TAZ Is a Negative Regulator of PPARγ Activity in Adipocytes and TAZ Deletion Improves Insulin Sensitivity and Glucose Tolerance.

Insulin resistance is a major factor in obesity-linked type 2 diabetes. PPARγ is a master regulator of adipogenesis, and small molecule agonists, termed thiazolidinediones, are potent therapeutic insulin sensitizers. Here, we studied the role of transcriptional co-activator with PDZ-binding motif (TAZ) as a transcriptional co-repressor of PPARγ. We found that adipocyte-specific TAZ knockout (TAZ AKO) mice demonstrate a constitutively active PPARγ state. Obese TAZ AKO mice show improved glucose tolerance and insulin sensitivity compared to littermate controls. PPARγ response genes are upregulated in adipose tissue from TAZ AKO mice and adipose tissue inflammation was also decreased. In vitro and in vivo mechanistic studies revealed that the TAZ-PPARγ interaction is partially dependent on ERK-mediated Ser112 PPARγ phosphorylation. As adipocyte PPARγ Ser112 phosphorylation is increased in obesity, repression of PPARγ activity by TAZ could contribute to insulin resistance. These results identify TAZ as a new factor in the development of obesity-induced insulin resistance.

Adipocyte PU.1 knockout promotes insulin sensitivity in HFD-fed obese mice.

Insulin resistance is a key feature of obesity and type 2 diabetes. PU.1 is a master transcription factor predominantly expressed in macrophages but after HFD feeding PU.1 expression is also significantly increased in adipocytes. We generated adipocyte specific PU.1 knockout mice using adiponectin cre to investigate the role of PU.1 in adipocyte biology, insulin and glucose homeostasis. In HFD-fed obese mice systemic glucose tolerance and insulin sensitivity were improved in PU.1 AKO mice and clamp studies indicated improvements in both adipose and liver insulin sensitivity. At the level of adipose tissue, macrophage infiltration and inflammation was decreased and glucose uptake was increased in PU.1 AKO mice compared with controls. While PU.1 deletion in adipocytes did not affect the gene expression of PPARg itself, we observed increased expression of PPARg target genes in eWAT from HFD fed PU.1 AKO mice compared with controls. Furthermore, we observed decreased phosphorylation at serine 273 in PU.1 AKO mice compared with fl/fl controls, indicating that PPARg is more active when PU.1 expression is reduced in adipocytes. Therefore, in obesity the increased expression of PU.1 in adipocytes modifies the adipocyte PPARg cistrome resulting in impaired glucose tolerance and insulin sensitivity.

RalA controls glucose homeostasis by regulating glucose uptake in brown fat.

Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo.

The role of dietary fat in obesity-induced insulin resistance.

Consumption of excess calories results in obesity and insulin resistance and has been intensively studied in mice and humans. The objective of this study was to determine the specific contribution of dietary fat rather than total caloric intake to the development of obesity-associated insulin resistance. We used an intragastric feeding method to overfeed excess calories from a low-fat diet (and an isocalorically matched high-fat diet) through a surgically implanted gastric feeding tube to generate obesity in wild-type mice followed by hyperinsulinemic-euglycemic clamp studies to assess the development of insulin resistance. We show that overfeeding a low-fat diet results in levels of obesity similar to high-fat diet feeding in mice. However, despite a similar body weight, obese high-fat diet-fed mice are more insulin resistant than mice fed an isocaloric low-fat diet. Therefore, increased proportion of calories from dietary fat further potentiates insulin resistance in the obese state. Furthermore, crossover diet studies revealed that reduction in dietary fat composition improves glucose tolerance in obesity. In the context of the current obesity and diabetes epidemic, it is particularly important to fully understand the role of dietary macronutrients in the potentiation and amelioration of disease.

Regulation of metabolism by the innate immune system.

Low-grade tissue inflammation induced by obesity can result in insulin resistance, which in turn is a key cause of type 2 diabetes mellitus. Cells of the innate immune system produce cytokines and other factors that impair insulin signalling, which contributes to the connection between obesity and the onset of type 2 diabetes mellitus. Here, we review the innate immune cells involved in secreting inflammatory factors in the obese state. In the adipose tissue, these cells include proinflammatory adipose tissue macrophages and natural killer cells. We also discuss the role of innate immune cells, such as anti-inflammatory adipose tissue macrophages, eosinophils, group 2 innate lymphoid cells and invariant natural killer T cells, in maintaining an anti-inflammatory and insulin-sensitive environment in the lean state. In the liver, both Kupffer cells and recruited hepatic macrophages can contribute to decreased hepatic insulin sensitivity. Proinflammatory macrophages might also adversely affect insulin sensitivity in the skeletal muscle and pancreatic ß-cell function. Finally, this Review provides an overview of the mechanisms for regulating proinflammatory immune responses that could lead to future therapeutic opportunities to improve insulin sensitivity.

Intestinal FXR agonism promotes adipose tissue browning and reduces obesity and insulin resistance.

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.

Endocrinization of FGF1 produces a neomorphic and potent insulin sensitizer.

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.

A Gpr120-selective agonist improves insulin resistance and chronic inflammation in obese mice.

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.

Prevalence of undiagnosed and inadequately treated type 2 diabetes mellitus, hypertension, and dyslipidemia in morbidly obese patients who present for bariatric surgery.

BACKGROUND: Pharmacotherapy is considered the primary treatment modality for diabetes mellitus (DM), hypertension (HTN), and dyslipidemia (DYS). We sought to investigate the status of DM, HTN, and DYS in patients who seek bariatric surgery. METHODS: Demographic and comorbidity history were prospectively collected on 1,508 patients referred for bariatric consultation at a single institution from February 2008 to March 2012. We utilized published consensus guidelines (GL) to benchmark the efficacy of standard pharmacotherapy for these metabolic diseases, and 881 patients met the study design criteria. RESULTS: Most patients exhibited at least one form of metabolic dysregulation (pre-DM or DM, 75.8%; pre-HTN or HTN, 91.1%; pre-DYS or DYS, 84.0%; metabolic syndrome, 76.0%). The majority of patients either did not meet GL treatment goals (DM, 45.7%; HTN, 39.5%; DYS, 22.3%) or were previously undiagnosed (DM, 15.8%; HTN, 13.7%; DYS, 41.7%). Non-GL pharmacotherapy was significantly less effective than GL pharmacotherapy at achieving treatment goals for DM (31.8 vs. 53.2%, p < 0.001) and HTN (43.6 vs. 63.2%, p = 0.007). Patients with concurrent DM, HTN, and DYS (35.5%) were less likely than patients with only one or two of these metabolic diseases to achieve GL treatment goals for HTN (38.1 vs. 72.6%, p < 0.001) and DYS (55.7 vs. 73.8%, p = 0.002). Only 8.0% of these patients achieved treatment goals for all three metabolic comorbidities. CONCLUSIONS: In this patient group, DM, HTN, and DYS were poorly compensated, even when pharmacotherapy was consistent with published GL. This may be due to disease burden in bariatric surgery candidates or to inadequate medical management prior to presentation.

Contributions of adipose tissue architectural and tensile properties toward defining healthy and unhealthy obesity.

The extracellular matrix (ECM) plays an important role in the maintenance of white adipose tissue (WAT) architecture and function, and proper ECM remodeling is critical to support WAT malleability to accommodate changes in energy storage needs. Obesity and adipocyte hypertrophy place a strain on the ECM remodeling machinery, which may promote disordered ECM and altered tissue integrity and could promote proinflammatory and cell stress signals. To explore these questions, new methods were developed to quantify omental and subcutaneous WAT tensile strength and WAT collagen content by three-dimensional confocal imaging, using collagen VI knockout mice as a methods validation tool. These methods, combined with comprehensive measurement of WAT ECM proteolytic enzymes, transcript, and blood analyte analyses, were used to identify unique pathophenotypes of metabolic syndrome and type 2 diabetes mellitus in obese women, using multivariate statistical modeling and univariate comparisons with weight-matched healthy obese individuals. In addition to the expected differences in inflammation and glycemic control, approximately 20 ECM-related factors, including omental tensile strength, collagen, and enzyme transcripts, helped discriminate metabolically compromised obesity. This is consistent with the hypothesis that WAT ECM physiology is intimately linked to metabolic health in obese humans, and the studies provide new tools to explore this relationship.

Glucagon regulates gluconeogenesis through KAT2B- and WDR5-mediated epigenetic effects.

Circulating pancreatic glucagon is increased during fasting and maintains glucose balance by stimulating hepatic gluconeogenesis. Glucagon triggering of the cAMP pathway upregulates the gluconeogenic program through the phosphorylation of cAMP response element-binding protein (CREB) and the dephosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic gene expression by promoting epigenetic changes that facilitate assembly of the transcriptional machinery. However, the nature of these modifications is unclear. Using mouse models and in vitro assays, we show that histone H3 acetylation at Lys 9 (H3K9Ac) was elevated over gluconeogenic genes and contributed to increased hepatic glucose production during fasting and in diabetes. Dephosphorylation of CRTC2 promoted increased H3K9Ac through recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat-containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle, whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Depletion of KAT2B or WDR5 decreased gluconeogenic gene expression, consequently breaking the cycle. Administration of a small-molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, suggesting that this enzyme may be a useful target for diabetes treatment.

Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity.

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Vitamin A upregulates matrix metalloproteinase-9 activity by murine myeloid dendritic cells through a nonclassical transcriptional mechanism.

Myeloid dendritic cells (DC) are specialized antigen-presenting immune cells. Upon activation in peripheral tissues, DC migrate to lymph nodes to activate T lymphocytes. Matrix metalloproteinase (MMP)-9 is a gelatinase essential for DC migration. We have previously shown that all-trans retinoic acid (atRA), a bioactive metabolite of vitamin A, significantly augmented DC MMP-9 mRNA and protein production. We investigated the mechanisms by which atRA increased MMP-9 activity in vitro. Mouse myeloid DC cultured with atRA demonstrated increased gelatinase activity compared with cells cultured with retinoic acid receptor (RAR)-alpha antagonist. Adding MMP-9 inhibitor significantly blocked DC gelatinase activity and increased adherence of DC in a dose-dependent manner. AtRA-induced Mmp-9 gene expression in DC was blocked by transcriptional inhibition. Because the Mmp-9 promoter contains no canonical retinoic acid response element (RARE), we performed additional studies to determine how atRA regulated DC Mmp-9 transcription. Electrophoretic mobility shift assays for the consensus Sp1, activating protein-1, and nuclear factor-kappaB binding sites located in the Mmp-9 promoter did not indicate greater nuclear protein binding in response to atRA. Chromatin immunoprecipitation assays indicated RARalpha and histone acetyltransferase p300 recruitment to, and acetylation of, histone H3 at the Mmp-9 promoter was greater after atRA treatment. These data suggest that atRA regulated DC adhesion in vitro partly through MMP-9 gelatinase activity. Mmp-9 expression was enhanced through a transcriptional mechanism involving greater RARalpha promoter binding, recruitment of p300, and subsequent histone H3 acetylation, despite the absence of a consensus RARE.

Vitamin A as a regulator of antigen presenting cells.

Vitamin A has been long associated with immune system competence. Vitamin A deficiency is known to compromise many aspects of both innate and adaptive immune responses. Recent advances in retinol uptake and metabolism have identified the antigen presenting cell (APC) as a central immune cell capable of vitamin A metabolism. APC are now known to express retinaldehyde dehydrogenase and secrete retinoic acid. The retinoic acid produced has both autocrine and paracrine effects. Autocrine effects include upregulation of CD1d nonclassical major histocompatibility class I-like molecule and matrix metalloproteinase-9. Paracrine effects influence multiple lymphocyte lineage cell populations. Specifically, retinoic acid increases IgA isotype class switching by B lymphocytes, enhances regulatory T cell differentiation, and directs homing of lymphocytes to mucosa. CD1d lipid antigen presentation expands natural killer T cell populations. Previously, the focus of vitamin A action in adaptive immunity was on lymphocytes, but these recent advances suggest the APC may be the central player in carrying out the immune system functions of vitamin A.

Retinoic acid decreases adherence of murine myeloid dendritic cells and increases production of matrix metalloproteinase-9.

Myeloid dendritic cells (DC) are professional antigen presenting cells (APC) that migrate to secondary lymphoid tissues upon antigen stimulation, where they activate naïve T cells. Vitamin A is essential for normal immune function. We investigated the ability of all-trans retinoic acid (atRA), a bioactive metabolite of vitamin A, to modulate DC adhesion in culture. Male BALB/cJ mouse bone marrow cells cultured with granulocyte-macrophage colony-stimulating factor in the presence of retinoic acid receptor (RAR) alpha-specific antagonist showed an increase in the percentage of developing DC that remained adherent compared with cells rescued with atRA treatment from d 8 to 10 of culture (P < 0.05). Replacement of the RARalpha antagonist with atRA on d 8 of the culture period decreased DC surface expression of the adhesion molecule CD11a (P < 0.0001) but not the gene expression. Rescue with atRA also dramatically increased gene and protein expression of pro-matrix metalloproteinase (MMP)-9 (P < 0.05). However, gene expression and protein production of tissue inhibitor of metalloproteinase (TIMP)-1 was unaffected by atRA rescue, altering the molar ratio of secreted pro-MMP-9:TIMP-1, resulting in a fold excess of pro-MMP-9 to its primary inhibitor (P < 0.05). These data suggest that atRA is essential to augment MMP-9 expression in myeloid DC and can alter their surface expression of adhesion molecules.