Yaacov Barak

PPARγ Is Required for Placental, Cardiac, and Adipose Tissue Development

The nuclear hormone receptor PPAR gamma promotes adipogenesis and macrophage differentiation and is a primary pharmacological target in the treatment of type II diabetes. Here, we show that PPAR gamma gene knockout results in two independent lethal phases. Initially, PPAR gamma deficiency interferes with terminal differentiation of the trophoblast and placental vascularization, leading to severe myocardial thinning and death by E10.0. Supplementing PPAR gamma null embryos with wild-type placentas via aggregation with tetraploid embryos corrects the cardiac defect, implicating a previously unrecognized dependence of the developing heart on a functional placenta. A tetraploid-rescued mutant surviving to term exhibited another lethal combination of pathologies, including lipodystrophy and multiple hemorrhages. These findings both confirm and expand the current known spectrum of physiological functions regulated by PPAR gamma.

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.

PPAR-|[gamma]| dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation

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.

Adipose-specific peroxisome proliferator-activated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle.

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.

Effects of peroxisome proliferator-activated receptor δ on placentation, adiposity, and colorectal cancer

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.

Muscle-specific Pparg deletion causes insulin resistance

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.

Improved insulin-sensitivity in mice heterozygous for PPAR-γ deficiency

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.

PPARδ is a very low-density lipoprotein sensor in macrophages

Although triglyceride-rich particles, such as very low-density lipoprotein (VLDL), contribute significantly to human atherogenesis, the molecular basis for lipoprotein-driven pathogenicity is poorly understood. We demonstrate that in macrophages, VLDL functions as a transcriptional regulator via the activation of the nuclear receptor peroxisome proliferator-activated receptor δ. The signaling components of native VLDL are its triglycerides, whose activity is enhanced by lipoprotein lipase. Generation of peroxisome proliferator-activated receptor δ null macrophages verifies the absolute requirement of this transcription factor in mediating the VLDL response. Thus, our data reveal a pathway through which dietary triglycerides and VLDL can directly regulate gene expression in atherosclerotic lesions.

STAT6 transcription factor is a facilitator of the nuclear receptor PPARγ-regulated gene expression in macrophages and dendritic cells.

Summary Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts

Peroxisome proliferator‐activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ over‐expression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre‐loxP technology to generate hepatocyte (PPARγΔhep)‐ and macrophage (PPARγΔmac)‐specific PPARγ‐knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high‐fat diet‐induced hepatic steatosis. Down‐regulated expression of genes involved in lipogenesis (SCD1, SREBP‐1c, and ACC), lipid transport (CD36/FAT, L‐FABP, and MTP), and β‐oxidation (PPARα and ACO) was observed in PPARγΔhep mice. Moreover, PPARγΔhep mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision‐cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγΔhep mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγΔmac mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.—Morán‐Salvador, E., López‐Parra, M., García‐Alonso, V., Titos, E., Martínez‐Clemente, M., González‐Périz, A., López‐Vicario, C., Barak, Y., Arroyo, V., Clària, J. Role for PPARγ in obesity‐induced hepatic steatosis as determined by hepatocyte‐ and macrophage‐specific conditional knockouts. FASEB J. 25, 2538–2550 (2011). www.fasebj.org