PPAR? signaling and emerging opportunities for improved therapeutics.
ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPAR?) is a ligand-activated nuclear receptor that regulates glucose and lipid metabolism, endothelial function and inflammation. Rosiglitazone (RGZ) and other thiazolidinedione (TZD) synthetic ligands of PPAR? are insulin sensitizers that have been used for the treatment of type 2 diabetes. However, undesirable side effects including weight gain, fluid retention, bone loss, congestive heart failure, and a possible increased risk of myocardial infarction and bladder cancer, have limited the use of TZDs. Therefore, there is a need to better understand PPAR? signaling and to develop safer and more effective PPAR?-directed therapeutics. In addition to PPAR? itself, many PPAR? ligands including TZDs bind to and activate G protein-coupled receptor 40 (GPR40), also known as free fatty acid receptor 1. GPR40 signaling activates stress kinase pathways that ultimately regulate downstream PPAR? responses. Recent studies in human endothelial cells have demonstrated that RGZ activation of GPR40 is essential to the optimal propagation of PPAR? genomic signaling. RGZ/GPR40/p38 MAPK signaling induces and activates PPAR? co-activator-1?, and recruits E1A binding protein p300 to the promoters of target genes, markedly enhancing PPAR?-dependent transcription. Therefore in endothelium, GPR40 and PPAR? function as an integrated signaling pathway. However, GPR40 can also activate ERK1/2, a proinflammatory kinase that directly phosphorylates and inactivates PPAR?. Thus the role of GPR40 in PPAR? signaling may have important implications for drug development. Ligands that strongly activate PPAR?, but do not bind to or activate GPR40 may be safer than currently approved PPAR? agonists. Alternatively, biased GPR40 agonists might be sought that activate both p38 MAPK and PPAR?, but not ERK1/2, avoiding its harmful effects on PPAR? signaling, insulin resistance and inflammation. Such next generation drugs might be useful in treating not only type 2 diabetes, but also diverse chronic and acute forms of vascular inflammation such as atherosclerosis and septic shock.
Project description:BACKGROUND:It has been reported that peroxisome proliferator-activated receptor (PPAR)-? and their synthetic ligands have direct effects on pancreatic ?-cells. We investigated whether PPAR-? activation stimulates insulin secretion through the up-regulation of GPR40 in pancreatic ?-cells. METHODS:Rat insulinoma INS-1 cells and primary rat islets were treated with rosiglitazone (RGZ) and/or adenoviral PPAR-? overexpression. OLETF rats were treated with RGZ. RESULTS:PPAR-? activation with RGZ and/or adenoviral PPAR-? overexpression increased free fatty acid (FFA) receptor GPR40 expression, and increased insulin secretion and intracellular calcium mobilization, and was blocked by the PLC inhibitors, GPR40 RNA interference, and GLUT2 RNA interference. As a downstream signaling pathway of intracellular calcium mobilization, the phosphorylated levels of CaMKII and CREB, and the downstream IRS-2 and phospho-Akt were significantly increased. Despite of insulin receptor RNA interference, the levels of IRS-2 and phospho-Akt was still maintained with PPAR-? activation. In addition, the ?-cell specific gene expression, including Pdx-1 and FoxA2, increased in a GPR40- and GLUT2-dependent manner. The levels of GPR40, phosphorylated CaMKII and CREB, and ?-cell specific genes induced by RGZ were blocked by GW9662, a PPAR-? antagonist. Finally, PPAR-? activation up-regulated ?-cell gene expressions through FoxO1 nuclear exclusion, independent of the insulin signaling pathway. Based on immunohistochemical staining, the GLUT2, IRS-2, Pdx-1, and GPR40 were more strongly expressed in islets from RGZ-treated OLETF rats compared to control islets. CONCLUSION:These observations suggest that PPAR-? activation with RGZ and/or adenoviral overexpression increased intracellular calcium mobilization, insulin secretion, and ?-cell gene expression through GPR40 and GLUT2 gene up-regulation.
Project description:Insulin resistance is the undisputed root cause of type 2 diabetes mellitus (T2DM). There is currently an unmet demand for safe and effective insulin sensitizers, owing to the restricted prescription or removal from market of certain approved insulin sensitizers, such as thiazolidinediones (TZDs), because of safety concerns. Effective insulin sensitizers without TZD-like side effects will therefore be invaluable to diabetic patients. The specific focus on peroxisome proliferator-activated receptor ?- (PPAR?-) based agents in the past decades may have impeded the search for novel and safer insulin sensitizers. This review discusses possible directions and promising strategies for future research and development of novel insulin sensitizers and describes the potential targets of these agents. Direct PPAR? agonists, selective PPAR? modulators (sPPAR?Ms), PPAR?-sparing compounds (including ligands of the mitochondrial target of TZDs), agents that target the downstream effectors of PPAR?, along with agents, such as heat shock protein (HSP) inducers, 5'-adenosine monophosphate-activated protein kinase (AMPK) activators, 11?-hydroxysteroid dehydrogenase type 1 (11?-HSD1) selective inhibitors, biguanides, and chloroquines, which may be safer than traditional TZDs, have been described. This minireview thus aims to provide fresh perspectives for the development of a new generation of safe insulin sensitizers.
Project description:The inhibition of apoptosis in cancer cells is the major pathological feature of hepatic carcinoma. Rosiglitazone (RGZ), a ligand for peroxisome proliferator-activated receptor ? (PPAR-?), has been shown to induce apoptosis in hepatic carcinoma cells. However, the mechanism underlying this effect remains to be elucidated. The present study aimed to investigate the effect of RGZ on cell viability and apoptosis, and its mechanisms in cultured HepG2 cells using MTT assay, flow cytometry and western blotting. The results revealed that treatment with RGZ may attenuate HepG2 cell viability and induce the apoptosis of the cells. The mechanism of RGZ-induced apoptosis involves an increase in the level of activated PPAR-? (p-PPAR-?) and a decrease in p85 and Akt expression. In addition, the PPAR-? antagonist GW9662 suppressed the effect of RGZ in the HepG2 cells. Taken together, the results suggest that RGZ induces the apoptosis of HepG2 cells through the activation of PPAR-?, suppressing the activation of the PI3K/Akt signaling pathway. Such mechanisms may contribute to the favorable effects of treatment using RGZ in HepG2 cells.
Project description:The peroxisome-proliferator receptor-? (PPAR?) is expressed in multiple cancer types. Recently, our group has shown that PPAR? is phosphorylated on serine 273 (S273), which selectively modulates the transcriptional program controlled by this protein. PPAR? ligands, including thiazolidinediones (TZDs), block S273 phosphorylation. This activity is chemically separable from the canonical activation of the receptor by agonist ligands and, importantly, these noncanonical agonist ligands do not cause some of the known side effects of TZDs. Here, we show that phosphorylation of S273 of PPAR? occurs in cancer cells on exposure to DNA damaging agents. Blocking this phosphorylation genetically or pharmacologically increases accumulation of DNA damage, resulting in apoptotic cell death. A genetic signature of PPAR? phosphorylation is associated with worse outcomes in response to chemotherapy in human patients. Noncanonical agonist ligands sensitize lung cancer xenografts and genetically induced lung tumors to carboplatin therapy. Moreover, inhibition of this phosphorylation results in deregulation of p53 signaling, and biochemical studies show that PPAR? physically interacts with p53 in a manner dependent on S273 phosphorylation. These data implicate a role for PPAR? in modifying the p53 response to cytotoxic therapy, which can be modulated for therapeutic gain using these compounds.
Project description:There is increasing evidence that thiazolidinediones (TZDs), antidiabetic compounds that are synthetic ligands for the peroxisome proliferator-activated receptor ? (PPAR?), have cardiovascular effects through as yet poorly defined mechanisms. We tested the effect of two TZD class drugs, rosiglitazone and pioglitazone, on human aortic smooth muscle cell (SMC) expression of insulin-like growth factor-1 receptor (IGF-1R). Both TZDs dose dependently up-regulated IGF-1R protein levels (rosiglitazone, 10 ?mol/liter, 67% increase, n = 4, p < 0.01; pioglitazone, 10 ?mol/liter, 41% increase, n = 4, p < 0.01) and increased IGF-1R signaling activity (36% increase in Akt phosphorylation). However, the endogenous PPAR? ligand, 15-deoxy-?(12,14)-prostaglandin J(2), dose dependently reduced IGF-1R (10 ?mol/liter, 80% decrease, n = 4, p < 0.01), and overexpression of PPAR? using an adenovirus likewise reduced IGF-1R (50% decrease versus SMC infected with control adenovirus), suggesting a PPAR?-independent action of TZDs. All three PPAR? ligands (rosiglitazone, pioglitazone, and 15-deoxy-?(12,14)-prostaglandin J(2)), however, did not change IGF-1R mRNA levels, indicating that their effects were posttranscriptional. Use of bicistronic constructs revealed that TZD induction of IGF-1R translation occurred via internal ribosomal entry. To examine the potential physiological relevance of TZD up-regulation of IGF-1R, we determined the effect of rosiglitazone on oxidized LDL (oxLDL)-induced apoptosis. 20 ?mol/liter of rosiglitazone reduced oxidized LDL-induced apoptosis by 40% and neutralizing antibody to IGF-1R (?IR3) counteracted this rescue, suggesting the rosiglitazone survival effect was, at least in part, mediated by IGF-1R. In conclusion, TZDs markedly up-regulate SMC IGF-1R expression and signaling, likely via a PPAR?-independent mechanism. This novel action of TZDs may play an important role in their cardiovascular effects.
Project description:Thiazolidinediones (TZDs) are PPAR? agonists with potent insulin-sensitizing effects. However, their use has been curtailed by substantial adverse effects on weight, bone, heart, and hemodynamic balance. TZDs induce the deacetylation of PPAR? on K268 and K293 to cause the browning of white adipocytes. Here, we show that targeted PPAR? mutations resulting in constitutive deacetylation (K268R/K293R, 2KR) increased energy expenditure and protected from visceral adiposity and diet-induced obesity by augmenting brown remodeling of white adipose tissues. Strikingly, when 2KR mice were treated with rosiglitazone, they maintained the insulin-sensitizing, glucose-lowering response to TZDs, while displaying little, if any, adverse effects on fat deposition, bone density, fluid retention, and cardiac hypertrophy. Thus, deacetylation appears to fulfill the goal of dissociating the metabolic benefits of PPAR? activation from its adverse effects. Strategies to leverage PPAR? deacetylation may lead to the design of safer, more effective agonists of this nuclear receptor in the treatment of metabolic diseases.
Project description:Diabetic nephropathy (DN) is a severe complication of diabetes and serves as the leading cause of chronic renal failure. In the past decades, angiotensin-converting enzyme inhibitors (ACEIs)/angiotensin II receptor blockers (ARBs) based first-line therapy can slow but cannot stop the progression of DN, which urgently requests the innovation of therapeutic strategies. Thiazolidinediones (TZDs), the synthetic exogenous ligands of nuclear receptor peroxisome proliferator-activated receptor- ? (PPAR ? ), had been thought to be a promising candidate for strengthening the therapy of DN. However, the severe adverse effects including fluid retention, cardiovascular complications, and bone loss greatly limited their use in clinic. Recently, numerous novel PPAR ? agonists involving the endogenous PPAR ? ligands and selective PPAR ? modulators (SPPARMs) are emerging as the promising candidates of the next generation of antidiabetic drugs instead of TZDs. Due to the higher selectivity of these novel PPAR ? agonists on the regulation of the antidiabetes-associated genes than that of the side effect-associated genes, they present fewer adverse effects than TZDs. The present review was undertaken to address the advancements and the therapeutic potential of these newly developed PPAR ? agonists in dealing with diabetic kidney disease. At the same time, the new insights into the therapeutic strategies of DN based on the PPAR ? agonists were fully addressed.
Project description:Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic receptor involved in the uptake of a variety of molecules, such as apoE, ?2-macroglobulin, and the amyloid ? peptide (A?), for either transcellular transport, protein trafficking or lysosomal degradation. The LRP1 gene can be transcribed upon activation of peroxisome proliferator receptor activated-? (PPAR?) by the potent PPAR? agonist, rosiglitazone (RGZ). In previous studies, RGZ was shown to upregulate LRP1 levels in concentrations between 0.1 and 5 ?M in HepG2 cells. In this study, we sought to replicate previous studies and to investigate the molecular mechanism by which high concentrations of RGZ reduce LRP1 levels in HepG2 cells. Our data confirmed that transcriptional activation of LRP1 occurred in response to RGZ at 3 and 10 ?M, in agreement with the study reported by Moon et al. (2012a). On the other hand, we found that high concentrations of RGZ decreased both mRNA and protein levels of LRP1. Mechanistically, transcriptional dysregulation of LRP1 was affected by the downregulation of PPAR? in a time- and concentration-dependent manner. However, downregulation of PPAR? was responsible for only 40% of the LRP1 reduction and thereby the remaining loss of LRP1 (60%) was found to be through degradation in the lysosomal system. In conclusion, our findings demonstrate the mechanisms by which high concentrations of RGZ caused LRP1 levels to be reduced in HepG2 cells. Taken together, this data will be helpful to better explain the pharmacological modulation of this pivotal membrane receptor by PPAR? agonists.
Project description:Peroxisome proliferator-activated receptor gamma (PPAR?) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR? agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR? target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR? ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR? ligands in the treatment of insulin resistance.
Project description:The nuclear receptor PPAR? regulates adipogenesis and plays a central role in lipid and glucose homeostasis, and is the molecular target of the glitazones (TZDs), therapeutics used to treat insulin resistance and type-2 diabetes (T2D). Although the TZDs, which are PPAR? agonists, demonstrated robust clinical efficacy in T2D, their use has been hampered by an array of untoward side effects. Paradoxically, partial agonists (e.g. MRL24), antagonists (e.g. SR1664), and inverse agonists (e.g. SR10171 and SR2595), possess similar insulin-sensitizing efficacy as the TZDs in obese diabetic mice. Given the unique pharmacology of these modulators, we sought to identify the components of the PPAR? transcriptional complex that is regulated by these ligands. To achieve this, we employed subcellular fractionation of adipocytes combined with either trapping of the receptor complex on biotinylated DNA oligonucleotide, or classical immunoprecipitation. Tandem mass spectrometry analysis revealed unique, partially overlapping, compound- and subcellular compartment-specific complexes. Components of these interactomes are putative coregulators of PPAR?. Interestingly, complexes isolated in the cytosol contain sets of proteins involve in cellular assembly and extracellular matrix. Furthermore, the interactome observed for cytosolic non-DNA bound receptor was distinct from that observed from nuclear chromatin associated PPAR?, suggesting cellular compartment-specific roles for this receptor.