Phosphorylation Of PPARγ Research Articles

Novel phosphorylation of PPARγ ameliorates obesity-induced adipose tissue inflammation and improves insulin sensitivity

Chronic inflammation in adipose tissue is highly associated with insulin resistance. Herein, we demonstrate that a novel modification of PPARγ is strongly associated with inflammatory responses in adipose tissue. c-Src kinase directly phosphorylated PPARγ at Tyr78, and this process was reversed by protein tyrosine phosphatase-1B (PTP-1B). In adipocytes, phosphorylation of PPARγ suppressed the expression of pro-inflammatory genes as well as the secretion of chemokines and cytokines, thus reducing macrophage migration. Importantly, pharmacological inhibition of c-Src kinase aggravated insulin resistance in obese mice with a concomitant increase in the expression of pro-inflammatory genes in adipose tissue. These data strongly suggest that PPARγ phosphorylation is the key regulatory mechanism of the inflammatory response in adipose tissue, which is highly associated with glucose tolerance and insulin sensitivity. Furthermore, these data increase our understanding of the mechanical aspects of developing novel anti-diabetic drugs targeting PPARγ phosphorylation.

L-Carnitine Ameliorates Cancer Cachexia in Mice Partly via the Carnitine Palmitoyltransferase-Associated PPAR-γ Signaling Pathway

Background:<smlcap>L</smlcap>-Carnitine has been demonstrated to ameliorate cachectic symptoms. In the present study, we sought to investigate the role of the peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway in the ameliorative effects of <smlcap>L</smlcap>-carnitine on cancer cachexia in a colon-26 tumor-bearing mouse model. Methods: The cachectic mice received <smlcap>L</smlcap>-carnitine (p.o.) or etomoxir (i.p.), or pioglitazone hydrochloride (p.o.) or GW9662 (i.p.). The physiological cachexia parameters, biochemical parameters, and serum cytokines were measured. The expression levels of representative molecules in the PPAR-γ signaling pathway were measured by using quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot analysis. Results: Oral administration of <smlcap>L</smlcap>-carnitine at 9 mg/kg/day improved the cachexia parameters and biochemical parameters in cancer cachectic mice. The elevated serum concentrations of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) were decreased by <smlcap>L</smlcap>-carnitine. These ameliorative effects of <smlcap>L</smlcap>-carnitine were lessened by the carnitine palmitoyltransferase I (CPT I) inhibitor, etomoxir. The mRNA and protein expression levels of PPAR-α and PPAR-γ were decreased in the livers of cancer cachectic mice and increased after <smlcap>L</smlcap>-carnitine administration, which attenuated the increased mRNA expression levels of sterol-regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS). Similar to pioglitazone, <smlcap>L</smlcap>-carnitine augmented the phosphorylation of PPAR-γ and attenuated the expression levels of phospho-p65 and cyclooxygenase (COX)-2. Additionally, the above-mentioned effects of <smlcap>L</smlcap>-carnitine were reversed by GW9662. Conclusion:<smlcap>L</smlcap>-Carnitine exerts its ameliorative effects in cancer cachexia in association with the PPAR-γ signaling pathway.

Selective targeting of PPARγ by the natural product chelerythrine with a unique binding mode and improved antidiabetic potency

Type 2 diabetes mellitus (T2DM) is a pervasive metabolic syndrome that is characterized by insulin resistance, hyperglycemia and dyslipidemia. As full agonists of PPARγ, thiazolidinedione (TZD) drugs elicit antidiabetic effects by targeting PPARγ but is accompanied by weight gain, fluid retention and cardiovascular risk associated with their transcriptional agonism potency. We here identify a natural product chelerythrine as a unique selective PPAR modulator (SPPARM) with a potent PPARγ binding activity but much less classical receptor transcriptional agonism. Structural analysis reveals that chelerythrine exhibits unique binding in parallel with H3 of PPARγ. Unlike TZDs, chelerythrine destabilizes helix 12, especially residue tyrosine 473, resulting in a loose configuration of AF-2 and a selective cofactor profile distinct from TZDs, leading to a differential target gene profile in adipogenesis in db/db diabetic mice. Moreover, chelerythrine improved insulin sensitivity by more potently blocking the phosphorylation of PPARγ by CDK5 compared to TZDs. These data fundamentally elucidate the mechanism by which chelerythrine retains the benefits of improving insulin sensitivity while reducing the adverse effects of TZDs, suggesting that the natural product chelerythrine is a very promising pharmacological agent by selectively targeting PPARγ for further development in the clinical treatment of insulin resistance.

Molecular Recognition of PPARγ by Kinase Cdk5/p25: Insights from a Combination of Protein-Protein Docking and Adaptive Biasing Force Simulations.

The peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that plays a major role in the regulation of glucose and lipid metabolisms and has, therefore, many implications in modern-life metabolic disorders such as diabetes, obesity, and cardiovascular diseases. Phosphorylation of PPARγ by the cyclin-dependent kinase 5 (Cdk5) has been recently proved to promote obesity and loss of insulin sensitivity. The inhibition of this reaction is currently being pursued to develop PPARγ ligands for type 2 diabetes treatments. The knowledge of the protein-protein interactions between Cdk5/p25 and PPARγ can be an important asset for better understanding of the molecular basis of the Cdk5-meditated phosphorylation of PPARγ and its inhibition. By means of a computational approach that combines protein-protein docking and adaptive biasing force molecular dynamics simulations, we obtained PPARγ-Cdk5/p25 structural models that are consistent with the mechanism of the enzymatic reaction and with overall structural features of the full length PPARγ-RXRα heterodimer bound to DNA. In addition to the active site, our model shows that the interacting regions between the two proteins should involve two distal docking sites, comprising the PPARγ Ω-loop and Cdk5 N-terminal lobe and the PPARγ β-sheet and Cdk5 C-terminal lobe. These sites are related to PPARγ transactivation and directly interact with PPARγ ligands. Our results suggest that β-sheets and Ω-loop stabilization promoted by PPARγ agonists could be important to inhibit Cdk5-mediated phosphorylation.

Hydrogen peroxide-induced changes of PPARγ in primary cultured cortical neurons

To investigate whether H₂O₂treatment negatively regulates PPARγ in primary cortical neurons by increasing PPARγ phosphorylation. Primary cultured cortical neurons were treated with H₂O₂(250, 500, and 750 µmol/L) for 2 h. 30 min before the H₂O₂(500 µmol/L), the specific inhibitor of ERK1/2 activation, U0126, was added to the culture. Morphological observation, MTT assay and the trypan blue exclusion method were used to detect cell damage. Western blot was carried out to evaluate the expressions of p-PPARγ (phospho-PPARγ) and total PPARγ, as well as to investigate the nuclear translocation of PPARγ (PPARγ activity). (1) Compared with the control group, cell survival rates were decreased by H₂O₂at concentrations of 250, 500, and 750 µmol/L (74.8% ± 5.2%, 53.6% ± 6.7% and 26.5% ± 5.8%, respectively, P < 0.05), while cell death rate were increased (ctrl group 6.6% ± 1.0%, H₂O₂-injured groups: 23.1% ± 2.8%, 48.2% ± 4.1% and 75.9% ± 4.4% respectively, P < 0.05). (2) Compared with the control group, the expression of total PPARγ failed to show significant change in H₂O₂-injured group, whereas the expression of p-PPARγ increased. Neurons injured by H₂O₂(500 µmol/L) also showed a reduction of PPARγ nuclear translocation (an increase in cytosol PPARγ and a simultaneous decrease in nuclear PPARγ). (3) Compared with H₂O₂-injured group, inhibition of ERK1/2 activation decreased p-PPARγ expression, and increased PPARγ nuclear translocation, as well as improved cell survival rate (53.6% ± 6.7% vs 70.8% ± 1.3%, P < 0.05) and decreased cell death rate (48.2% ± 4.1% vs 29.8%±3.4%, P < 0.05). Phosphorylation of PPARγ may be involved in cell death induced by hydrogen peroxide in primary cultured cortical neurons.

Phosphorylation of PPARγ Affects the Collective Motions of the PPARγ-RXRα-DNA Complex.

Peroxisome-proliferator activated receptor-γ (PPARγ) is a nuclear hormone receptor that forms a heterodimeric complex with retinoid X receptor-α (RXRα) to regulate transcription of genes involved in fatty acid storage and glucose metabolism. PPARγ is a target for pharmaceutical intervention in type 2 diabetes, and insight into interactions between PPARγ, RXRα, and DNA is of interest in understanding the function and regulation of this complex. Phosphorylation of PPARγ by cyclin-dependent kinase 5 (Cdk5) has been shown to dysregulate the expression of metabolic regulation genes, an effect that is counteracted by PPARγ ligands. We applied molecular dynamics (MD) simulations to study the relationship between the ligand-binding domains of PPARγ and RXRα with their respective DNA-binding domains. Our results reveal that phosphorylation alters collective motions within the PPARγ-RXRα complex that affect the LBD-LBD dimerization interface and the AF-2 coactivator binding region of PPARγ.

Cell Signaling Mechanisms by which Geniposide Regulates Insulin- Degrading Enzyme Expression in Primary Cortical Neurons.

An increasing number of studies have demonstrated that insulin-degrading enzyme (IDE) plays an essential role in both the degradation and its activity of β-amyloid (Aβ). Therefore, the regulation of IDE expression and/or modification of IDE-dependent actions are two emerging strategies for the treatment of Alzheimer's disease (AD). We previously observed that geniposide, a novel agonist of glucagon-like peptide 1 receptor (GLP-1R), could attenuate Aβ-induced neurotoxicity by regulating the expression of IDE in primary cortical neurons. However, the signal transduction mechanisms underlying this effect were not elucidated. The present study, therefore examined and explored the cell signaling transduction and molecular mechanisms by which geniposide induces the expression of IDE in primary cortical neurons. The current study revealed that LY294002 (an inhibitor for phosphatidyl inositol 3-kinase, PI3K), PP1 (inhibitor for c-Src), GW9662 (antagonist for peroxisome proliferator-activated receptor γ, PPARγ), H89 (an inhibitor for protein kinase A, PKA) and AG1478 (an antagonist for epidermal growth factor receptor, EGFR) prohibited the up-regulation of IDE induced by geniposide in primary cortical neurons. Further, geniposide also enhanced the phosphorylation of PPARγ and accelerated the release of phosphorylated FoxO1 (forkhead box O1) from nuclear fraction to the cytosol. Moreover, geniposide directly activated the activity of IDE promoter in PC12 cells, which confirmed the presence of the GLP-1 receptor. Taken together, our findings reveal for the first time the cell signaling transduction pathway of geniposide regulating the expression of IDE in neurons.

22 IL-34 Antibody Ameliorates Experimental Colitis by Alternating IL-12p40 Expression in Macrophages

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor widely expressed and best known for its role in glucose metabolism. Within the intestine, PPARγ signaling has been implicated in both colon cancer and inflammation. A trial of the PPARγ full agonist rosiglitazone for the treatment of ulcerative colitis showed efficacy similar to that of 5-aminosalicylic acid but its use has been limited by its adverse side effect profile. It has recently been demonstrated that, in addition to receptor agonism, rosiglitazone has a second biological function—to block PPARγ phosphorylation at Serine 273. To assess the role of PPARγ phosphorylation in the pathogenesis of experimental colitis, we generated mice lacking the ability to phosphorylate PPAR γ at S273 by deleting the kinase, CDK5, in either macrophages (CDK5-MKO) or intestinal epithelial cells (CDK5-EKO) using the CreloxP system. When challenged with oral Dextran Sodium Sulfate (DSS) for one week, mice lacking CDK5 in either tissue lose weight and develop bloody diarrhea similar to wild type mice. However, upon withdrawal of DSS, the CDK5-MKO and CDK5-EKO mice demonstrate enhanced recovery compared to wild type controls with improved clinical severity and histology scores. Colonic tissue from CDK5-MKO mice recovering from colitis have increased expression of genes consistent with alternatively activated (M2) macrophages. Colonic tissue from CDK5-EKO mice exhibit evidence for decreased macrophage infiltration as well as preserved epithelial barrier integrity when compared to control mice. To further test our hypothesis, we generated global knock-in mice that contain an allele of PPAR γ that cannot be regulated by phosphorylation (Serine 273 to Alanine, S273A). Since inhibition of PPAR γ phosphorylation at S273 has anti-inflammatory effects, we hypothesized that these knockin mice would exhibit enhanced recovery similar to our macrophage and epithelial specific knock-outs. The knock-in mice are a virtual phenocopy of the tissue specific CDK5 deletion with comparable acute colitis but dramatically improved recovery. These data suggest that there may be a role for safer and more targeted PPAR γactivators in the treatment of inflammatory bowel disease.

Identification of a novel selective agonist of PPARγ with no promotion of adipogenesis and less inhibition of osteoblastogenesis.

Nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in the regulation of glucose homeostasis and lipid metabolism. However, current PPARγ-targeting drugs such as thiazolidinediones (TZDs) are associated with undesirable side effects. We identified a small molecular compound, F12016, as a selective PPARγ agonist by virtual screening, which showed moderate PPARγ agonistic activity and binding ability for PPARγ. F12016 did not activate other PPAR subtypes at 30 μM and selectively modulated PPARγ target gene expression. In diabetic KKAy mice, F12016 had insulin-sensitizing and glucose-lowering properties, and suppressed weight gain. In vitro, F12016 effectively increased glucose uptake and blocked cyclin-dependent kinase 5-mediated phosphorylation of PPARγ at Ser273, but slightly triggered adipogenesis and less inhibited osteoblastogenesis than rosiglitazone. Moreover, compared with the full agonist rosiglitazone, F12016 had a distinct group of coregulators and a different predicted binding mode for the PPARγ ligand-binding domain. A site mutation assay confirmed the key epitopes, especially Tyr473 in AF-2. In summary, our study shows that F12016 is a non-TZD, novel selective PPARγ agonist without the classical lipogenic side effects, which may provide a new structural strategy for designing PPARγ ligands with advantages over TZDs.

21 Genetic Modulation of PPARγ Phosphorylation Decreases the Severity of Experimental Colitis in Mice

21 Genetic Modulation of PPARγ Phosphorylation Decreases the Severity of Experimental Colitis in Mice

흑삼의 열수 및 에탄올 추출물의 항비만 효과

Black ginseng was made by steaming raw white ginseng nine times at 100°C for 2 h and drying. We then performed pilot experiments using the nine black ginseng extracts for different steaming and drying times to determine their anti-obesity effects. Two ginseng extracts, steaming and drying five times (FSFD) and steaming and drying nine times (NSND), prepared in water or ethanol solution decreased lipid accumulation of 3T3-L1 cells. FSFD in water and ethanol extracts showed higher levels of ginsenosides, in particular, Rh1, Rg2, and Rb1 than NSND, and levels of the three ginsenosides were higher in ethanol extracts than in water extracts. Treatment with FSFD and/or NSND in ethanol extracts significantly regulated PPARγ, C/EBPα and AMPK phosphorylation in 3T3-L1 cells. We verified doubling time of stem cells from both abdominal fat and subcutaneous fat after FSFD and NSND in ethanol and water extracts were added. Although addition of FSFD and NSFD in water extracts had no effects on proliferation, ethanol extracts with FSFD and NSND increased doubling time of stem cells in subcutaneous fat. FSFD and NSND in ethanol extracts more effectively reduced adipogenesis compared to those in water extracts. FSFD in ethanol extracts promoted secretion of anti-inflammatory cytokine such as IL-10 and depressed MCP-1 infiltration in 3T3-L1 preadipocytes co-cultured with RAW264.7 cells. We concluded that FSFD and NSND ethanol extracts may be developed as a functional food for its anti-obesity effect, but anti-inflammatory effect was shown in ethanol extracted FSFD rather than in NSND.

Inhibition of adipogenic differentiation of bone marrow mesenchymal stem cells by erythropoietin via activating ERK and P38 MAPK.

We examined whether erythropoietin (EPO) can inhibit adipogenic differentiation of mesenchymal stem cells (MSCs) in the mouse bone marrow and its underlying mechanism. We separated and extracted mouse bone marrow MSCs and induced adipogenic differen-tiation using 3-isobutyl-1-methylxanthine, insulin, and dexamethasone. Different concentrations of EPO were added to the cells and observed by Oil Red O staining on the 20th day to quantitatively analyze the degree of cell differentiation. mRNA expression levels of peroxysome proliferator-activated receptor γ (PPARγ), CCAAT enhancer binding protein α, and adiponectin were analyzed by real-time quantitative polymerase chain reaction, and the activity of PPARγ, extracellular sig-nal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) were determined by western blotting. EPO significantly inhibited adipogenic differentiation of MSCs after 20 days and reduced absorbance values by Oil Red O staining without affecting proliferation activity. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponec-tin during adipogenesis and increased protein phosphorylation of ERK, p38 MAPK, and PPARγ during differentiation. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponectin by increasing protein phosphor-ylation of ERK, p38 MAPK, and PPARγ during differentiation, which inhibited adipogenic differentiation of MSCs.

Expression of p-PPARγ in the aging thoracic aorta of spontaneously hypertensive rat and inhibitory effect of rosiglitazone

ABSTRACT Objective To investigate the expression of phosphorylated peroxisome proliferators-activated receptor γ (p-PPARγ) in the aging thoracic aorta of spontaneously hypertensive rat (SHR) and the inhibitory effect of rosiglitazone on the phosphorylation of PPARγ. Methods 16, 32 and 64 week-old Wistar-Kyoto rats (WKY) and SHR were randomly and respectively divided into WKY, SHR and SHR+rosiglitazone group (9 in each group). The rats in SHR+rosiglitazone group were treated with rosiglitazone (5 mg/kg, intragastrically) for 56 d, whereas normal saline was applied in WKY and SHR groups. Systolic blood pressure (SBP) of rats was measured by tail cuff method. Histopathological damage of thoracic aorta was analyzed using Hematoxylin-Eosin (HE) staining. Immunohistochemical staining and western blot were performed to test the level of p-PPARγ protein in the thoracic aorta arising from each group. Results The SBP in 16, 32 and 64 week-old SHR were significantly higher as compared with those in matched WKY rats (P Conclusions The PPARγ phosphorylation was observed in the thoracic aorta of SHR and its expression was increased by the increase of age. Furthermore, rosiglitazone inhibited the PPARγ phosphorylation and suppressed vascular aging in SHR.

An ERK/Cdk5 axis controls the diabetogenic actions of PPARγ.

Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ at serine 273 by Cdk5 stimulates diabetogenic gene expression in adipose tissues1. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic PPARγ ligand drugs, such as the thiazolidinediones and PPARγ partial/non-agonists2. To better understand the importance of this obesity-linked PPARγ phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. Surprisingly, these mice have both a paradoxical increase in PPARγ phosphorylation at S273 and worsened insulin resistance. Unbiased proteomic studies show that ERK kinases are activated in these KO animals. We show here that ERK directly phosphorylates S273 of PPARγ in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MEK, the ERK kinase. Importantly, pharmacological MEK and ERK inhibition markedly improves insulin resistance in both obese wild type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPARγ function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes.

Angiotensin-I converting enzyme inhibitory peptides from antihypertensive skate (Okamejei kenojei) skin gelatin hydrolysate in spontaneously hypertensive rats

The aim of this study was to investigate antihypertensive effect of bioactive peptides from skate (Okamejei kenojei) skin gelatin. The Alcalase/protease gelatin hydrolysate below 1kDa (SAP) exhibited the highest angiotensin-I converting enzyme (ACE) inhibition compared to other hydrolysates. SAP can decrease systolic blood pressure significantly in spontaneously hypertensive rats. SAP inhibited vasoconstriction via PPAR-γ expression, activation and phosphorylation of eNOS in lungs. Moreover, the expression levels of endothelin-1, RhoA, α-smooth muscle actin, cleaved caspase 3 and MAPK were decreased by SAP in lungs. Vascularity, muscularization and cellular proliferation in lungs were detected by immunohistochemical staining. Finally, two purified peptides (LGPLGHQ, 720Da and MVGSAPGVL, 829Da) showed potent ACE inhibition with IC50 values of 4.22 and 3.09μM, respectively. These results indicate that bioactive peptides isolated from skate skin gelatin may serve as candidates against hypertension and could be used as functional food ingredients.

Thrap3 docks on phosphoserine 273 of PPARγ and controls diabetic gene programming.

Phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) at Ser273 by cyclin-dependent kinase 5 (CDK5) in adipose tissue stimulates insulin resistance, but the underlying molecular mechanisms are unclear. We show here that Thrap3 (thyroid hormone receptor-associated protein 3) can directly interact with PPARγ when it is phosphorylated at Ser273, and this interaction controls the diabetic gene programming mediated by the phosphorylation of PPARγ. Knockdown of Thrap3 restores most of the genes dysregulated by CDK5 action on PPARγ in cultured adipocytes. Importantly, reduced expression of Thrap3 in fat tissue by antisense oligonucleotides (ASOs) regulates a specific set of genes, including the key adipokines adiponectin and adipsin, and effectively improves hyperglycemia and insulin resistance in high-fat-fed mice without affecting body weight. These data indicate that Thrap3 plays a crucial role in controlling diabetic gene programming and may provide opportunities for the development of new therapeutics for obesity and type 2 diabetes.

L312, a novel PPARγ ligand with potent anti-diabetic activity by selective regulation

BackgroundSelective PPARγ modulators (sPPARγM) retains insulin sensitizing activity but with minimal side effects compared to traditional TZDs agents, is thought as a promising strategy for development of safer insulin sensitizer. MethodsWe used a combination of virtual docking, SPR-based binding, luciferase reporter and adipogenesis assays to analyze the interaction mode, affinity and agonistic activity of L312 to PPARγ in vitro, respectively. And the anti-diabetic effects and underlying molecular mechanisms of L312 was studied in db/db mice. ResultsL312 interacted with PPARγ-LBD in a manner similar to known sPPARγM. L312 showed similar PPARγ binding affinity, but displayed partial PPARγ agonistic activity compared to PPARγ full agonist pioglitazone. In addition, L312 displayed partial recruitment of coactivator CBP yet equal disassociation of corepressor NCoR1 compared to pioglitazone. In db/db mice, L312 (30mg/kg·day) treatment considerably improved insulin resistance with the regard to OGTT, ITT, fasted blood glucose, HOMA-IR and serum lipids, but elicited less weight gain, adipogenesis and hemodilution compared with pioglitazone. Further studies demonstrated that L312 is a potent inhibitor of CDK5-mediated PPARγ phosphorylation and displayed a selective gene expression profile in epididymal WAT. ConclusionsL312 is a novel sPPARγM. General significanceL312 may represent a novel lead for designing ideal sPPARγM for T2DM treatment with advantages over current TZDs.

A Novel Non-agonist Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand UHC1 Blocks PPARγ Phosphorylation by Cyclin-dependent Kinase 5 (CDK5) and Improves Insulin Sensitivity

Thiazolidinedione class of anti-diabetic drugs which are known as peroxisome proliferator-activated receptor γ (PPARγ) ligands have been used to treat metabolic disorders, but thiazolidinediones can also cause several severe side effects, including congestive heart failure, fluid retention, and weight gain. In this study, we describe a novel synthetic PPARγ ligand UNIST HYUNDAI Compound 1 (UHC1) that binds tightly to PPARγ without the classical agonism and which blocks cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation. We modified the non-agonist PPARγ ligand SR1664 chemically to improve its solubility and then developed a novel PPARγ ligand, UHC1. According to our docking simulation, UHC1 occupied the ligand-binding site of PPARγ with a higher docking score than SR1664. In addition, UHC1 more potently blocked CDK5-mediated PPARγ phosphorylation at Ser-273. Surprisingly, UHC1 treatment effectively ameliorated the inflammatory response both in vitro and in high-fat diet-fed mice. Furthermore, UHC1 treatment dramatically improved insulin sensitivity in high-fat diet-fed mice without causing fluid retention and weight gain. Taken together, compared with SR1664, UHC1 exhibited greater beneficial effects on glucose and lipid metabolism by blocking CDK5-mediated PPARγ phosphorylation, and these data indicate that UHC1 could be a novel therapeutic agent for use in type 2 diabetes and related metabolic disorders.

Oxidative stress-induced alterations in PPAR-γ and associated mitochondrial destabilization contribute to kidney cell apoptosis.

The mechanism(s) underlying renoprotection by peroxisome proliferator-activated receptor (PPAR)-γ agonists in diabetic and nondiabetic kidney disease are not well understood. Mitochondrial dysfunction and oxidative stress contribute to kidney disease. PPAR-γ upregulates proteins required for mitochondrial biogenesis. Our aim was to determine whether PPAR-γ has a role in protecting the kidney proximal tubular epithelium (PTE) against mitochondrial destabilisation and oxidative stress. HK-2 PTE cells were subjected to oxidative stress (0.2-1.0 mM H₂O₂) for 2 and 18 h and compared with untreated cells for apoptosis, mitosis (morphology/biomarkers), cell viability (MTT), superoxide (dihydroethidium), mitochondrial function (MitoTracker red and JC-1), ATP (luminescence), and mitochondrial ultrastructure. PPAR-γ, phospho-PPAR-γ, PPAR-γ coactivator (PGC)-1α, Parkin (Park2), p62, and light chain (LC)3β were investigated using Western blots. PPAR-γ was modulated using the agonists rosiglitazone, pioglitazone, and troglitazone. Mitochondrial destabilization increased with H₂O₂concentration, ATP decreased (2 and 18 h; P < 0.05), Mitotracker red and JC-1 fluorescence indicated loss of mitochondrial membrane potential, and superoxide increased (18 h, P < 0.05). Electron microscopy indicated sparse mitochondria, with disrupted cristae. Mitophagy was evident at 2 h (Park2 and LC3β increased; p62 decreased). Impaired mitophagy was indicated by p62 accumulation at 18 h (P < 0.05). PPAR-γ expression decreased, phospho-PPAR-γ increased, and PGC-1α decreased (2 h), indicating aberrant PPAR-γ activation and reduced mitochondrial biogenesis. Cell viability decreased (2 and 18 h, P < 0.05). PPAR-γ agonists promoted further apoptosis. In summary, oxidative stress promoted mitochondrial destabilisation in kidney PTE, in association with increased PPAR-γ phosphorylation. PPAR-γ agonists failed to protect PTE. Despite positive effects in other tissues, PPAR-γ activation appears to be detrimental to kidney PTE health when oxidative stress induces damage.

FKBP51 Controls Cellular Adipogenesis through p38 Kinase-Mediated Phosphorylation of GRα and PPARγ

Glucocorticoid receptor-α (GRα) and peroxisome proliferator-activated receptor-γ (PPARγ) are critical regulators of adipogenic responses. We have shown that FK506-binding protein 51 (FKBP51) represses the Akt-p38 kinase pathway to reciprocally inhibit GRα but stimulate PPARγ by targeting serine 112 (PPARγ) and serines 220 and 234 (GRα). Here, this mechanism is shown to be essential for GRα and PPARγ control of cellular adipogenesis. In 3T3-L1 cells, FKBP51 was a prominent marker of the differentiated state and knockdown of FKBP51 showed reduced lipid accumulation and expression of adipogenic genes. Compared with wild-type (WT), FKBP51 knockout (51KO) mouse embryonic fibroblasts (MEFs) showed dramatic resistance to differentiation, with almost no lipid accumulation and greatly reduced adipogenic gene expression. These features were rescued by reexpression of FKBP51 in 51KO cells. 51KO MEFs exhibited reduced fatty acid synthase activity, increased sensitivity to GRα-induced lipolysis, and reduced PPARγ activity at adipogenic genes (adiponectin, CD36, and perilipin) but elevated GRα transrepression at these same genes. A p38 kinase inhibitor increased lipid content in WT cells and also restored lipid levels in 51KO cells, showing that elevated p38 kinase activity is a major contributor to adipogenic resistance in the 51KO cells. In 51KO cells, the S112A mutant of PPARγ and the triple S212A/S220A/S234A mutant of GRα both increased lipid accumulation, identifying these residues as targets of the FKBP51/p38 axis. Our combined investigations have uncovered FKBP51 as a key regulator of adipogenesis via the Akt-p38 pathway and as a potential target in the treatment of obesity and related disorders.