Genetic Manipulations of PPARs: Effects on Obesity and Metabolic Disease

The interest in genetic manipulations of PPARs is as old as their discovery as receptors of ligands with beneficial clinical activities. Considering the effects of PPAR ligands on critical aspects of systemic physiology, including obesity, lipid metabolism, insulin resistance, and diabetes, gene knockout (KO) in mice is the ideal platform for both hypothesis testing and discovery of new PPAR functions in vivo. With the fervent pursuit of the magic bullet to eradicate the obesity epidemic, special emphasis has been placed on the impacts of PPARs on obesity and its associated diseases. As detailed in this review, understanding how PPARs regulate gene expression and basic metabolic pathways is a necessary intermediate en route to deciphering their effects on obesity. Over a decade and dozens of genetic modifications of PPARs into this effort, valuable lessons have been learned, but we are left with more questions to be answered. These lessons and future prospects are the subject of this review.

Related papers:

title: Hepatic de novo synthesis of glucose-6-phosphate is not affected in PPAR α-deficient mice but is preferentially directed towards hepatic glycogen stores after a short-term fast
abstract: Apart from impaired β-oxidation, Pparα-deficient (Pparα) mice suffer from hypoglycemia during prolonged fasting, suggesting alterations in hepatic glucose metabolism. We compared hepatic glucose metabolism in vivo in wild-type (WT) and Pparα mice after a short-term fast, applying novel isotopic methods. After a 9h fast mice were infused with [U-C] glucose, [2-C]glycerol, [1-H]galactose and paracetamol for 6 h and blood and urine was collected in timed intervals. Plasma glucose concentrations remained constant and were not different between the groups. Hepatic glycogen content was 69 ± 11 and 90 ± 31 μmol/g liver after 15 hr of fasting in WT and Pparα mice, respectively. The gluconeogenic flux towards glucose-6-phosphate was not different between the groups, i.e., 157 ± 9 and 153 ± 9 μmol/kg/min in WT and Pparα mice, respectively. The gluconeogenic flux towards plasma glucose, however, was decreased in Pparα mice, i.e., 142 ± 9 vs 124 ± 13 μmol/kg/min (P < 0.05), accounting for the observed decrease (-15 %) in hepatic glucose production in Pparα mice. Expression of the gene encoding glucose-6-phosphate hydrolase (G6ph) was lower in the Pparα mice compared to WT mice. In conclusion, Pparα mice were able to maintain a normal total gluconeogenic flux to glucose-6-phosphate during moderate fasting, despite their inability to upregulate β-oxidation. However, this gluconeogenic flux was directed more towards glycogen leading to a decreased hepatic glucose output. This was associated with a downregulation of the expression of G6ph in Pparα-deficient mice. Role of PPARα in gluconeogenesis 133

title: Nuclear receptor signaling and cardiac energetics.
abstract: The heart has a tremendous capacity for ATP generation, allowing it to function as an efficient pump throughout the life of the organism. The adult myocardium uses either fatty acid or glucose oxidation as its main energy source. Under normal conditions, the adult heart derives most of its energy through oxidation of fatty acids in mitochondria. However, the myocardium has a remarkable ability to switch between carbohydrate and fat fuel sources so that ATP production is maintained at a constant rate in diverse physiological and dietary conditions. This fuel selection flexibility is important for normal cardiac function. Although cardiac energy conversion capacity and metabolic flux is modulated at many levels, an important mechanism of regulation occurs at the level of gene expression. The expression of genes involved in multiple energy transduction pathways is dynamically regulated in response to developmental, physiological, and pathophysiological cues. This review is focused on gene transcription pathways involved in short- and long-term regulation of myocardial energy metabolism. Much of our knowledge about cardiac metabolic regulation comes from studies focused on mitochondrial fatty acid oxidation. The genes involved in this key energy metabolic pathway are transcriptionally regulated by members of the nuclear receptor superfamily, specifically the fatty acid-activated peroxisome proliferator-activated receptors (PPARs) and the nuclear receptor coactivator, PPARgamma coactivator-1alpha (PGC-1alpha). The dynamic regulation of the cardiac PPAR/PGC-1 complex in accordance with physiological and pathophysiological states will be described.

title: A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders.
abstract: We hypothesized that the lipid-activated transcription factor, the peroxisome proliferator-activated receptor alpha (PPARalpha), plays a pivotal role in the cellular metabolic response to fasting. Short-term starvation caused hepatic steatosis, myocardial lipid accumulation, and hypoglycemia, with an inadequate ketogenic response in adult mice lacking PPARalpha (PPARalpha-/-), a phenotype that bears remarkable similarity to that of humans with genetic defects in mitochondrial fatty acid oxidation enzymes. In PPARalpha+/+ mice, fasting induced the hepatic and cardiac expression of PPARalpha target genes encoding key mitochondrial (medium-chain acyl-CoA dehydrogenase, carnitine palmitoyltransferase I) and extramitochondrial (acyl-CoA oxidase, cytochrome P450 4A3) enzymes. In striking contrast, the hepatic and cardiac expression of most PPARalpha target genes was not induced by fasting in PPARalpha-/- mice. These results define a critical role for PPARalpha in a transcriptional regulatory response to fasting and identify the PPARalpha-/- mouse as a potentially useful murine model of inborn and acquired abnormalities of human fatty acid utilization.

title: Constitutive Regulation of Cardiac Fatty Acid Metabolism through Peroxisome Proliferator-activated Receptor α Associated with Age-dependent Cardiac Toxicity*
abstract: The peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor superfamily and mediates the biological effects of peroxisome proliferators. To determine the physiological role of PPARα in cardiac fatty acid metabolism, we examined the regulation of expression of cardiac fatty acid-metabolizing proteins using PPARα-null mice. The capacity for constitutive myocardial β-oxidation of the medium and long chain fatty acids, octanoic acid and palmitic acid, was markedly reduced in the PPARα-null mice as compared with the wild-type mice, indicating that mitochondrial fatty acid catabolism is impaired in the absence of PPARα. In contrast, constitutive β-oxidation of the very long chain fatty acid, lignoceric acid, did not differ between the mice, suggesting that the constitutive expression of enzymes involved in peroxisomal β-oxidation is independent of PPARα.Indeed, PPARα-null mice had normal levels of the peroxisomal β-oxidation enzymes except the D-type bifunctional protein. At least seven mitochondrial fatty acid-metabolizing enzymes were expressed at much lower levels in the PPARα-null mice, whereas other fatty acid-metabolizing enzymes were present at similar or slightly lower levels in the PPARα-null, as compared with wild-type mice. Additionally, lower constitutive mRNA expression levels of fatty acid transporters were found in the PPARα-null mice, suggesting a role for PPARα in fatty acid transport and catabolism. Indeed, in fatty acid metabolism experiments in vivo, myocardial uptake of iodophenyl 9-methylpentadecanoic acid and its conversion to 3-methylnonanoic acid were reduced in the PPARα-null mice. Interestingly, a decreased ATP concentration after exposure to stress, abnormal cristae of the mitochondria, abnormal caveolae, and fibrosis were observed only in the myocardium of the PPARα-null mice. These cardiac abnormalities appeared to proceed in an age-dependent manner. Taken together, the results presented here indicate that PPARα controls constitutive fatty acid oxidation, thus establishing a role for the receptor in cardiac fatty acid homeostasis. Furthermore, altered expression of fatty acid-metabolizing proteins seems to lead to myocardial damage and fibrosis, as inflammation and abnormal cell growth control can cause these conditions.

title: A gender-related defect in lipid metabolism and glucose homeostasis in peroxisome proliferator- activated receptor alpha- deficient mice.
abstract: The peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor implicated in the control of cellular lipid utilization. To test the hypothesis that PPARalpha is activated as a component of the cellular lipid homeostatic response, the expression of PPARalpha target genes was characterized in response to a perturbation in cellular lipid oxidative flux caused by pharmacologic inhibition of mitochondrial fatty acid import. Inhibition of fatty acid oxidative flux caused a feedback induction of PPARalpha target genes encoding fatty acid oxidation enzymes in liver and heart. In mice lacking PPARalpha (PPARalpha-/-), inhibition of cellular fatty acid flux caused massive hepatic and cardiac lipid accumulation, hypoglycemia, and death in 100% of male, but only 25% of female PPARalpha-/- mice. The metabolic phenotype of male PPARalpha-/- mice was rescued by a 2-wk pretreatment with beta-estradiol. These results demonstrate a pivotal role for PPARalpha in lipid and glucose homeostasis in vivo and implicate estrogen signaling pathways in the regulation of cardiac and hepatic lipid metabolism.

title: The cardiac phenotype induced by PPARalpha overexpression mimics that caused by diabetes mellitus.
abstract: Recent evidence has defined an important role for PPARalpha in the transcriptional control of cardiac energy metabolism. To investigate the role of PPARalpha in the genesis of the metabolic and functional derangements of diabetic cardiomyopathy, mice with cardiac-restricted overexpression of PPARalpha (MHC-PPAR) were produced and characterized. The expression of PPARalpha target genes involved in cardiac fatty acid uptake and oxidation pathways was increased in MHC-PPAR mice. Surprisingly, the expression of genes involved in glucose transport and utilization was reciprocally repressed in MHC-PPAR hearts. Consistent with the gene expression profile, myocardial fatty acid oxidation rates were increased and glucose uptake and oxidation decreased in MHC-PPAR mice, a metabolic phenotype strikingly similar to that of the diabetic heart. MHC-PPAR hearts exhibited signatures of diabetic cardiomyopathy including ventricular hypertrophy, activation of gene markers of pathologic hypertrophic growth, and transgene expression-dependent alteration in systolic ventricular dysfunction. These results demonstrate that (a) PPARalpha is a critical regulator of myocardial fatty acid uptake and utilization, (b) activation of cardiac PPARalpha regulatory pathways results in a reciprocal repression of glucose uptake and utilization pathways, and (c) derangements in myocardial energy metabolism typical of the diabetic heart can become maladaptive, leading to cardiomyopathy.

title: PPAR-α–Null Mice Are Protected From High-Fat Diet–Induced Insulin Resistance
abstract: Peroxisome proliferator–activated receptor (PPAR)-α controls the expression of genes involved in lipid metabolism. PPAR-α furthermore participates to maintain blood glucose during acute metabolic stress, as shown in PPAR-α–null mice, which develop severe hypoglycemia when fasted. Here, we assessed a potential role for PPAR-α in glucose homeostasis in response to long-term high-fat feeding. When subjected to this nutritional challenge, PPAR-α–null mice remained normoglycemic and normoinsulinemic, whereas wild-type mice became hyperinsulinemic (190%; P

title: Dexamethasone induction of hypertension and diabetes is PPAR-α dependent in LDL receptor–null mice
abstract: Hypertension and diabetes are common side effects of glucocorticoid treatment. To determine whether peroxisome proliferator–activated receptor-α (PPAR-α) mediates these sequelae, mice deficient in low-density lipoprotein receptor (Ldlr−/−), with (Ppara+/+) or without (Ppara−/−) PPAR-α, were treated chronically with dexamethasone. Ppara+/+, but not Ppara−/−, mice developed hyperglycemia, hyperinsulinemia and hypertension. Similar effects on glucose metabolism were seen in a different model using C57BL/6 mice. Hepatic gluconeogenic gene expression was increased and insulin-mediated suppression of endogenous glucose production was less effective in dexamethasone-treated Ppara+/+ mice. Adenoviral reconstitution of PPAR-α in the livers of nondiabetic, normotensive, dexamethasone-treated Ppara−/− mice induced hyperglycemia, hyperinsulinemia and increased gluconeogenic gene expression. It also increased blood pressure, renin activity, sympathetic nervous activity and renal sodium retention. Human hepatocytes treated with dexamethasone and the PPAR-α agonist Wy14,643 induced PPARA and gluconeogenic gene expression. These results identify hepatic activation of PPAR-α as a mechanism underlying glucocorticoid-induced insulin resistance.

title: Peroxisome proliferator-activated receptor-alpha deficiency does not alter insulin sensitivity in mice maintained on regular or high-fat diet: hyperinsulinemic-euglycemic clamp studies.
abstract: Chronic peroxisome proliferator-activated receptor (PPAR)-alpha activation improves glucose metabolism in rodent models of insulin resistance and diabetes; however, PPAR-alpha deficiency was also reported to protect against high-fat diet (HFD)-induced insulin resistance. The aim of this study was to clarify the role of PPAR-alpha in the development of insulin resistance using PPAR-alpha knockout (KO) mice and wild-type controls (WT). Both WT and PPAR-alpha KO mice on HFD gained significantly more weight relative to chow-fed groups and displayed an increase in insulin levels and a decrease in adiponectin levels. Hyperinsulinemic-euglycemic clamp performed in the nonfasting state demonstrated that HFD caused a marked reduction in whole body, muscle, and white and brown adipose tissue glucose uptake in both WT and PPAR-alpha KO mice relative to chow-fed groups. Suppression of endogenous glucose production during the clamp was markedly blunted in both WT and PPAR-alpha KO HFD-fed mice, indicating liver insulin resistance. The magnitude of HFD-induced changes in the clamp parameters of insulin sensitivity was comparable in PPAR-alpha KO and WT mice. In conclusion, these data show that PPAR-alpha deficiency does not alter insulin sensitivity in mice fed normal chow diet and does not protect against HFD-induced insulin resistance as measured by hyperinsulinemic-euglycemic clamp in nonfasted state.

title: Deficiency of PPARα disturbs the response of lipogenic flux and of lipogenic and cholesterogenic gene expression to dietary cholesterol in mouse white adipose tissue
abstract: PPARα-deficiency in mice fed a high-carbohydrate, low-cholesterol diet was associated with a decreased weight of epididymal adipose tissue and an increased concentration of adipose tissue cholesterol. Consumption of a high (2% w/w) cholesterol diet resulted in a further increase in the concentration of cholesterol and a further decrease in epididymal fat pad weight in PPARα-null mice, but had no effect in the wild-type. These reductions in fat pad weight were associated with an increase in hepatic triacylglycerol content, indicating that both PPARα-deficiency and cholesterol altered the distribution of triacylglycerol in the body. Adipose tissue de novo lipogenesis was increased in PPARα-null mice and was further enhanced when they were fed a cholesterol-rich diet; no such effect was observed in the wild-type mice. The increased lipogenesis in the chow-fed PPARα-null mice was accompanied paradoxically by lower mRNA expression of SREBP-1c and its target genes, acetyl-CoA carboxylase and fatty acid synthase. Consumption of a high-cholesterol diet increased the mRNA expression of these genes in the PPARα-deficient mice but not in the wild-type. De novo cholesterol synthesis was not detectable in the adipose tissue of either genotype despite a relatively high expression of the mRNA's encoding SREBP-2 and 3-hydroxy-3-methylglutaryl Coenzyme A reductase. The mRNA expression of these genes and of the LDL-receptor in adipose tissue of the PPARα-deficient mice was lower than that of the wild-type and was not downregulated by cholesterol feeding. The results suggest that PPARα plays a role in adipose tissue cholesterol and triacylglycerol homeostasis and prevents cholesterol-mediated changes in de novo lipogenesis.

title: Fatty Acid Homeostasis and Induction of Lipid Regulatory Genes in Skeletal Muscles of Peroxisome Proliferator-activated Receptor (PPAR) α Knock-out Mice
abstract: Ablation of peroxisome proliferator activated receptor (PPAR) α, a lipid-activated transcription factor that regulates expression of β-oxidative genes, results in profound metabolic abnormalities in liver and heart. In the present study we used PPARα knockout (KO) mice to determine whether this transcription factor is essential for regulating fuel metabolism in skeletal muscle. When animals were challenged with exhaustive exercise or starvation, KO mice exhibited lower serum levels of glucose, lactate, and ketones and higher nonesterified fatty acids than wild type (WT) littermates. During exercise, KO mice exhausted earlier than WT and exhibited greater rates of glycogen depletion in liver but not skeletal muscle. Fatty acid oxidative capacity was similar between muscles of WT and KO when animals were fed and only 28% lower in KO muscles when animals were starved. Exercise-induced regulation and starvation-induced regulation of pyruvate-dehydrogenase kinase 4 and uncoupling protein 3, two classical and robustly responsive PPARα target genes, were similar between WT and KO in skeletal muscle but markedly different between genotypes in heart. Real time quantitative PCR analyses showed that unlike in liver and heart, in mouse skeletal muscle PPARδ is severalfold more abundant than either PPARα or PPARγ. In both human and rodent myocytes, the highly selective PPARδ agonist GW742 increased fatty acid oxidation about 2-fold and induced expression of several lipid regulatory genes, including pyruvate-dehydrogenase kinase 4 and uncoupling protein 3, responses that were similar to those elicited by the PPARα agonist GW647. These results show redundancy in the functions of PPARs α and δ as transcriptional regulators of fatty acid homeostasis and suggest that in skeletal muscle high levels of the δ-subtype can compensate for deficiency of PPARα.

title: A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes.
abstract: The role of the peroxisome proliferator-activated receptor-alpha (PPARalpha) in the development of insulin-resistant diabetes was evaluated using gain- and loss-of-function approaches. Transgenic mice overexpressing PPARalpha in muscle (MCK-PPARalpha mice) developed glucose intolerance despite being protected from diet-induced obesity. Conversely, PPARalpha null mice were protected from diet-induced insulin resistance in the context of obesity. In skeletal muscle, MCK-PPARalpha mice exhibited increased fatty acid oxidation rates, diminished AMP-activated protein kinase activity, and reduced insulin-stimulated glucose uptake without alterations in the phosphorylation status of key insulin-signaling proteins. These effects on muscle glucose uptake involved transcriptional repression of the GLUT4 gene. Pharmacologic inhibition of fatty acid oxidation or mitochondrial respiratory coupling prevented the effects of PPARalpha on GLUT4 expression and glucose homeostasis. These results identify PPARalpha-driven alterations in muscle fatty acid oxidation and energetics as a potential link between obesity and the development of glucose intolerance and insulin resistance.

title: Peroxisome Proliferator-Activated Receptor-- Null Mice Have Increased White Adipose Tissue Glucose Utilization , GLUT 4 , and Fat Mass : Role in Liver and Brain
abstract: Unité Mixté de Recherche 5018 (C.K., P.D.C., E.M., M.B., A.W., R.B.), Centre National de la Recherche Scientifique, University Paul Sabatier, 31403 Toulouse, France; Institut de Biologie Animale (J.R., S.K., B.D., S.G., W.W.), Université de Lausanne, CH-1015 Lausanne, Switzerland; Département de Physiologie (J.S.), Faculté de Médecine, Université de Genève, CH-1211 Genève, Switzerland; Institut de Pharmacologie et de Toxicologie (M.F., M.U., M.H., B.T.), Université de Lausanne, CH-1005 Lausanne, Switzerland; and Unit of Pharmacokinetics, Metabolism, Nutrition, and Toxicology (P.D.C., N.M.D.), Université Catholique de Louvain, B-1348 Brussels, Belgium

title: An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor γ (PPARγ) (*)
abstract: Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro, thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however, the molecular basis for this adipogenic effect has remained unclear. Here, we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents, BRL49653, binds to PPARγ with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPARγ is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore, these data raise the intriguing possibility that PPARγ is a target for the therapeutic actions of this class of compounds.

title: 15-Deoxy-Δ 12,14-Prostaglandin J 2 is a ligand for the adipocyte determination factor PPARγ
abstract: Regulation of adipose cell mass is a critical homeostatic process in higher vertebrates. The conversion of fibroblasts into cells of the adipose lineage is induced by expression of the orphan nuclear receptor PPARyγ. This suggests that an endogenous PPARγ ligand may be an important regulator of adipogenesis. By assaying arachidonate metabolites for their capacity to activate PPAR response elements, we have identified 15-deoxy- Δ 12,14 -prostaglandin J 2 as both a PPARγ ligand and an inducer of adipogenesis. Similarly, the thiazolidinedione class of antidiabetic drugs also bind to PPARγ and act as potent regulators of adipocyte development. Thus, adipogenic prostanoids and antidiabetic thiazolidinediones initiate key transcriptional events through a common nuclear receptor signaling pathway. These findings suggest a pivotal role for PPARγ and its endogenous ligand in adipocyte development and glucose homeostasis and as a target for intervention in metabolic disorders.

title: Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor
abstract: Peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) is an adipocyte-specific nuclear hormone receptor that has recently been identified as a key regulator of two fat cell enhancers. Transcriptional activation by PPAR gamma 2 is potentiated by a variety of lipids and lipid-like compounds, including naturally occurring polyunsaturated fatty acids. We demonstrate here that retroviral expression of PPAR gamma 2 stimulates adipose differentiation of cultured fibroblasts. PPAR activators promote the differentiation of PPAR gamma 2-expressing cells in a dose-dependent manner. C/EBP alpha, a second transcription factor induced during adipocyte differentiation, can cooperate with PPAR gamma 2 to stimulate the adipocyte program dramatically. Our results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.

title: PPARγ Is Required for the Differentiation of Adipose Tissue In Vivo and In Vitro
abstract: The process of adipogenesis is known to involve the interplay of several transcription factors. Activation of one of these factors, the nuclear hormone receptor PPARγ, is known to promote fat cell differentiation in vitro. Whether PPARγ is required for this process in vivo has remained an open question because a viable loss-of-function model for PPARγ has been lacking. We demonstrate here that mice chimeric for wild-type and PPARγ null cells show little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPARγ for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPARγ gene dosage. These data provide direct evidence that PPARγ is essential for the formation of fat.

title: Adipose-specific peroxisome proliferator-activated receptor γ knockout causes insulin resistance in fat and liver but not in muscle
abstract: Syndrome X, typified by obesity, insulin resistance (IR), dyslipidemia, and other metabolic abnormalities, is responsive to antidiabetic thiazolidinediones (TZDs). Peroxisome proliferator-activated receptor (PPAR) γ, a target of TZDs, is expressed abundantly in adipocytes, suggesting an important role for this tissue in the etiology and treatment of IR. Targeted deletion of PPARγ in adipose tissue resulted in marked adipocyte hypocellularity and hypertrophy, elevated levels of plasma free fatty acids and triglyceride, and decreased levels of plasma leptin and ACRP30. In addition, increased hepatic glucogenesis and IR were observed. Despite these defects, blood glucose, glucose and insulin tolerance, and insulin-stimulated muscle glucose uptake were all comparable to those of control mice. However, targeted mice were significantly more susceptible to high-fat diet-induced steatosis, hyperinsulinemia, and IR. Surprisingly, TZD treatment effectively reversed liver IR, whereas it failed to lower plasma free fatty acids. These results suggest that syndrome X may be comprised of separable PPARγ-dependent components whose origins and therapeutic sites may reside in distinct tissues.

title: PPARγ and the Treatment of Insulin Resistance
abstract: Numerous studies across several population groups have indicated that insulin resistance plays a central role in the development of type 2 diabetes mellitus (T2DM). Moreover, this disorder is also strongly associated with other metabolic syndromes, including hypertension, dyslipidemias and polycystic ovarian syndrome (PCOS). Recent advances have demonstrated that pharmacological agents of the thiazolidinedione class can reverse insulin resistance and profoundly improve many of these associated symptoms. These effects have been documented in a variety of genetic and acquired animal models of insulin resistance, as well as in numerous clinical trials in patients with insulin resistance. These compounds appear to enhance insulin action by modulating the activity of the nuclear receptor peroxisome proliferator-activated receptor (PPAR) gamma. This activation results in changes in the expression of a number of genes that are critically involved in glucose and lipid metabolism, as well as in insulin signal transduction. While precise events that occur downstream from PPAR gamma modulation remain uncertain, new insights are emerging from knockout studies in mice and the identification of genetic variants in humans. These findings indicate that there is still much to learn about the molecular biology and physiology of these interesting receptors, and that research in this area can lead to more effective and safer drugs to treat insulin resistance and associated syndromes.

title: The potential role of peroxisome proliferator-activated receptors on inflammation in type 2 diabetes mellitus and atherosclerosis.
abstract: Increasing attention has focused on the role of inflammation in various chronic diseases, including atherosclerosis. Recent compelling data have begun to unite work from various arenas, such as epidemiology and vascular biology, and even clinical trials to provide evidence for inflammation as a mechanism underlying cardiovascular disease. Inflammation has been implicated in the pathogenesis, progression, and complications of both atherosclerosis and diabetes mellitus-2 complex disorders often found intertwined in patients. Although this story continues to evolve, peroxisome proliferator-activated receptors (PPARs) have been implicated as a molecular pathway involved in both these disease processes. In vitro data, animal work, and some human studies suggest that synthetic PPAR agonists in clinical use, such as thiazolidinediones, may not only regulate metabolic processes but may also limit inflammatory responses, including some involved in atherosclerosis.

title: Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain
abstract: We have generated a transgenic line that expresses the Cre gene product under the regulation of a 12.5 kb upstream regulatory sequence from the Sox2 gene. Using a R26R reporter line, we show that this transgenic line induces recombination in all epiblast cells by embryonic day (E) 6.5 but little or no activity in other extraembryonic cell types at this time. When crossed to a conditional allele of the Sonic hedgehog gene (Shhc), all Sox2Cre;Shhn/Shhc embryos displayed a phenotype indistinguishable from that of the Shh null mutant. Sox2Cre functioned more efficiently in epiblast-mediated recombination than the Mox2Cre (MORE) transgenic line, which has also been shown to drive Cre-mediated recombination exclusively in the embryonic component of the early mouse embryo. Although most MORE; shhh/shhc embryos have a shh hull phenotype, 33% displayed a milder skeletal phenotype, most likely result of incomplete recombination at egg cylinder stages. In agreement with these findings, Sox2Cre was active earlier and Sox2Cre-mediated recombination was more advanced than MORE-mediated recombination at early gastrulation stages. The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.

title: Improved insulin-sensitivity in mice heterozygous for PPAR-γ deficiency
abstract: The thiazolidinedione class of insulin-sensitizing, antidiabetic drugs interacts with peroxisome proliferator-activated receptor gamma (PPAR-gamma). To gain insight into the role of this nuclear receptor in insulin resistance and diabetes, we conducted metabolic studies in the PPAR-gamma gene knockout mouse model. Because homozygous PPAR-gamma-null mice die in development, we studied glucose metabolism in mice heterozygous for the mutation (PPAR-gamma(+/-) mice). We identified no statistically significant differences in body weight, basal glucose, insulin, or FFA levels between the wild-type (WT) and PPAR-gamma(+/-) groups. Nor was there a difference in glucose excursion between the groups of mice during oral glucose tolerance test, but insulin concentrations of the WT group were greater than those of the PPAR-gamma(+/-) group, and insulin-induced increase in glucose disposal rate was significantly increased in PPAR-gamma(+/-) mice. Likewise, the insulin-induced suppression of hepatic glucose production was significantly greater in the PPAR-gamma(+/-) mice than in the WT mice. Taken together, these results indicate that - counterintuitively - although pharmacological activation of PPAR-gamma improves insulin sensitivity, a similar effect is obtained by genetically reducing the expression levels of the receptor.

title: Effect of heterozygous PPARγ deficiency and TZD treatment on insulin resistance associated with age and high-fat feeding
abstract: 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.

title: Inhibition of RXR and PPARgamma ameliorates diet-induced obesity and type 2 diabetes.
abstract: PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptin's effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

title: The Mechanisms by Which Both Heterozygous Peroxisome Proliferator-activated Receptor γ (PPARγ) Deficiency and PPARγ Agonist Improve Insulin Resistance*
abstract: Peroxisome proliferator-activated receptor (PPAR) γ is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARγ and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARγ by PPARγ agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARγ activity by heterozygous PPARγ deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARγ deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARγ deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor α, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARγ deficiency and PPARγ agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy.

title: Deletion of PPARγ in adipose tissues of mice protects against high fat diet-induced obesity and insulin resistance
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) plays a crucial role in adipocyte differentiation, glucose metabolism, and other physiological processes. To further explore the role of PPARγ in adipose tissues, we used a Cre/loxP strategy to generate adipose-specific PPARγ knockout mice. These animals exhibited marked abnormalities in the formation and function of both brown and white adipose tissues. When fed a high-fat diet, adipose-specific PPARγ knockout mice displayed diminished weight gain despite hyperphagia, had diminished serum concentrations of both leptin and adiponectin, and did not develop glucose intolerance or insulin resistance. Characterization of in vivo glucose dynamics pointed to improved hepatic glucose metabolism as the basis for preventing high-fat diet-induced insulin resistance. Our findings further illustrate the essential role for PPARγ in the development of adipose tissues and suggest that a compensatory induction of hepatic PPARγ may stimulate an increase in glucose disposal by the liver.

title: Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy
abstract: We describe the generation and characterization of the first inducible 'fatless' model system, the FAT-ATTAC mouse (fat apoptosis through targeted activation of caspase 8). This transgenic mouse develops identically to wild-type littermates. Apoptosis of adipocytes can be induced at any developmental stage by administration of a FK1012 analog leading to the dimerization of a membrane-bound, adipocyte-specific caspase 8–FKBP fusion protein. Within 2 weeks of dimerizer administration, FAT-ATTAC mice show near-knockout levels of circulating adipokines and markedly reduced levels of adipose tissue. FAT-ATTAC mice are glucose intolerant, have diminished basal and endotoxin-stimulated systemic inflammation, are less responsive to glucose-stimulated insulin secretion and show increased food intake independent of the effects of leptin. Most importantly, we show that functional adipocytes can be recovered upon cessation of treatment, allowing the study of adipogenesis in vivo, as well as a detailed examination of the importance of the adipocyte in the regulation of multiple physiological functions and pathological states.

title: Muscle-specific PPARgamma-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones.
abstract: Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by thiazolidinediones (TZDs) improves insulin resistance by increasing insulin-stimulated glucose disposal in skeletal muscle. It remains debatable whether the effect of TZDs on muscle is direct or indirect via adipose tissue. We therefore generated mice with muscle-specific PPARgamma knockout (MuPPARgammaKO) using Cre/loxP recombination. Interestingly, MuPPARgammaKO mice developed excess adiposity despite reduced dietary intake. Although insulin-stimulated glucose uptake in muscle was not impaired, MuPPARgammaKO mice had whole-body insulin resistance with a 36% reduction (P < 0.05) in the glucose infusion rate required to maintain euglycemia during hyperinsulinemic clamp, primarily due to dramatic impairment in hepatic insulin action. When placed on a high-fat diet, MuPPARgammaKO mice developed hyperinsulinemia and impaired glucose homeostasis identical to controls. Simultaneous treatment with TZD ameliorated these high fat-induced defects in MuPPARgammaKO mice to a degree identical to controls. There was also altered expression of several lipid metabolism genes in the muscle of MuPPARgammaKO mice. Thus, muscle PPARgamma is not required for the antidiabetic effects of TZDs, but has a hitherto unsuspected role for maintenance of normal adiposity, whole-body insulin sensitivity, and hepatic insulin action. The tissue crosstalk mediating these effects is perhaps due to altered lipid metabolism in muscle.

title: Muscle-specific Pparg deletion causes insulin resistance
abstract: Thiazolidinediones (TZDs) are insulin-sensitizing drugs and are potent agonists of the nuclear peroxisome proliferator-activated receptor-γ (PPAR-γ). Although muscle is the major organ responsible for insulin-stimulated glucose disposal, PPAR-γ is more highly expressed in adipose tissue than in muscle. To address this issue, we used the Cre-loxP system to knock out Pparg, the gene encoding PPAR-γ, in mouse skeletal muscle. As early as 4 months of age, mice with targeted disruption of PPAR-γ in muscle showed glucose intolerance and progressive insulin resistance. Using the hyperinsulinemic-euglycemic clamp technique, the in vivo insulin-stimulated glucose disposal rate (IS-GDR) was reduced by ∼80% and was unchanged by 3 weeks of TZD treatment. These effects reveal a crucial role for muscle PPAR-γ in the maintenance of skeletal muscle insulin action, the etiology of insulin resistance and the action of TZDs.

title: Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes.
abstract: To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.

title: Liver Peroxisome Proliferator-activated Receptor γ Contributes to Hepatic Steatosis, Triglyceride Clearance, and Regulation of Body Fat Mass*
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that mediates the antidiabetic effects of thiazolidinediones. PPARγ 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 PPARγ in both lipoatrophic A-ZIP/F-1 (AZIP) and wild type mice. In AZIP mice, ablation of liver PPARγ reduced the hepatic steatosis but worsened the hyperlipidemia, triglyceride clearance, and muscle insulin resistance. Inactivation of AZIP liver PPARγ 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 PPARγ, suggesting that adipose tissue is the major site of thiazolidinedione action in typical mice with adipose tissue. Interestingly, mice without liver PPARγ, but with adipose tissue, developed relative fat intolerance, increased adiposity, hyperlipidemia, and insulin resistance. Thus, liver PPARγ regulates triglyceride homeostasis, contributing to hepatic steatosis, but protecting other tissues from triglyceride accumulation and insulin resistance.

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

title: PPARγ Promotes Monocyte/Macrophage Differentiation and Uptake of Oxidized LDL
abstract: The formation of foam cells from macrophages in the arterial wall is characterized by dramatic changes in lipid metabolism, including increased expression of scavenger receptors and the uptake of oxidized low-density lipoprotein (oxLDL). We demonstrate here that the nuclear receptor PPARγ is induced in human monocytes following exposure to oxLDL and is expressed at high levels in the foam cells of atherosclerotic lesions. Ligand activation of the PPARγ:RXRα heterodimer in myelomonocytic cell lines induces changes characteristic of monocytic differentiation and promotes uptake of oxLDL through transcriptional induction of the scavenger receptor CD36. These results reveal a novel signaling pathway controlling differentiation and lipid metabolism in monocytic cells, and suggest that endogenous PPARγ ligands may be important regulators of gene expression during atherogenesis.

title: The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation
abstract: The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-γ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) bind to PPAR-γ and stimulate transcription of target genes. Prostaglandin D2metabolites have not yet been identified in adipose tissue, butaremajor products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-γ ligands in this cell type. Here we show that PPAR-γ is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-γ ligands. PPAR-γ inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-κB. These observations suggest that PPAR-γ and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-γ ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

title: Peroxisome proliferator–activated receptor γ ligands inhibit development of atherosclerosis in LDL receptor–deficient mice
abstract: The peroxisome proliferator‐activated receptor γ (PPARγ) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARγ is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARγ-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor‐deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-α and gelatinase B, indicating both systemic and local actions of PPARγ. These findings suggest that PPARγ agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARγ ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.  (2000).

title: PPAR-γ dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation
abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is highly expressed in lipid-accumulating macrophages of the coronary artery. In light of this, the wide-spread clinical use of thiazolidinediones (TZDs) in the treatment of type II diabetes raises concerns about the role of PPAR-γ in macrophage function and disease progression. To define the role of PPAR-γ in macrophage biology, we used homologous recombination to create embryonic stem cells that were homozygous for a null mutation in the PPAR-γ gene. We demonstrate here that PPAR-γ is neither essential for nor substantially affects the development of the macrophage lineage both in vitro and in vivo. In contrast, we show it is an important regulator of the scavenger receptor CD36, which has been genetically linked to lipid accumulation in macrophages. Both 15-deoxy-Δ12,14prostaglandin J2 and thiazolidinediones have anti-inflammatory effects that are independent of PPAR-γ. We show that PPAR-γ is required for positive effects of its ligands in modulating macrophage lipid metabolism, but that inhibitory effects on cytokine production and inflammation may be receptor independent.

title: A PPARγ-LXR-ABCA1 Pathway in Macrophages Is Involved in Cholesterol Efflux and Atherogenesis
abstract: Previous work has implicated PPARγ in the regulation of CD36 expression and macrophage uptake of oxidized LDL (oxLDL). We provide evidence here that in addition to lipid uptake, PPARγ regulates a pathway of cholesterol efflux. PPARγ induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by the nuclear receptor LXRα. Ligand activation of PPARγ leads to primary induction of LXRα and to coupled induction of ABCA1. Transplantation of PPARγ null bone marrow into LDLR −/− mice results in a significant increase in atherosclerosis, consistent with the hypothesis that regulation of LXRα and ABCA1 expression is protective in vivo. Thus, we propose that PPARγ coordinates a complex physiologic response to oxLDL that involves particle uptake, processing, and cholesterol removal through ABCA1.

title: Conditional Knockout of Macrophage PPARγ Increases Atherosclerosis in C57BL/6 and Low-Density Lipoprotein Receptor–Deficient Mice
abstract: Peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in macrophage-derived foam cells of atherosclerotic lesions, and its expression may have a dramatic impact on atherosclerosis. To investigate the contribution of macrophage PPARγ expression on atherogenesis in vivo, we generated macrophage-specific PPARγ knockout (MacPPARγKO) mice. C57BL/6 and low-density lipoprotein (LDL) receptor–deficient (LDLR−/−) mice were reconstituted with MacPPARγKO or wild-type marrow and challenged with an atherogenic diet. No differences were found in serum lipids between recipients reconstituted with MacPPARγKO and wild-type marrow. In contrast, both C57BL/6 and LDLR−/− mice transplanted with MacPPARγKO marrow had significantly larger atherosclerotic lesions than control recipients. In addition, MacPPARγKO→LDLR−/− mice had higher numbers of macrophages in atherosclerotic lesions compared with controls. Peritoneal macrophages isolated from the MacPPARγKO mice had decreased uptake of oxidized but not acetylated LDL and showed no changes in either cholesterol efflux or inflammatory cytokine expression. Macrophages from MacPPARγKO mice had increased levels of migration and CC chemokine receptor 2 (CCR2) expression compared with wild-type macrophages. Thus, macrophage PPARγ deficiency increases atherosclerosis under conditions of mild and severe hypercholesterolemia, indicating an antiatherogenic role for PPARγ, which may be caused, at least in part, by modulation of CCR2 expression and monocyte recruitment.

title: Considerations for management of fluid dynamic issues associated with thiazolidinediones.
abstract: Thiazolidinediones (TZDs) can cause weight gain and fluid retention in some patients. In most cases, fluid retention is expressed as mild hemodilution. The incidence of clinically evident edema is relatively uncommon. In large clinical trials with rosiglitazone and pioglitazone, the frequency of edema in TZD-treated patients was about 3 to 4 times higher than in placebo-treated patients. The precise mechanisms responsible for weight gain, fluid retention, and edema associated with TZD therapy are unclear but appear to be both dose- and host-related. Weight gain is most likely multifactorial, reflecting increased body fat and fluid retention. Available data are conflicting and do not completely support the concept that increased body weight and decreased hemoglobin/hematocrit are linked with evidence of fluid retention and hemodilution. As uncommon as edema is, new-onset heart failure is even less common in patients treated with a TZD. In controlled clinical trials, the frequency of congestive heart failure (CHF) was identical in rosiglitazone- and placebo-treated patients. The incidence of CHF is higher in patients receiving combination therapy with insulin and a TZD. Patients in the insulin-treated population who develop CHF tend to be older, have a longer history of type 2 diabetes mellitus, and have risk factors for heart failure in addition to diabetes. TZDs do not necessarily require discontinuation in patients who develop fluid retention or weight gain. Mild fluid retention can be treated by decreasing the TZD dose and/or adding a diuretic. Patients who are taking a TZD should be monitored for signs and symptoms of CHF, including excessive weight gain, edema, and dyspnea. Patients with New York Heart Association (NYHA) class I or II CHF can be treated with TZDs. Therapy should be initiated at low doses and slowly titrated to the lowest effective dose. If CHF worsens or becomes refractory to treatment, it may be necessary to discontinue the TZD. Diagnoses of NYHA class III and IV CHF were not studied in clinical trials of TZDs, and thus TZDs are not recommended for patients with CHF of this severity.

title: Collecting duct-specific deletion of peroxisome proliferator-activated receptor gamma blocks thiazolidinedione-induced fluid retention.
abstract: The peroxisome proliferator-activated receptor subtype gamma (PPARgamma) ligands, namely the synthetic insulin-sensitizing thiazolidinedione (TZD) compounds, have demonstrated great potential in the treatment of type II diabetes. However, their clinical applicability is limited by a common and serious side effect of edema. To address the mechanism of TZD-induced edema, we generated mice with collecting duct (CD)-specific disruption of the PPARgamma gene. We found that mice with CD knockout of this receptor were resistant to the rosiglitazone- (RGZ) induced increases in body weight and plasma volume expansion found in control mice expressing PPARgamma in the CD. RGZ reduced urinary sodium excretion in control and not in conditional knockout mice. Furthermore, RGZ stimulated sodium transport in primary cultures of CD cells expressing PPARgamma and not in cells lacking this receptor. These findings demonstrate a PPARgamma-dependent pathway in regulation of sodium transport in the CD that underlies TZD-induced fluid retention.

title: Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption
abstract: Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-γ (PPARγ, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct–specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg flox/flox mice. Deletion of collecting duct Pparg decreased renal Na+ avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na+ absorption and Scnn1g mRNA (encoding the epithelial Na+ channel ENaCγ) expression through a PPARγ-dependent pathway. These studies identify Scnn1g as a PPARγ target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.

title: PPARγ in endothelial cells influences high fat diet-induced hypertension
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) ligands improve human hypertension. However, the mechanism and site of this effect remains unknown, confounded by PPARγ expression in many cell types, including endothelial cells (ECs).  To evaluate the vascular role of PPARγ we used a conditional null mouse model. Specific disruption of PPARγ in ECs was created by crossing Tie2-Cre+ transgenic (T2T+) and PPARγ-floxed (fl/fl) mice to generate PPARγ (fl/fl)T2T+ (PPARγ E-null) mice. Conscious 8- to 12-week-old congenic PPARγ (fl/fl)Cre− (wild type) and PPARγ E-null mice were examined for changes in systolic blood pressure (BP) and heart rate (HR), untreated, after 2 months of salt-loading (drinking water), and after treatment for 3 months with high fat (HF) diet alone or supplemented during the last 2 weeks with rosiglitazone (3 mg/kg/d).  Untreated PPARγ E-nulls were phenotypically indistinguishable from wild-type littermates. However, compared to similarly treated wild types, HF-treated PPARγ Ẽ-nulls had significantly elevated systolic BP not seen after normal diet or salt-loading. Despite sex-dependent baseline differences, salt-loaded and HF-treated PPARγ Ẽ-nulls of either sex had significantly elevated HR versus wild types. Interestingly, rosiglitazone improved serum insulin levels, but not HF diet-induced hypertension, in PPARγ Ẽ-null mice.  These results suggest that PPARγ in ECs not only is an important regulator of hypertension and HR under stressed conditions mimicking those arising in type 2 diabetics, but also mediates the antihypertensive effects of rosiglitazone. These data add evidence supporting a beneficial role for PPARγ-specific ligands in the treatment of hypertension, and suggest therapeutic strategies targeting ECs may prove useful.

title: Cardiomyocyte-Specific Knockout and Agonist of Peroxisome Proliferator–Activated Receptor-γ Both Induce Cardiac Hypertrophy in Mice
abstract: Peroxisome proliferator–activated receptor (PPAR)-γ is required for adipogenesis but is also found in the cardiovascular system, where it has been proposed to oppose inflammatory pathways and act as a growth suppressor. PPAR-γ agonists, thiazolidinediones (TZDs), inhibit cardiomyocyte growth in vitro and in pressure overload models. Paradoxically, TZDs also induce cardiac hypertrophy in animal models. To directly determine the role of cardiomyocyte PPAR-γ, we have developed a cardiomyocyte-specific PPAR-γ–knockout (CM-PGKO) mouse model. CM-PGKO mice developed cardiac hypertrophy with preserved systolic cardiac function. Treatment with a TZD, rosiglitazone, induced cardiac hypertrophy in both littermate control mice and CM-PGKO mice and activated distinctly different hypertrophic pathways from CM-PGKO. CM-PGKO mice were found to have increased expression of cardiac embryonic genes (atrial natriuretic peptide and β-myosin heavy chain) and elevated nuclear factor &kgr;B activity in the heart, effects not found by rosiglitazone treatment. Rosiglitazone increased cardiac phosphorylation of p38 mitogen-activated protein kinase independent of PPAR-γ, whereas rosiglitazone induced phosphorylation of extracellular signal–related kinase 1/2 in the heart dependent of PPAR-γ. Phosphorylation of c-Jun N-terminal kinases was not affected by rosiglitazone or CM-PGKO. Surprisingly, despite hypertrophy, Akt phosphorylation was suppressed in CM-PGKO mouse heart. These data show that cardiomyocyte PPAR-γ suppresses cardiac growth and embryonic gene expression and inhibits nuclear factor &kgr;B activity in vivo. Further, rosiglitazone causes cardiac hypertrophy at least partially independent of PPAR-γ in cardiomyocytes and through different mechanisms from CM-PGKO.

title: Differential expression and activation of a family of murine peroxisome proliferator-activated receptors.
abstract: To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in mammals, we have cloned and characterized two PPAR alpha-related cDNAs (designated PPAR gamma and -delta, respectively) from mouse. The three PPAR isoforms display widely divergent patterns of expression during embryogenesis and in the adult. Surprisingly, PPAR gamma and -delta are not activated by pirinixic acid (Wy 14,643), a potent peroxisome proliferator and activator of PPAR alpha. However, PPAR gamma and -delta are activated by the structurally distinct peroxisome proliferator LY-171883 and linoleic acid, respectively, indicating that each of the isoforms can act as a regulated activator of transcription. These data suggest that tissue-specific responsiveness to peroxisome proliferators, including certain fatty acids, is in part a consequence of differential expression of multiple, pharmacologically distinct PPAR isoforms.

title: Selective disruption of PPARγ2 impairs the development of adipose tissue and insulin sensitivity
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that plays a pivotal role in obesity and diabetes. PPARγ has two isoforms, PPARγ1 and PPARγ2. We investigated the functional differences between PPARγ1 and PPARγ2 by selectively disrupting PPARγ2 in mice. In contrast to the embryonic lethality of PPARγ-deficient mice, PPARγ2-/- mice survived. Although normal development was identified in other tissues we examined, PPARγ2-/- mice exhibited an overall reduction in white adipose tissue, less lipid accumulation, and decreased expression of adipogenic genes in adipose tissue. In addition, insulin sensitivity was impaired in male PPARγ2-/- mice, with dramatically decreased expression of insulin receptor substrate 1 and glucose transporter 4 in the skeletal muscle, but thiazolidinediones were able to normalize this insulin resistance. Consistent with in vivo data, PPARγ2-/- mouse embryonic fibroblasts showed a dramatically reduced capacity for adipogenesis in vitro compared with wild-type mouse embryonic fibroblasts. Taken together, our data demonstrate that PPARγ2 deficiency impairs the development of adipose tissue and insulin sensitivity. PPARγ2-/- mice may provide a tool to study the role of PPARγ2 in obesity and diabetes.

title: The link between nutritional status and insulin sensitivity is dependent on the adipocyte-specific peroxisome proliferator-activated receptor-gamma2 isoform.
abstract: The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) is critically required for adipogenesis. PPARgamma exists as two isoforms, gamma1 and gamma2. PPARgamma2 is the more potent adipogenic isoform in vitro and is normally restricted to adipose tissues, where it is regulated more by nutritional state than PPARgamma1. To elucidate the relevance of the PPARgamma2 in vivo, we generated a mouse model in which the PPARgamma2 isoform was specifically disrupted. Despite similar weight, body composition, food intake, energy expenditure, and adipose tissue morphology, male mice lacking the gamma2 isoform were more insulin resistant than wild-type animals when fed a regular diet. These results indicate that insulin resistance associated with ablation of PPARgamma2 is not the result of lipodystrophy and suggests a specific role for PPARgamma2 in maintaining insulin sensitivity independently of its effects on adipogenesis. Furthermore, PPARgamma2 knockout mice fed a high-fat diet did not become more insulin resistant than those on a normal diet, despite a marked increase in their mean adipocyte cell size. These findings suggest that PPARgamma2 is required for the maintenance of normal insulin sensitivity in mice but also raises the intriguing notion that PPARgamma2 may be necessary for the adverse effects of a high-fat diet on carbohydrate metabolism.

title: Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPARγ hypomorphic mice
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor, which controls adipocyte differentiation. We targeted with homologous recombination the PPARγ2-specific exon B, resulting in a white adipose tissue knockdown of PPARγ. Although homozygous (PPARγhyp/hyp) mice are born with similar weight as the WT mice, the PPARγhyp/hyp animals become growth retarded and develop severe lipodystrophy and hyperlipidemia. Almost half of these PPARγhyp/hyp mice die before adulthood, whereas the surviving PPARγhyp/hyp animals overcome the growth retardation, yet remain lipodystrophic. In contrast to most lipodystrophic models, the adult PPARγhyp/hyp mice only have mild glucose intolerance and do not have a fatty liver. These metabolic consequences of the lipodystrophy are relatively benign because of the induction of a compensatory gene expression program in the muscle that enables efficient oxidation of excess lipids. The PPARγhyp/hyp mice unequivocally demonstrate that PPARγ is the master regulator of adipogenesis in vivo and establish that lipid and glucose homeostasis can be relatively well maintained in the absence of white adipose tissue.

title: Dominant negative mutations in human PPARγ associated with severe insulin resistance, diabetes mellitus and hypertension
abstract: Thiazolidinediones are a new class of antidiabetic agent that improve insulin sensitivity and reduce plasma glucose and blood pressure in subjects with type 2 diabetes. Although these agents can bind and activate an orphan nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ), there is no direct evidence to conclusively implicate this receptor in the regulation of mammalian glucose homeostasis. Here we report two different heterozygous mutations in the ligand-binding domain of PPARγ in three subjects with severe insulin resistance. In the PPARγ crystal structure, the mutations destabilize helix 12 which mediates transactivation. Consistent with this, both receptor mutants are markedly transcriptionally impaired and, moreover, are able to inhibit the action of coexpressed wild-type PPARγ in a dominant negative manner. In addition to insulin resistance, all three subjects developed type 2 diabetes mellitus and hypertension at an unusually early age. Our findings represent the first germline loss-of-function mutations in PPARγ and provide compelling genetic evidence that this receptor is important in the control of insulin sensitivity, glucose homeostasis and blood pressure in man.

title: A Novel Heterozygous Mutation in Peroxisome Proliferator-Activated Receptor-γ Gene in a Patient with Familial Partial Lipodystrophy
abstract: Familial partial lipodystrophies (FPL) are a heterogeneous group of genetic disorders characterized by marked loss of subcutaneous (sc) fat from the extremities. Affected individuals show an increased preponderance of insulin resistance, diabetes mellitus and dyslipidemia. Recently, lamin A/C gene mutations were found in patients with FPL, Dunnigan variety. However, the genetic basis of other phenotypes remains unknown. We studied peroxisome proliferator-activated receptor-gamma (PPARgamma) gene as a candidate gene in seven FPL patients who did not appear to have Dunnigan variety. Analysis of the coding region of PPARG revealed C to T heterozygous mutation at nucleotide 1273 in exon 6 which changes a highly conserved residue, arginine at position 425 to cysteine (R425C) in the patient FX200.21. The patient is a 64-year-old nonHispanic white woman who developed diabetes mellitus and hypertriglyceridemia at age 32 years and lipodystrophy of the extremities and face at age 50 years. She also had hirsutism. Anthropometry and whole body magnetic resonance imaging revealed marked loss of sc fat particularly from the extremities but sc truncal fat was slightly increased. None of the four unaffected family members harbored the mutation. We conclude that heterozygous, R425C, mutation in PPARG could be the molecular basis for one of the familial partial lipodystrophy phenotypes.

title: PPARG F388L, a transactivation-deficient mutant, in familial partial lipodystrophy.
abstract: Autosomal dominant familial partial lipodystrophy (FPLD) due to mutant LMNA encoding nuclear lamin A/C is characterized by adipose tissue repartitioning together with multiple metabolic disturbances, including insulin resistance and dyslipidemia. There is emerging evidence that some rare mutations in peroxisome proliferator-activated receptor-gamma (PPAR-gamma), encoded by PPARG, might be associated with human lipodystrophy. We report a three-generation Canadian kindred ascertained based upon partial lipodystrophy, with a normal LMNA gene sequence. Candidate gene sequencing showed that all four affected subjects were heterozygous for a novel T-->A mutation at PPARG nucleotide 1164 in exon 5 that predicted substitution of phenylalanine at codon 388 by leucine (F388L). The mutation was absent from normal family members and normal unrelated subjects, and altered a highly conserved residue within helix 8 of the predicted ligand-binding pocket of PPAR-gamma. The mutant receptor had significantly decreased basal transcriptional activity and impaired stimulation by a synthetic ligand. The germline transmission of a transactivation-deficient mutation in PPARG suggests that autosomal dominant partial lipodystrophy is genetically heterogeneous. Our findings are consistent with the idea that mutant PPARG can underlie the partial lipodystrophy phenotype.

title: Digenic inheritance of severe insulin resistance in a human pedigree
abstract: Impaired insulin action is a key feature of type 2 diabetes and is also found, to a more extreme degree, in familial syndromes of insulin resistance. Although inherited susceptibility to insulin resistance may involve the interplay of several genetic loci, no clear examples of interactions among genes have yet been reported. Here we describe a family in which five individuals with severe insulin resistance, but no unaffected family members, were doubly heterozygous with respect to frameshift/premature stop mutations in two unlinked genes, PPARG and PPP1R3A these encode peroxisome proliferator activated receptor γ, which is highly expressed in adipocytes, and protein phosphatase 1, regulatory subunit 3, the muscle-specific regulatory subunit of protein phosphatase 1, which are centrally involved in the regulation of carbohydrate and lipid metabolism, respectively. That mutant molecules primarily involved in either carbohydrate or lipid metabolism can combine to produce a phenotype of extreme insulin resistance provides a model of interactions among genes that may underlie common human metabolic disorders such as type 2 diabetes.

title: The metabolic syndrome: peroxisome proliferator-activated receptor gamma and its therapeutic modulation.
abstract: By the end of this decade, it has been estimated that between 200 million and 300 million people worldwide will meet World Health Organization diagnostic criteria for diabetes mellitus. This epidemic of predominantly type 2 diabetes has largely been mediated by our shift toward a more sedentary lifestyle predisposing to obesity and insulin resistance. Affected individuals can also exhibit an array of associated undesirable traits such as hypertension, dyslipidemia, and hypercoagulability, leading to morbidity and mortality from atherosclerotic vascular disease. The coexistence of several of these traits with insulin resistance constitutes the metabolic syndrome. Accordingly, improving insulin sensitivity in this group, and thereby potentially ameliorating the excess vascular risk, is a primary goal of treatment. Recent interest has focused on the thiazolidinediones, a novel class of antidiabetic agents, which act as insulin sensitizers and, therefore, potentially target the underlying metabolic disturbance. These agents are high-affinity ligands for the nuclear receptor peroxisome proliferator-activated receptor gamma, and a large body of in vitro and in vivo data has evolved to support their increasing clinical use. Importantly, clinical and laboratory findings in human subjects harboring natural mutations and polymorphisms within the receptor have provided additional insights. Here, we focus on the consequences of inherited variation in the human peroxisome proliferator-activated receptor gamma gene, linking this receptor to disordered glucose homeostasis, adipogenesis, lipid metabolism, and blood pressure regulation. These studies provide further support for the future development of more selective receptor modulators, targeting specific pathways to ameliorate facets of the metabolic syndrome.

title: Hypertension and abnormal fat distribution but not insulin resistance in mice with P465L PPARgamma.
abstract: Peroxisome proliferator-activated receptor gamma (PPARgamma), the molecular target of a class of insulin sensitizers, regulates adipocyte differentiation and lipid metabolism. A dominant negative P467L mutation in the ligand-binding domain of PPARgamma in humans is associated with severe insulin resistance and hypertension. Homozygous mice with the equivalent P465L mutation die in utero. Heterozygous mice grow normally and have normal total adipose tissue weight. However, they have reduced interscapular brown adipose tissue and intra-abdominal fat mass, and increased extra-abdominal subcutaneous fat, compared with wild-type mice. They have normal plasma glucose levels and insulin sensitivity, and increased glucose tolerance. However, during high-fat feeding, their plasma insulin levels are mildly elevated in association with a significant increase in pancreatic islet mass. They are hypertensive, and expression of the angiotensinogen gene is increased in their subcutaneous adipose tissues. The effects of P465L on blood pressure, fat distribution, and insulin sensitivity are the same in both male and female mice regardless of diet and age. Thus the P465L mutation alone is sufficient to cause abnormal fat distribution and hypertension but not insulin resistance in mice. These results provide genetic evidence for a critical role for PPARgamma in blood pressure regulation that is not dependent on altered insulin sensitivity.

title: A Dominant Negative Peroxisome Proliferator-activated Receptor-γ Knock-in Mouse Exhibits Features of the Metabolic Syndrome*
abstract: Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear hormone receptor family, is a master regulator of adipogenesis. Humans with dominant negative PPARγ mutations have features of the metabolic syndrome (severe insulin resistance, dyslipidemia, and hypertension). We created a knock-in mouse model containing a potent dominant negative PPARγ L466A mutation, shown previously to inhibit wild-type PPARγ action in vitro. Homozygous PPARγ L466A knock-in mice die in utero. Heterozygous PPARγ L466A knock-in (PPARKI) mice exhibit hypoplastic adipocytes, hypoadiponectinemia, increased serum-free fatty acids, and hepatic steatosis. When subjected to high fat diet feeding, PPARKI mice gain significantly less weight than controls. Hyperinsulinemic-euglycemic clamp studies in PPARKI mice revealed insulin resistance and reduced glucose uptake into skeletal muscle. Female PPARKI mice exhibit hypertension independent of diet. The PPARKI mouse provides a novel model for studying the relationship between impaired PPARγ function and the metabolic syndrome.

title: Obesity associated with a mutation in a genetic regulator of adipocyte differentiation.
abstract: There is increasing evidence of genetic factors leading to obesity, but the exact genes involved have not been defined. Peroxisome-proliferator-activated receptor gamma2 (PPARgamma2) is a transcription factor that has a key role in adipocyte differentiation, and therefore mutations of the gene for this factor might predispose people to obesity.



We studied 358 unrelated German subjects, including 121 obese subjects (defined as those with a body-mass index [the weight in kilograms divided by the square of the height in meters] of more than 29). We evaluated these subjects for mutations in the gene for PPARgamma2 at or near a site of serine phosphorylation at position 114 that negatively regulates the transcriptional activity of the protein, using a polymerase-chain-reaction-based assay coupled with specific endonuclease digestion. The activity of the mutation identified was analyzed by retroviral transfection and overexpression in murine fibroblasts.



Four of the 121 obese subjects had a missense mutation in the gene for PPARgamma2 that resulted in the conversion of proline to glutamine at position 115, as compared with none of the 237 subjects of normal weight (P=0.01). All the subjects with the mutant allele were markedly obese, with body-mass-index values ranging from 37.9 to 47.3, as compared with a mean of 33.6 in the other obese subjects. Overexpression of the mutant gene in murine fibroblasts led to the production of a protein in which the phosphorylation of serine at position 114 was defective, as well as to accelerated differentiation of the cells into adipocytes and greater cellular accumulation of triglyceride than with the wild-type PPARgamma2. These effects were similar to those of an in vitro mutation created directly at the Ser114 phosphorylation site.



A Pro115Gln mutation in PPARgamma2 accelerates the differentiation of adipocytes and may cause obesity.

title: A Pro12Ala substitution in PPARγ2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity
abstract: The peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor that has a pivotal role in adipocyte differentiation and expression of adipocyte-specific genes. The PPARγ1 and γ2 isoforms result from alternative splicing and have ligand-dependent and -independent activation domains. PPARγ2 has an additional 28 amino acids at its amino terminus that renders its ligand-independent activation domain 5-10-fold more effective than that of PPARγ1. Insulin stimulates the ligand-independent activation of PPARγ1 and γ2 (ref. 5), however, obesity and nutritional factors only influence the expression of PPARγ2 in human adipocytes. Here, we report that a relatively common Pro12Ala substitution in PPARγ2 is associated with lower body mass index (BMI; P=0.027; 0.015) and improved insulin sensitivity among middle-aged and elderly Finns. A significant odds ratio (4.35, P=0.028) for the association of the Pro/Pro genotype with type 2 diabetes was observed among Japanese Americans. The PPARγ2 Ala allele showed decreased binding affinity to the cognate promoter element and reduced ability to transactivate responsive promoters. These findings suggest that the PPARγ2 Pro12Ala variant may contribute to the observed variability in BMI and insulin sensitivity in the general population.

title: The common PPARγ Pro12Ala polymorphism is associated with decreased risk of type 2 diabetes
abstract: Genetic association studies are viewed as problematic and plagued by irreproducibility. Many associations have been reported for type 2 diabetes, but none have been confirmed in multiple samples and with comprehensive controls. We evaluated 16 published genetic associations to type 2 diabetes and related sub-phenotypes using a family-based design to control for population stratification, and replication samples to increase power. We were able to confirm only one association, that of the common Pro12Ala polymorphism in peroxisome proliferator-activated receptor-γ (PPARγ) with type 2 diabetes. By analysing over 3,000 individuals, we found a modest (1.25-fold) but significant (P=0.002) increase in diabetes risk associated with the more common proline allele (∼85% frequency). Moreover, our results resolve a controversy about common variation in PPARγ. An initial study found a threefold effect, but four of five subsequent publications failed to confirm the association. All six studies are consistent with the odds ratio we describe. The data implicate inherited variation in PPARγ in the pathogenesis of type 2 diabetes. Because the risk allele occurs at such high frequency, its modest effect translates into a large population attributable risk—influencing as much as 25% of type 2 diabetes in the general population.

title: Genetic Modulation of PPARγ Phosphorylation Regulates Insulin Sensitivity
abstract: Obesity-associated diabetes is epidemic in industrialized societies. The nuclear receptor p eroxisome p roliferator- a ctivated r eceptor γ (PPARγ) is highly expressed in adipose tissue and the presumed molecular target for antidiabetic thiazolidinedione drugs that reverse insulin resistance but also promote weight gain. Phosphorylation reduces the activity of PPARγ in vitro, but physiological relevance has not been demonstrated. We have studied mice homozygous for a mutation (S112A) that prevents PPARγ phosphorylation. Surprisingly, the weights and adipose mass of PPARγ-S112A mice are not greater than wild-type. Remarkably, however, genetic prevention of PPARγ phosphorylation preserves insulin sensitivity in the setting of diet-induced obesity. Underlying this protection are smaller fat cells, elevated serum adiponectin, and reduced free fatty acid levels. Thus, the phosphorylation state of PPARγ modulates insulin sensitivity. Compounds that prevent PPARγ phosphorylation or ligands that induce the conformation of nonphosphorylated PPARγ may selectively enhance insulin sensitivity without increasing body weight.

title: Effects of peroxisome proliferator-activated receptor δ on placentation, adiposity, and colorectal cancer
abstract: Targeting of the nuclear prostaglandin receptor peroxisome proliferator-activated receptor δ (PPARδ) by homologous recombination results in placental defects and frequent (>90%) midgestation lethality. Surviving PPARδ−/− mice exhibit a striking reduction in adiposity relative to wild-type levels. This effect is not reproduced in mice harboring an adipose tissue-specific deletion of PPARδ, and thus likely reflects peripheral PPARδ functions in systemic lipid metabolism. Finally, we observe that PPARδ is dispensable for polyp formation in the intestine and colon of APCmin mice, inconsistent with its recently proposed role in the establishment of colorectal tumors. Together, these observations reveal specific roles for PPARδ in embryo development and adipocyte physiology, but not cancer.

title: Differentiation of Trophoblast Giant Cells and Their Metabolic Functions Are Dependent on Peroxisome Proliferator-Activated Receptor β/δ
abstract: Mutation of the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) severely affects placenta development, leading to embryonic death at embryonic day 9.5 (E9.5) to E10.5 of most, but not all, PPARβ/δ-null mutant embryos. While very little is known at present about the pathway governed by PPARβ/δ in the developing placenta, this paper demonstrates that the main alteration of the placenta of PPARβ/δ-null embryos is found in the giant cell layer. PPARβ/δ activity is in fact essential for the differentiation of the Rcho-1 cells in giant cells, as shown by the severe inhibition of differentiation once PPARβ/δ is silenced. Conversely, exposure of Rcho-1 cells to a PPARβ/δ agonist triggers a massive differentiation via increased expression of 3-phosphoinositide-dependent kinase 1 and integrin-linked kinase and subsequent phosphorylation of Akt. The links between PPARβ/δ activity in giant cells and its role on Akt activity are further strengthened by the remarkable pattern of phospho-Akt expression in vivo at E9.5, specifically in the nucleus of the giant cells. In addition to this phosphatidylinositol 3-kinase/Akt main pathway, PPARβ/δ also induced giant cell differentiation via increased expression of I-mfa, an inhibitor of Mash-2 activity. Finally, giant cell differentiation at E9.5 is accompanied by a PPARβ/δ-dependent accumulation of lipid droplets and an increased expression of the adipose differentiation-related protein (also called adipophilin), which may participate to lipid metabolism and/or steroidogenesis. Altogether, this important role of PPARβ/δ in placenta development and giant cell differentiation should be considered when contemplating the potency of PPARβ/δ agonist as therapeutic agents of broad application.

title: PGC1α expression is controlled in skeletal muscles by PPARβ, whose ablation results in fiber-type switching, obesity, and type 2 diabetes
abstract: Mice in which peroxisome proliferator-activated receptor beta (PPARbeta) is selectively ablated in skeletal muscle myocytes were generated to elucidate the role played by PPARbeta signaling in these myocytes. These somatic mutant mice exhibited a muscle fiber-type switching toward lower oxidative capacity that preceded the development of obesity and diabetes, thus demonstrating that PPARbeta is instrumental in myocytes to the maintenance of oxidative fibers and that fiber-type switching is likely to be the cause and not the consequence of these metabolic disorders. We also show that PPARbeta stimulates in myocytes the expression of PGC1alpha, a coactivator of various transcription factors, known to play an important role in slow muscle fiber formation. Moreover, as the PGC1alpha promoter contains a PPAR response element, the effect of PPARbeta on the formation and/or maintenance of slow muscle fibers can be ascribed, at least in part, to a stimulation of PGC1alpha expression at the transcriptional level.

title: Peroxisome Proliferator-activated Receptor β/δ Regulates Very Low Density Lipoprotein Production and Catabolism in Mice on a Western Diet*
abstract: The results of recent studies using selective agonists for peroxisome proliferator-activated receptor β (PPARβ) suggest that this receptor may have a role in regulating levels of serum lipids in animal models of obesity and insulin resistance. To further examine this possibility, serum lipid profiles of mice lacking a functional PPARβ receptor were determined. PPARβ-null mice maintained on either normal chow or a 10-week high fat (HF) diet, a condition that has been shown to induce insulin resistance and obesity in mice, have elevated levels of serum triglycerides primarily associated with very low density lipoprotein (VLDL) with no difference in either total cholesterol or phospholipids. Consistent with this finding, PPARβ-null mice on a HF-diet were shown to have an increased rate of hepatic VLDL production as well as lowered lipoprotein lipase activity in serum compared with wild-type controls. The latter parallels an increase in the hepatic expression of the genes encoding angiopoietin-like proteins 3 and 4 in PPARβ-null mice on a HF diet, both proteins of which have recently been shown to inhibit lipoprotein lipase (LPL) activity in vivo. Consistent with elevated VLDL production, a marked increase in plasma VLDL apoB48, -E, -AI, and -AII, as well as a sharp depletion of the hepatic lipid stores was also found in PPARβ-null mice. In addition, PPARβ-null mice on a HF diet were shown to have increased adiposity, despite lower total body weight. Together, these results indicate a clear role for PPARβ in regulating levels of serum triglycerides in mice on a high fat Western diet by modulating both VLDL production and LPL-mediated catabolism of VLDL-triglycerides and also suggest a potential therapeutic role for PPARβ in the improvement of serum lipids in the setting of metabolic syndrome.

title: Peroxisome-Proliferator-Activated Receptor δ Activates Fat Metabolism to Prevent Obesity
abstract: In contrast to the well-established roles of PPARγ and PPARα in lipid metabolism, little is known for PPARδ in this process. We show here that targeted activation of PPARδ in adipose tissue specifically induces expression of genes required for fatty acid oxidation and energy dissipation, which in turn leads to improved lipid profiles and reduced adiposity. Importantly, these animals are completely resistant to both high-fat diet-induced and genetically predisposed ( Lepr db/db ) obesity. As predicted, acute treatment of Lepr db/db mice with a PPARδ agonist depletes lipid accumulation. In parallel, PPARδ-deficient mice challenged with high-fat diet show reduced energy uncoupling and are prone to obesity. In vitro, activation of PPARδ in adipocytes and skeletal muscle cells promotes fatty acid oxidation and utilization. Our findings suggest that PPARδ serves as a widespread regulator of fat burning and identify PPARδ as a potential target in treatment of obesity and its associated disorders.

title: Regulation of Muscle Fiber Type and Running Endurance by PPARδ
abstract: Endurance exercise training can promote an adaptive muscle fiber transformation and an increase of mitochondrial biogenesis by triggering scripted changes in gene expression. However, no transcription factor has yet been identified that can direct this process. We describe the engineering of a mouse capable of continuous running of up to twice the distance of a wild-type littermate. This was achieved by targeted expression of an activated form of peroxisome proliferator-activated receptor δ (PPARδ) in skeletal muscle, which induces a switch to form increased numbers of type I muscle fibers. Treatment of wild-type mice with PPARδ agonist elicits a similar type I fiber gene expression profile in muscle. Moreover, these genetically generated fibers confer resistance to obesity with improved metabolic profiles, even in the absence of exercise. These results demonstrate that complex physiologic properties such as fatigue, endurance, and running capacity can be molecularly analyzed and manipulated.

title: Peroxisome proliferator‐activated receptor δ controls muscle development and oxydative capability
abstract: Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors exerting several functions in development and metabolism. The physiological functions of PPARδ remain elusive. By using a CRE‐Lox recombination approach, we generated an animal model for muscle‐specific PPARδ overexpression to investigate the role of PPARδ in this tissue. Muscle‐ specific PPARδ overexpression results in a profound change in fiber composition due to hyperplasia and/or shift to more oxidative fiber and, as a consequence, leads to the increase of both enzymatic activities and genes implicated in oxidative metabolism. These changes in muscle are accompanied by a reduction of body fat mass, mainly due to a large reduction of adipose cell size. Furthermore, we demonstrate that endurance exercise promotes an accumulation of PPARδ protein in muscle of wild‐type animals. Collectively, these results suggest that PPARδ plays an important role in muscle development and adaptive response to environmental changes, such as training exercise. They strongly support the idea that activation of PPARδ could be beneficial in prevention of metabolic disorders, such as obesity or type 2 diabetes.

title: PPARδ regulates glucose metabolism and insulin sensitivity
abstract: The metabolic syndrome is a collection of obesity-related disorders. The peroxisome proliferator-activated receptors (PPARs) regulate transcription in response to fatty acids and, as such, are potential therapeutic targets for these diseases. We show that PPARδ (NR1C2) knockout mice are metabolically less active and glucose-intolerant, whereas receptor activation in db/db mice improves insulin sensitivity. Euglycemic–hyperinsulinemic-clamp experiments further demonstrate that a PPARδ-specific agonist suppresses hepatic glucose output, increases glucose disposal, and inhibits free fatty acid release from adipocytes. Unexpectedly, gene array and functional analyses suggest that PPARδ ameliorates hyperglycemia by increasing glucose flux through the pentose phosphate pathway and enhancing fatty acid synthesis. Coupling increased hepatic carbohydrate catabolism with its ability to promote β-oxidation in muscle allows PPARδ to regulate metabolic homeostasis and enhance insulin action by complementary effects in distinct tissues. The combined hepatic and peripheral actions of PPARδ suggest new therapeutic approaches to treat type II diabetes.

title: Cardiomyocyte-restricted peroxisome proliferator-activated receptor-δ deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy
abstract: Fatty acid oxidation (FAO) is a primary energy source for meeting the heart's energy requirements. Peroxisome proliferator-activated receptor-δ (PPAR-δ) may have important roles in FAO. But it remains unclear whether PPAR-δ is required for maintaining basal myocardial FAO. We show that cre-loxP-mediated cardiomyocyte-restricted deletion of PPAR-δ in mice downregulates constitutive expression of key FAO genes and decreases basal myocardial FAO. These mice have cardiac dysfunction, progressive myocardial lipid accumulation, cardiac hypertrophy and congestive heart failure with reduced survival. Thus, chronic myocardial PPAR-δ deficiency leads to lipotoxic cardiomyopathy. Together, our data show that PPAR-δ is a crucial determinant of constitutive myocardial FAO and is necessary to maintain energy balance and normal cardiac function. We suggest that PPAR-δ is a potential therapeutic target in treating lipotoxic cardiomyopathy and other heart diseases.

title: Transcriptional Repression of Atherogenic Inflammation: Modulation by PPARδ
abstract: The formation of an atherosclerotic lesion is mediated by lipid-laden macrophages (foam cells), which also establish chronic inflammation associated with lesion progression. The peroxisome proliferator-activated receptor (PPAR) γ promotes lipid uptake and efflux in these atherogenic cells. In contrast, we found that the closely related receptor PPARδ controls the inflammatory status of the macrophage. Deletion of PPARδ from foam cells increased the availability of inflammatory suppressors, which in turn reduced atherosclerotic lesion area by more than 50%. We propose an unconventional ligand-dependent transcriptional pathway in which PPARδ controls an inflammatory switch through its association and disassociation with transcriptional repressors. PPARδ and its ligands may thus serve as therapeutic targets to attenuate inflammation and slow the progression of atherosclerosis.