suppresses FSHβ expression via a SirT1-independent pathway., (Published by Elsevier Inc.)

In adipose tissue, muscle, liver and macrophages, signaling by the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) is a determinant of insulin sensitivity and this receptor mediates the insulin-sensitizing effects of thiazolidinediones (TZDs). As PPAR-γ is also expressed in neurons, we generated mice with neuron-specific Pparg knockout (Pparg brain knockout (BKO)) to determine whether neuronal PPAR-γ signaling contributes to either weight gain or insulin sensitivity. During high-fat diet (HFD) feeding, food intake was reduced and energy expenditure increased in Pparg-BKO mice compared to Pparg(f/f) mice, resulting in reduced weight gain. Pparg-BKO mice also responded better to leptin administration than Pparg(f/f) mice. When treated with the TZD rosiglitazone, Pparg-BKO mice were resistant to rosiglitazone-induced hyperphagia and weight gain and, relative to rosiglitazone-treated Pparg(f/f) mice, experienced only a marginal improvement in glucose metabolism. Hyperinsulinemic euglycemic clamp studies showed that the increase in hepatic insulin sensitivity induced by rosiglitazone treatment during HFD feeding was completely abolished in Pparg-BKO mice, an effect associated with the failure of rosiglitazone to improve liver insulin receptor signal transduction. We conclude that excess weight gain induced by HFD feeding depends in part on the effect of neuronal PPAR-γ signaling to limit thermogenesis and increase food intake. Neuronal PPAR-γ signaling is also required for the hepatic insulin sensitizing effects of TZDs.

Acid sphingomyelinase (ASM) regulates the homeostasis of sphingolipids, including ceramides and sphingosine-1-phosphate (S1P). Because sphingolipids regulate AKT activation, we investigated the role of ASM in hepatic glucose and lipid metabolism. Initially, we overexpressed ASM in the livers of wild-type and diabetic db/db mice by adenovirus vector (Ad5ASM). In these mice, glucose tolerance was improved, and glycogen and lipid accumulation in the liver were increased. Using primary cultured hepatocytes, we confirmed that ASM increased glucose uptake, glycogen deposition, and lipid accumulation through activation of AKT and glycogen synthase kinase-3β. In addition, ASM induced up-regulation of glucose transporter 2 accompanied by suppression of AMP-activated protein kinase (AMPK) phosphorylation. Loss of sphingosine kinase-1 (SphK1) diminished ASM-mediated AKT phosphorylation, but exogenous S1P induced AKT activation in hepatocytes. In contrast, SphK1 deficiency did not affect AMPK activation. These results suggest that the SphK/S1P pathway is required for ASM-mediated AKT activation but not for AMPK inactivation. Finally, we found that treatment with high-dose glucose increased glycogen deposition and lipid accumulation in wild-type hepatocytes but not in ASM(-/-) cells. This result is consistent with glucose intolerance in ASM(-/-) mice. In conclusion, ASM modulates AKT activation and AMPK inactivation, thus regulating glucose and lipid metabolism in the liver.

Peroxisome proliferators-activated receptor gamma (PPARG) ligands improve insulin sensitivity in type 2 diabetes and polycystic ovarian syndrome (PCOS). Despite clinical studies showing normalization of pituitary responsiveness to gonadotropin-releasing hormone (GnRH) in patients with PCOS, the precise role of PPARG in regulating the hypothalamic-pituitary-gonadal axis remains unclear. In the present study, we tested the hypothesis that the PPARG agonist rosiglitazone has a direct effect on the pituitary. In mouse LbetaT2 immortalized gonadotrophs, rosiglitazone treatment inhibited GnRH stimulation of the stress kinases p38MAPK and MAPKs/JNKs, but did not alter activation of ERKs, both in the presence and absence of activin. Furthermore, p38MAPK signaling was critical for both Lhb and Fshb promoter activity, and rosiglitazone suppressed the GnRH-mediated induction of Lhb and Fshb mRNA. Depletion of PPARG using a lentivirally encoded short hairpin RNA abolishes the effect of rosiglitazone to suppress activation of JNKs and induction of the transcription factors EGR1 and FOS as well as the gonadotropin genes Lhb and Fshb. Lastly, we show conditional knockout of Pparg in pituitary gonadotrophs caused an increase in luteinizing hormone levels in female mice, a decrease in follicle-stimulating hormone in male mice, and a fertility defect characterized by reduced litter size. Taken together, our data support a direct role for PPARG in modulating pituitary function in vitro and in vivo.

The macrophage-mediated inflammatory response is a key etiologic component of obesity-related tissue inflammation and insulin resistance. The transcriptional factor FoxO1 is a key regulator of cell metabolism, cell cycle and cell death. Its activity is tightly regulated by the phosphoinositide-3-kinase-AKT (PI3K-Akt) pathway, which leads to phosphorylation, cytoplasmic retention and inactivation of FoxO1. Here, we show that FoxO1 promotes inflammation by enhancing Tlr4-mediated signalling in mature macrophages. By means of chromatin immunoprecipitation (ChIP) combined with massively parallel sequencing (ChIP-Seq), we show that FoxO1 binds to multiple enhancer-like elements within the Tlr4 gene itself, as well as to sites in a number of Tlr4 signalling pathway genes. While FoxO1 potentiates Tlr4 signalling, activation of the latter induces AKT and subsequently inactivates FoxO1, establishing a self-limiting mechanism of inflammation. Given the central role of macrophage Tlr4 in transducing extrinsic proinflammatory signals, the novel functions for FoxO1 in macrophages as a transcriptional regulator of the Tlr4 gene and its inflammatory pathway, highlights FoxO1 as a key molecular adaptor integrating inflammatory responses in the context of obesity and insulin resistance.

Increased dietary saturated fat intake can lead to detrimental effects on human health. In this issue of Cell Metabolism, Lichtenstein et al. (2010) show that by inhibiting lipoprotein lipase (LPL) activity in mesenteric lymph nodes, Angiopoietin-like protein 4 (Angptl4) protects resident macrophages from dietary saturated fatty acid (SFA)-induced inflammation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Omega-3 fatty acids (omega-3 FAs), DHA and EPA, exert anti-inflammatory effects, but the mechanisms are poorly understood. Here, we show that the G protein-coupled receptor 120 (GPR120) functions as an omega-3 FA receptor/sensor. Stimulation of GPR120 with omega-3 FAs or a chemical agonist causes broad anti-inflammatory effects in monocytic RAW 264.7 cells and in primary intraperitoneal macrophages. All of these effects are abrogated by GPR120 knockdown. Since chronic macrophage-mediated tissue inflammation is a key mechanism for insulin resistance in obesity, we fed obese WT and GPR120 knockout mice a high-fat diet with or without omega-3 FA supplementation. The omega-3 FA treatment inhibited inflammation and enhanced systemic insulin sensitivity in WT mice, but was without effect in GPR120 knockout mice. In conclusion, GPR120 is a functional omega-3 FA receptor/sensor and mediates potent insulin sensitizing and antidiabetic effects in vivo by repressing macrophage-induced tissue inflammation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Recent findings denote an important contribution of macrophage inflammatory pathways in causing obesity-related insulin resistance. Inducible nitric oxide synthase (iNOS) is activated in proinflammatory macrophages and modestly elevated in insulin-responsive tissues. Although the benefits of systemic iNOS inhibition in insulin-resistant models have been demonstrated, the role of macrophage iNOS in metabolic disorders is not clear. In the current work, we used bone marrow transplantation (BMT) to generate mice with myeloid iNOS deficiency [iNOS BMT knockout (KO)]. Interestingly, disruption of iNOS in myeloid cells did not protect mice from high-fat diet-induced obesity and insulin resistance. When mice were treated with the iNOS inhibitor, N6-(1-Iminoethyl)-L-lysine hydrochloride (L-NIL), we observed a significant and comparable improvement of glucose homeostasis and insulin sensitivity in both wild-type and iNOS BMT KO mice. We further demonstrated that absence of iNOS in primary macrophages did not affect acute TLR4 signaling pathways and had only a modest and mixed effect on inflammatory gene expression. With respect to TNFalpha treatment, iNOS KO macrophages showed, if anything, a greater inflammatory response. In summary, we conclude that iNOS inhibition in tissues other than myeloid cells is responsible for the beneficial effects in obesity/insulin resistance.

Background & Aims: Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)-containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1beta (IL-1beta) in steatohepatitis, fibrosis, and insulin resistance., Methods: Wild-type (WT), TLR9(-/-), IL-1 receptor (IL-1R)(-/-), and MyD88(-/-) mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively., Results: The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9(-/-) mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1beta production was suppressed in TLR9(-/-) mice. Kupffer cells produced IL-1beta in response to CpG oligodeoxynucleotide. IL-1beta but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-kappaB inactivation, resulting in cell death in response to IL-1beta. IL-1beta induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R(-/-) mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9(-/-), IL-1R(-/-), and MyD88(-/-) mice had less insulin resistance than WT mice on the CDAA diet., Conclusions: In a mouse model of NASH, TLR9 signaling induces production of IL-1beta by Kupffer cells, leading to steatosis, inflammation, and fibrosis., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Obesity represents a state of chronic, low grade inflammation and is associated with infiltration of increased numbers of adipose tissue macrophages (ATMs). Diet-induced obesity leads to an increase in non-inflammatory M1-like ATMs displaying the CD11c surface marker. We assessed the function of CD11c-positive ATMs when insulin resistant high fat diet (HFD) mice become insulin-sensitive after switching from HFD to normal chow (NC). HFD mice rapidly become insulin-sensitive in all major insulin-target tissues, including muscle, liver, and adipose tissue, after the diet switch. In adipose tissue the CD11c-positive macrophages remain constant in number despite the presence of insulin sensitivity, but these macrophages now assume a new phenotype in which they no longer exhibit increased inflammatory pathway markers. Adipose tissue markers of apoptosis and necrosis were elevated on HFD and remain high after the HFD --> NC diet switch. Furthermore, ATM accumulation preceded detectable adipocyte necrosis at the early phase of HFD. Together, these results indicate that 1) CD11c-positive M1-like ATMs can exhibit phenotypic plasticity and that the polarization of these cells between inflammatory and non-inflammatory states is well correlated to the presence of absence of insulin resistance, and 2) adipocyte necrosis and apoptosis can be dissociated from ATM accumulation.

Tissue macrophage inflammatory pathways contribute to obesity-associated insulin resistance. Here, we have examined the efficacy and mechanisms of action of a novel anti-inflammatory compound (HE3286) in vitro and in vivo. In primary murine macrophages, HE3286 attenuates LPS- and TNFalpha-stimulated inflammation. In Zucker diabetic fatty rats, inflammatory cytokine/chemokine expression was downregulated in liver and adipose tissue by HE3286 treatment, as was macrophage infiltration into adipose tissue. In line with reduced inflammation, HE3286 treatment normalized fasting and fed glucose levels, improved glucose tolerance, and enhanced skeletal muscle and liver insulin sensitivity, as assessed by hyperinsulinemic euglycemic clamp studies. In phase 2 clinical trials, HE3286 treatment led to an enhancement in insulin sensitivity in humans. Gluconeogenic capacity was also reduced by HE3286 treatment, as evidenced by a reduced glycemic response during pyruvate tolerance tests and decreased basal hepatic glucose production (HGP) rates. Since serum levels of gluconeogenic substrates were decreased by HE3286, it indicates that the reduction of both intrinsic gluconeogenic capacity and substrate availability contributes to the decrease in HGP. Lipidomic analysis revealed that HE3286 treatment reduced liver cholesterol and triglyceride content, leading to a feedback elevation of LDL receptor and HMG-CoA reductase expression. Accordingly, HE3286 treatment markedly decreased total serum cholesterol. In conclusion, HE3286 is a novel anti-inflammatory compound, which displays both glucose-lowering and cholesterol-lowering effects.

Insulin resistance, the major metabolic abnormality underlying type 2 diabetes, is associated with chronic inflammation and heavy macrophage infiltration in white adipose tissue (WAT). The therapeutic properties of the synthetic adrenal steroid Delta(5)-androstene-17alpha-ethynyl-3beta,7beta,17beta-triol (HE3286) were characterized in metabolic disease models. Treatment of diabetic db/db mice with HE3286 suppressed progression to hyperglycemia and markedly improved glucose clearance. Similar effects were also observed in insulin-resistant, diet-induced obese C57BL/6J mice and genetically obese ob/ob mice. This effect appeared to be a consequence of reduced insulin resistance because HE3286 lowered blood insulin levels in db/db and ob/ob mice. Treatment with HE3286 was accompanied by suppressed expression of the prototype macrophage-attracting chemokine monocyte chemoattractant protein-1 in WAT, along with its cognate receptor C-C motif chemokine receptor-2. Exposure of mouse macrophages to HE3286 in vitro caused partial suppression of endotoxin (lipopolysaccharide)-induced nuclear factor kappa-B (NF-kappaB)-sensitive reporter gene expression, NF-kappaB nuclear translocation, and NF-kappaB/p65 serine phosphorylation. Proinflammatory kinases, including IkappaB kinase, c-Jun NH2-terminal kinase, and p38, were also inhibited by HE3286. In ligand competition experiments HE3286 did not bind to classical sex steroid or corticosteroid receptors, including androgen receptor (AR), progesterone receptor, estrogen receptor (ER) alpha or ERbeta, and glucocorticoid receptor (GR). Likewise, in cells expressing nuclear receptor-sensitive reporter genes HE3286 did not substantially stimulate transactivation of AR, ER, GR, or peroxisome proliferator-activated receptor (PPAR) alpha, PPARdelta, and PPARgamma. These findings indicate that HE3286 improves glucose homeostasis in diabetic and insulin-resistant mice and suggest that the observed therapeutic effects result from attenuation of proinflammatory pathways, independent of classic sex steroid receptors, corticosteroid receptors, or PPARs.