Glycogen synthase 2 is a novel target gene of peroxisome proliferator-activated receptors.

Glycogen synthase 2 (Gys-2) is the ratelimiting enzyme in the storage of glycogen in liver and adipose tissue, yet little is known about regulation of Gys-2 transcription. The peroxisome proliferator-activated receptors (PPARs) are transcription factors involved in the regulation of lipid and glucose metabolism and might be hypothesized to govern glycogen synthesis as well. Here, we show that Gys-2 is a direct target gene of PPARalpha, PPARbeta/delta and PPARgamma. Expression of Gys-2 is significantly reduced in adipose tissue of PPARalpha-/-, PPARbeta/delta-/- and PPARgamma+/- mice. Furthermore, synthetic PPARbeta/delta, and gamma agonists markedly up-regulate Gys-2 mRNA and protein expression in mouse 3T3-L1 adipocytes. In liver, PPARalpha deletion leads to decreased glycogen levels in the refed state, which is paralleled by decreased expression of Gys-2 in fasted and refed state. Two putative PPAR response elements (PPREs) were identified in the mouse Gys-2 gene: one in the upstream promoter (DR-1prom) and one in intron 1 (DR-1int). It is shown that DR-1int is the response element for PPARs, while DR-1prom is the response element for Hepatic Nuclear Factor 4 alpha (HNF4alpha). In adipose tissue, which does not express HNF4alpha, DR-1prom is occupied by PPARbeta/delta and PPARgamma, yet binding does not translate into transcriptional activation of Gys-2. Overall, we conclude that mouse Gys-2 is a novel PPAR target gene and that transactivation by PPARs and HNF4alpha is mediated by two distinct response elements.

A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) plays a key role in adipocyte differentiation and insulin sensitivity. Its synthetic ligands, the thiazolidinediones (TZD), are used as insulin sensitizers in the treatment of type 2 diabetes. These compounds induce both adipocyte differentiation in cell culture models and promote weight gain in rodents and humans. Here, we report on the identification of a new synthetic PPARgamma antagonist, the phosphonophosphate SR-202, which inhibits both TZD-stimulated recruitment of the coactivator steroid receptor coactivator-1 and TZD-induced transcriptional activity of the receptor. In cell culture, SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreasing PPARgamma activity, either by treatment with SR-202 or by invalidation of one allele of the PPARgamma gene, leads to a reduction of both high fat diet-induced adipocyte hypertrophy and insulin resistance. These effects are accompanied by a smaller size of the adipocytes and a reduction of TNFalpha and leptin secretion. Treatment with SR-202 also dramatically improves insulin sensitivity in the diabetic ob/ob mice. Thus, although we cannot exclude that its actions involve additional signaling mechanisms, SR-202 represents a new selective PPARgamma antagonist that is effective both in vitro and in vivo. Because it yields both antiobesity and antidiabetic effects, SR-202 may be a lead for new compounds to be used in the treatment of obesity and type 2 diabetes.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Role of genetic polymorphism peroxisome proliferator-activated receptor-gamma2 Pro12Ala on ethnic susceptibility to diabetes in South-Asian and Caucasian subjects: Evidence for heterogeneity

OBJECTIVE: To determine whether the peroxisome proliferator-activated receptor (PPAR)-gamma Pro12ala polymorphism modulates susceptibility to diabetes in South Asians. RESEARCH DESIGN AND METHODS: South Asians (n = 697) and Caucasians (n = 457) living in Dallas/Forth Worth, Texas, and South Asians living in Chennai, India (n = 1,619), were enrolled for this study. PPAR-gamma Pro12Ala was determined using restriction fragment-length polymorphism. Insulin responsiveness to an oral glucose tolerance test (OGTT) was measured in nondiabetic subjects. RESULTS: The Caucasian diabetic subjects had significantly lower prevalence of PPAR-gamma 12Ala when compared with the Caucasian nondiabetic subjects (20 vs. 9%, P = 0.006). However, there were no significant differences between diabetic and nondiabetic subjects with reference to the Pro12Ala polymorphism among the South Asians living in Dallas (20 vs. 23%) and in India (19 vs. 19.3%). Although Caucasians carrying PPAR-gamma Pro12Ala had lower plasma insulin levels at 2 h of OGTT than the wild-type (Pro/Pro) carriers (76 +/- 68 and 54 +/- 33 microU/ml, respectively, P = 0.01), no differences in either fasting or 2-h plasma insulin concentrations were found between South Asians carrying the PPAR-gamma Pro12Ala polymorphism and those with the wild-type genotype at either Chennai or Dallas. CONCLUSIONS: Although further replication studies are necessary to test the validity of the described genotype-phenotype relationship, our study supports the hypothesis that the PPAR-gamma Pro12Ala polymorphism is protective against diabetes in Caucasians but not in South Asians.

Peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians

AIMS: The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-gamma co-activator-1 alpha (PGC-1alpha) gene with Type 2 diabetes in Asian Indians. METHODS: The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Haplotype frequencies were estimated using an expectation-maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. RESULTS: The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264-2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. CONCLUSIONS: The A allele of Thr394Thr (G --> A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population.

Ibuprofen inhibits migration and proliferation of human coronary artery smooth muscle cells by inducing a differentiated phenotype: role of peroxisome proliferator-activated receptor y

Objectives: The search for agents that are capable of preventing restenosis and reduce the risk of late thrombosis is of utmost importance. In this study we aim to evaluate the in vitro effects of ibuprofen on proliferation and migration of human coronary artery smooth muscle cells (HCASMCs) and on human coronary artery endothelial cells (HCAECs) migration. Methods: Cell proliferation was evaluated by direct cell counting using trypan blue exclusion. Cell migration was assessed by wound healing “scratch” assay and by time lapse video-microscopy. Protein expression was assessed by immunoblotting, and morphological changes were studied by immunocytochemistry. The involvement of the PPARγ pathway was studied with the selective agonist troglitazone, and the use of highly selective antagonists of PPARγ such as PGF2α and GW9662. Results: We demonstrate that ibuprofen inhibits proliferation and migration of HCASMCs and induces a switch in HCASMCs towards a differentiated and contractile phenotype, and that these effects are mediated through the PPARγ pathway. Importantly we also show that the effects of ibuprofen are cell type specific as it does not affect migration and proliferation of endothelial cells. Conclusions: Taken together, our results suggest that ibuprofen could be an effective drug for the development of novel drug eluting stents, which could lead reduced rates of restenosis and potentially other complications of DES stent implantation.

Endurance training in humans leads to fiber type-specific increases in levels of peroxisome proliferator-activated receptor-[gamma] coactivator-1 and peroxisome proliferator-activated receptor-[alpha] in skeletal muscle

The peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PGC-1) can induce mitochondria biogenesis and has been implicated in the development of oxidative type I muscle fibers. The PPAR isoforms α, β/δ, and γ control the transcription of genes involved in fatty acid and glucose metabolism. As endurance training increases skeletal muscle mitochondria and type I fiber content and fatty acid oxidative capacity, our aim was to determine whether these increases could be mediated by possible effects on PGC-1 or PPAR-α, -β/δ, and -γ. Seven healthy men performed 6 weeks of endurance training and the expression levels of PGC-1 and PPAR-α, -β/δ, and -γ mRNA as well as the fiber type distribution of the PGC-1 and PPAR-α proteins were measured in biopsies from their vastus lateralis muscle. PGC-1 and PPAR-α mRNA expression increased by 2.7- and 2.2-fold (P < 0.01), respectively, after endurance training. PGC-1 expression was 2.2- and 6-fold greater in the type IIa than in the type I and IIx fibers, respectively. It increased by 2.8-fold in the type IIa fibers and by 1.5-fold in both the type I and IIx fibers after endurance training (P < 0.015). PPAR-α was 1.9-fold greater in type I than in the II fibers and increased by 3.0-fold and 1.5-fold in these respective fibers after endurance training (P < 0.001). The increases in PGC-1 and PPAR-α levels reported in this study may play an important role in the changes in muscle mitochondria content, oxidative phenotype, and sensitivity to insulin known to be induced by endurance training.

Upregulation of peroxisome proliferator-activated receptor gamma coactivator gene (PGC1A) during weight loss is related to insulin sensitivity but not to energy expenditure

Aims/hypothesis We investigated whether skeletal muscle peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1A; also known as PPARGC1A) and its target mitofusin-2 (MFN2), as well as carnitine palmitoyltransferase-1 (CPT1; also known as carnitine palmitoyltransferase 1A [liver] [CPT1A]) and uncoupling protein (UCP)3, are involved in the improvement of insulin resistance and/or in the modification of energy expenditure during surgically induced massive weight loss.
Materials and methods Seventeen morbidly obese women (mean BMI: 45.9 ± 4 kg/m2) were investigated before, and 3 and 12 months after, Roux-en-Y gastric bypass (RYGB). We evaluated insulin sensitivity by the euglycaemic–hyperinsulinaemic clamp, energy expenditure and substrate oxidation by indirect calorimetry, and muscle mRNA expression by PCR.
Results Post-operatively, PGC1A was enhanced at 3 (p = 0.02) and 12 months (p = 0.03) as was MFN2 (p = 0.008 and p = 0.03 at 3 and 12 months respectively), whereas UCP3 was reduced (p = 0.03) at 12 months. CPT1 did not change. The expression of PGC1A and MFN2 were strongly (p < 0.0001) related. Insulin sensitivity, which increased after surgery (p = 0.002 at 3, p = 0.003 at 12 months), was significantly related to PGC1A and MFN2, but only MFN2 showed an independent influence in a multiple regression analysis. Energy expenditure was reduced at 3 months post-operatively (p = 0.001 vs before RYGB), remaining unchanged thereafter until 12 months. CPT1 and UCP3 were not significantly related to the modifications of energy expenditure or of lipid oxidation rate.
Conclusions/interpretation Weight loss upregulates PGC1A, which in turn stimulates MFN2 expression. MFN2 expression significantly and independently contributes to the improvement of insulin sensitivity. UCP3 and CPT1 do not seem to influence energy expenditure after RYGB.

17beta-estradiol upregulates the expression of peroxisome proliferator-activated receptor alpha and lipid oxidative genes in skeletal muscle

This study examined the actions of 17β-estradiol (E2) and progesterone on the regulation of the peroxisome proliferator-activated receptors (PPARα and PPARγ) family of nuclear transcription factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically,
carnitine palmitoyltransferase I (CPT I), β-3-hydroxyacyl CoA dehydrogenase (β-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague–Dawley rats were ovariectomized and treated with placebo (Ovx), E2, progesterone, or both hormones in combination (E+P). Additionally,
sham-operated rats were treated with placebo (Sham) to serve as controls. Hormone (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E2 treatment increased PPARα mRNA expression and protein content (P<0·05), compared with Ovx treatment. E2 also resulted in upregulated mRNA of CPT I and PDK4 (P<0·05). PPARγ mRNA expression was also increased (P<0·05) by E2 treatment, although protein content remained unaltered. These data
demonstrate the novel regulation of E2 on PPARα and genes encoding key proteins that are pivotal in regulating skeletal muscle lipid oxidative flux.

Peroxisome proliferator-activated receptor-γ coactivator-1 and insulin resistance: acute effect of fatty acids

Aims/hypothesis Peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PPARGC1), a coactivator regulating the transcription of genes involved in oxidative metabolism, is downregulated in patients with type 2 diabetes and in their first-degree relatives. Whether this downregulation is a cause or effect of early aberrations in the development of insulin resistance, such as disturbances in fat metabolism, is unknown. We examined whether lipid-induced insulin resistance was associated with downregulation of expression of skeletal muscle genes involved in oxidative metabolism and mitochondrial biogenesis in humans.
Materials and methods Nine healthy lean male subjects underwent a 6-h hyperinsulinaemic–euglycaemic clamp with simultaneous infusion of either a lipid emulsion or glycerol as a control. Blood was sampled at regular time points and muscle biopsies were taken before and after every test. Intramuscular triacylglycerol (IMTG) content was determined by Oil Red O staining and gene expression was measured by quantitative PCR.
Results Lipid infusion resulted in a ∼2.7-fold increase in plasma NEFA levels and a 31±6% decrease in insulin sensitivity (p=0.001). The infusion of lipids resulted in a ∼1.6-fold increase in IMTG (p=0.02), whereas during the clamp with glycerol infusion IMTG tended to decrease to ∼53% of preinfusion levels (p=0.065). Lipid infusion decreased PPARGC1A, PPARGC1B and PPARA expression to ∼61, 77 and ∼52% of basal values respectively, whereas expression of uncoupling protein 3 was upregulated 1.8-fold (all p<0.05).
Conclusions/interpretation Acute elevation of plasma NEFA levels, leading to muscular fat accumulation and insulin resistance, downregulates PPARGC1A, PPARGC1B and PPARA expression, suggesting that the decrease in PPARGC1 expression observed in the (pre)diabetic state may be the result, rather than the cause of lipid-induced insulin resistance.

Ligand-enhanced expression and in-cell assay of human peroxisome proliferator-activated receptor alpha ligand binding domain

A human peroxisome proliferator-activated receptor alpha ligand binding domain (PPARαLBD)-maltose binding protein fusion construct was expressed in Escherichia coli. A codon optimized DNA sequence encoding human PPARαLBD (aa196–468) was synthesized and ligated into the pDEST17 E. coli expression vector downstream of a MBP solubility fusion tag and an intermittent TEV protease cleavage site. Following auto-induction at 28 °C, PPARαLBD protein was purified to electrophoretic homogeneity by a nickel affinity chromatographic step, on-column TEV protease cleavage followed by Sephacryl S200 size exclusion chromatography. The recombinant protein displayed cross-reactivity with goat anti-(human PPARα) polyclonal antibody and was identified as human PPARα by trypic peptide mass finger-printing. The addition of a PPARα specific ligand (fenofibric acid, GW7647 or GW590735) to the growth media significantly stabilized the PPARαLBD structure and enhanced the expression of soluble protein. In-cell ligand binding was examined by monitoring the enhancement of PPARαLBD expression as a function of the concentration of ligand in the growth media. The efficient expression and in-cell assay of the reported PPARαLBD construct make it amenable to high through-put screening assays in drug discovery programs.

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) gamma Activators and Pan-PPAR Partial Agonists

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Graves ophthalmopathy: low-dose glucocorticoid increases peroxisome proliferator-activated receptor-gamma gene expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)