Disruption of inducible 6-phosphofructo-2-kinase impairs the suppressive effect of PPARγ activation on diet-induced intestine inflammatory response

PFKFB3 is a target gene of peroxisome proliferator-activated receptor gamma (PPARγ) and encodes for inducible 6-phosphofructo-2-kinase (iPFK2). As a key regulatory enzyme that stimulates glycolysis, PFKFB3/iPFK2 links adipocyte metabolic and inflammatory responses. Additionally, PFKFB3/iPFK2 is involved in the effect of active PPARγ on suppressing overnutrition-induced adipose tissue inflammatory response, which accounts for the insulin-sensitizing and antidiabetic effects of PPARγ activation. Using PFKFB3/iPFK2-disrupted mice, the present study investigated the role of PFKFB3/iPFK2 in regulating overnutrition-associated intestine inflammatory response and in mediating the effects of PPARγ activation. In wild-type mice, intestine PFKFB3/iPFK2 was increased in response to high-fat diet (HFD) feeding compared with that in mice fed a low-fat diet. However, intestine PFKFB3/iPFK2 was decreased in PFKFB3/iPFK2-disrupted mice and did not respond to HFD feeding. Furthermore, on an HFD, PFKFB3/iPFK2-disrupted mice displayed a significant increase in major intestine proinflammatory indicators such as toll-like receptor 4 expression, c-Jun N-terminal kinase 1 and nuclear factor kappa B phosphorylation, and proinflammatory cytokine expression compared with wild-type littermates. Upon treatment with rosiglitazone, an agonist of PPARγ, intestine proinflammatory indicators were markedly decreased in wild-type mice, but to a much lesser degree in PFKFB3/iPFK2-disrupted mice. Overall, the status of HFD-induced intestine inflammatory response in all treated mice correlated inversely with systemic insulin sensitivity, indicated by the homeostasis model assessment of insulin resistance data. Together, these results suggest that PFKFB3/iPFK2 is critically involved in the effect of PPARγ activation on suppressing diet-induced intestine inflammatory response.     

Molecular mechanism of insulin resistance and obesity

Obesity and insulin resistance have been recognized as leading causes of major health issues. We have endeavored to depict the molecular mechanism of insulin resistance, focusing on the function of adipocyte. We have investigated a role of PPARgamma on the pathogenesis of Type II diabetes. Heterozygous PPARgamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. Moreover, a Pro12Ala polymorphism in the human PPARgamma2 gene was associated with decreased risk of Type II diabetes in Japanese. Taken together with these results, PPARgamma is proved to be a thrifty gene mediating Type II diabetes. Pharmacological inhibitors of PPARgamma/RXR ameliorate high-fat diet-induced insulin resistance in animal models of Type II diabetes. We have performed a genome-wide scan of Japanese Type 2 diabetic families using affected sib pair analysis. Our genome scan reveals at least 9 chromosomal regions potentially harbor susceptibility genes of Type II diabetes in Japanese. Among these regions, 3q26-q28 appeared to be very attractive one, because of the gene encoding adiponectin, the expression of which we had found enhanced in insulin-sensitive PPARgamma-deficient mice. Indeed, the subjects with the G/G genotype of SNP276 in the adiponectin gene were at increased risk for Type II diabetes compared with those having the T/T genotype. The plasma adiponectin levels were lower in the subjects with the G allele, suggesting that genetically inherited decrease in adiponectin levels predispose subjects to insulin resistance and Type II diabetes. Our work also confirmed that replenishment of adiponectin represents a novel treatment strategy for insulin resistance and Type II diabetes using animal models. Further investigation will be needed to clarify how adiponectin exerts its effect and to discover the molecular target of therapies.     

Effect of heterozygous PPARgamma deficiency and TZD treatment on insulin resistance associated with age and high-fat feeding

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is the target receptor for thiazolidinedione (TZD) compounds, which are a class of insulin-sensitizing drugs used in the treatment of type 2 diabetes. Paradoxically, however, mice deficient in PPARgamma (PPARgamma(+/-)) are more insulin sensitive than their wild-type (WT) littermates, not less, as would be predicted. To determine whether PPARgamma deficiency could prevent the development of the insulin resistance associated with increasing age or high-fat (HF) feeding, insulin sensitivity was assessed in PPARgamma(+/-) and WT mice at 2, 4, and 8 mo of age and in animals fed an HF diet. Because TZDs elicit their effect through PPARgamma receptor, we also examined the effect of troglitazone (a TZD) in these mice. Glucose metabolism was assessed by hyperinsulinemic euglycemic clamp and oral glucose tolerance test. Insulin sensitivity declined with age for both groups. However, the decline in the PPARgamma(+/-) animals was substantially less than that of the WT animals, such that, by 8 mo of age, the PPARgamma(+/-) mice were markedly more insulin sensitive than the WT mice. This greater sensitivity in PPARgamma(+/-) mice was lost with TZD treatment. HF feeding led to marked adipocyte hypertrophy and peripheral tissue and hepatic insulin resistance in WT mice but also in PPARgamma(+/-) mice. Treatment of these mice with troglitazone completely prevented the adipocyte hypertrophy and normalized insulin action. In conclusion, PPARgamma deficiency partially protects against age-related insulin resistance but does not protect against HF diet-induced insulin resistance.     

A Novel Peroxisome Proliferator-activated Receptor (PPAR)α Agonist and PPARγ Antagonist,Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice

A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia.     

Targeted elimination of peroxisome proliferator-activated receptor gamma in beta cells leads to abnormalities in islet mass without compromising glucose homeostasis

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is an important regulator of lipid and glucose homeostasis and cellular differentiation. Studies of many cell types in vitro and in vivo have demonstrated that activation of PPAR gamma can reduce cellular proliferation. We show here that activation of PPAR gamma is sufficient to reduce the proliferation of cultured insulinoma cell lines. We created a model with mice in which the expression of the PPARG gene in beta cells was eliminated (beta gamma KO mice), and these mice were found to have significant islet hyperplasia on a chow diet. Interestingly, the normal expansion of beta-cell mass that occurs in control mice in response to high-fat feeding is markedly blunted in these animals. Despite this alteration in beta-cell mass, no effect on glucose homeostasis in beta gamma KO mice was noted. Additionally, while thiazolidinediones enhanced insulin secretion from cultured wild-type islets, administration of rosiglitazone to insulin-resistant control and beta gamma KO mice revealed that PPAR gamma in beta cells is not required for the antidiabetic actions of these compounds. These data demonstrate a critical physiological role for PPAR gamma function in beta-cell proliferation and also indicate that the mechanisms controlling beta-cell hyperplasia in obesity are different from those that regulate baseline cell mass in the islet.     

Cystathionine γ lyase–hydrogen sulfide increases peroxisome proliferator-activated receptor γ activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes

Adipocytes express the cystathionine γ lyase (CSE)–hydrogen sulfide (H₂S) system. CSE–H₂S promotes adipogenesis but ameliorates adipocyte insulin resistance. We investigated the mechanism of how CSE–H₂S induces these paradoxical effects. First, we confirmed that an H₂S donor or CSE overexpression promoted adipocyte differentiation. Second, we found that H₂S donor inhibited but CSE inhibition increased phosphodiesterase (PDE) activity. H₂S replacing isobutylmethylxanthine in the differentiation program induced adipocyte differentiation in part. Inhibiting PDE activity by H₂S induced peroxisome proliferator activated receptor γ (PPARγ) protein and mRNA expression. Of note, H₂S directly sulfhydrated PPARγ protein. Sulfhydrated PPARγ increased its nuclear accumulation, DNA binding activity and adipogenesis gene expression, thereby increasing glucose uptake and lipid storage, which were blocked by the desulfhydration reagent DTT. H₂S induced PPARγ sulfhydration, which was blocked by mutation of the C139 site of PPARγ. In mice fed a high-fat diet (HFD) for 4 weeks, the CSE inhibitor decreased but H₂S donor increased adipocyte numbers. In obese mice fed an HFD for 13 weeks, H₂S treatment increased PPARγ sulfhydration in adipose tissues and attenuated insulin resistance but did not increase obesity. In conclusion, CSE–H₂S increased PPARγ activity by direct sulfhydration at the C139 site, thereby changing glucose into triglyceride storage in adipocytes. CSE–H₂S-mediated PPARγ activation might be a new therapeutic target for diabetes associated with obesity.     

Quercetin attenuates adipose hypertrophy,in part through activation of adipogenesis in rats fed a high-fat diet

An impaired capacity of adipose tissue expansion leads to adipocyte hypertrophy, inflammation and insulin resistance (IR) under positive energy balance. We previously showed that a grape pomace extract, rich in flavonoids including quercetin (Q), attenuates adipose hypertrophy. This study investigated whether dietary Q supplementation promotes adipogenesis in the epididymal white adipose tissue (eWAT) of rats consuming a high-fat diet, characterizing key adipogenic regulators in 3T3-L1 pre-adipocytes. Consumption of a high-fat diet for 6 weeks caused IR, increased plasma TNFα concentrations, eWAT weight, adipocyte size and the eWAT/brown adipose tissue (BAT) ratio. These changes were accompanied by decreased levels of proteins involved in angiogenesis, VEGF-A and its receptor 2 (VEGF-R2), and of two central adipogenic regulators, i.e. PPARγ and C/EBPα, and proteins involved in mature adipocyte formation, i.e. fatty acid synthase (FAS) and adiponectin. Q significantly reduced adipocyte size and enhanced angiogenesis and adipogenesis without changes in eWAT weight and attenuated systemic IR and inflammation. In addition, high-fat diet consumption increased eWAT hypoxia inducible factor-1 alpha (HIF-1α) levels and those of proteins involved in adipose inflammation (TLR-4, CD68, MCP-1, JNK) and activation of endoplasmic reticulum (ER) stress, i.e. ATF-6 and XBP-1. Q mitigated all these events. Q and quercetin 3-glucoronide prevented TNFα-mediated downregulation of adipogenesis during 3T3-L1 pre-adipocytes early differentiation. Together, Q capacity to promote a healthy adipose expansion enhancing angiogenesis and adipogenesis may contribute to reduced adipose hypertrophy, inflammation and IR. Consumption of diets rich in Q could be useful to counteract the adverse effects of high-fat diet-induced adipose dysfunction.     

Liver peroxisome proliferator-activated receptor gamma contributes to hepatic steatosis,triglyceride clearance,and regulation of body fat mass

Peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor that mediates the antidiabetic effects of thiazolidinediones. PPAR gamma is present in adipose tissue and becomes elevated in fatty livers, but the roles of specific tissues in thiazolidinedione actions are unclear. We studied the function of liver PPAR gamma in both lipoatrophic A-ZIP/F-1 (AZIP) and wild type mice. In AZIP mice, ablation of liver PPAR gamma reduced the hepatic steatosis but worsened the hyperlipidemia, triglyceride clearance, and muscle insulin resistance. Inactivation of AZIP liver PPAR gamma also abolished the hypoglycemic and hypolipidemic effects of rosiglitazone, demonstrating that, in the absence of adipose tissue, the liver is a primary and major site of thiazolidinedione action. In contrast, rosiglitazone remained effective in non-lipoatrophic mice lacking liver PPAR gamma, suggesting that adipose tissue is the major site of thiazolidinedione action in typical mice with adipose tissue. Interestingly, mice without liver PPAR gamma, but with adipose tissue, developed relative fat intolerance, increased adiposity, hyperlipidemia, and insulin resistance. Thus, liver PPAR gamma regulates triglyceride homeostasis, contributing to hepatic steatosis, but protecting other tissues from triglyceride accumulation and insulin resistance.     

Dok1 mediates high-fat diet-induced adipocyte hypertrophy and obesity through modulation of PPAR-gamma phosphorylation

Insulin receptor substrate (IRS)-1 and IRS-2 have dominant roles in the action of insulin, but other substrates of the insulin receptor kinase, such as Gab1, c-Cbl, SH2-B and APS, are also of physiological relevance. Although the protein downstream of tyrosine kinases-1 (Dok1) is known to function as a multisite adapter molecule in insulin signaling, its role in energy homeostasis has remained unclear. Here we show that Dok1 regulates adiposity. Expression of Dok1 in white adipose tissue was markedly increased in mice fed a high-fat diet, whereas adipocytes lacking this adapter were smaller and showed a reduced hypertrophic response to this dietary manipulation. Dok1-deficient mice were leaner and showed improved glucose tolerance and insulin sensitivity compared with wild-type mice. Embryonic fibroblasts from Dok1-deficient mice were impaired in adipogenic differentiation, and this defect was accompanied by an increased activity of the protein kinase ERK and a consequent increase in the phosphorylation of peroxisome proliferator-activated receptor (PPAR)-gamma on Ser112. Mutation of this negative regulatory site for the transactivation activity of PPAR-gamma blocked development of the lean phenotype caused by Dok1 ablation. These results indicate that Dok1 promotes adipocyte hypertrophy by counteracting the inhibitory effect of ERK on PPAR-gamma and may thus confer predisposition to diet-induced obesity.     

Impairment of energy sensors,SIRT1 and AMPK,in lipid induced inflamed adipocyte is regulated by Fetuin A

Although several reports demonstrated that accumulation of excess lipid in adipose tissue produces defects in adipocyte which leads to the disruption of energy homeostasis causing severe metabolic problems, underlying mechanism of this event remains yet unclear. Here we demonstrate that FetuinA (FetA) plays a critical role in the impairment of two metabolic sensors, SIRT1 and AMPK, in inflamed adipocytes of high fat diet (HFD) mice. A linear increase in adipocyte hypertrophy from 10 to 16 week was in tandem with the increase in FetA and that coincided with SIRT1 cleavage and decrease in pAMPK which adversely affects PGC1α activation. Knock down (KD) of FetA gene in HFD mice could significantly improve this situation indicating FetA's contribution in the damage of energy sensors in inflamed adipocyte. However, FetA effect was not direct, it was mediated through TNF-α which again is dependent on FetA as FetA augments TNF-α expression. FetA being an upstream regulator of TNF-α, its suppression prevented TNF-α mediated Caspase-1 activation and cleavage of SIRT1. FetA induced inactivation of PGC1α due to SIRT1 cleavage decreased PPARϒ, adiponectin, NRF1 and Tfam expression. All these together caused a significant fall in mitochondrial biogenesis and bioenergetics that disrupted energy homeostasis resulting loss of insulin sensitivity. Taken together, our findings revealed a new dimension of FetA, it not only induced inflammation in adipocyte but also acts as an upstream regulator of SIRT1 cleavage and AMPK activation. Intervention of FetA may be worthwhile to prevent metabolic imbalance that causes insulin resistance and type 2 diabetes.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.     

Adipocyte NCoR knockout decreases PPARγ phosphorylation and enhances PPARγ activity and insulin sensitivity

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.