Regulation of chemokine and chemokine receptor expression by PPARγ in adipocytes and macrophages

Background

PPARγ plays a key role in adipocyte biology, and Rosiglitazone (Rosi), a thiazolidinedione (TZD)/PPARγ agonist, is a potent insulin-sensitizing agent. Recent evidences demonstrate that adipose tissue inflammation links obesity with insulin resistance and that the insulin-sensitizing effects of TZDs result, in part, from their anti-inflammatory properties. However the underlying mechanisms are unclear.

Methodology and Principal Findings

In this study, we establish a link between free fatty acids (FFAs) and PPARγ in the context of obesity-associated inflammation. We show that treatment of adipocytes with FFAs, in particular Arachidonic Acid (ARA), downregulates PPARγ protein and mRNA levels. Furthermore, we demonstrate that the downregulation of PPARγ by ARA requires the activation the of Endoplamsic Reticulum (ER) stress by the TLR4 pathway. Knockdown of adipocyte PPARγ resulted in upregulation of MCP1 gene expression and secretion, leading to enhanced macrophage chemotaxis. Rosi inhibited these effects. In a high fat feeding mouse model, we show that Rosi treatment decreases recruitment of proinflammatory macrophages to epididymal fat. This correlates with decreased chemokine and decreased chemokine receptor expression in adipocytes and macrophages, respectively.

Conclusions and Significance

In summary, we describe a novel link between FAs, the TLR4/ER stress pathway and PPARγ, and adipocyte-driven recruitment of macrophages. We thus both describe an additional potential mechanism for the anti-inflammatory and insulin-sensitizing actions of TZDs and an additional detrimental property associated with the activation of the TLR4 pathway by FA.     

The gene expression of the myocardial lipid droplet protein is highly regulated by PPARγ in adipocytes differentiated from MEFs or SVCs

We demonstrate that expression of the myocardial lipid droplet protein (MLDP) and ERα observed in adipose tissues is undetectable in 3T3-L1 cells but detectable in mouse embryonic fibroblasts (MEFs) and stromal-vascular cells (SVCs) during adipocyte differentiation. MLDP gene expression in MEFs or SVCs is induced by treatment with a PPARγ agonist or forced expression of PPARγ, indicating that PPARγ enhances MLDP expression during adipogenesis. PCR analyses reveal the dual expression of SREBP-1a and SREBP-1c in MEFs and SVCs as well as white adipose tissues unlike the predominant expression of SREBP-1a in 3T3-L1 cells. These results suggest that MEFs and SVCs are useful model cells for examining function of MLDP in lipid droplet formation and adipocyte differentiation.     

Cardioprotection from oxidative stress in the newborn heart by activation of PPARγ is mediated by catalase

Regulation of catalase (CAT) by peroxisome proliferator-activated receptor-γ (PPARγ) was investigated to determine if PPARγ activation provides cardioprotection from oxidative stress caused by hydrogen peroxide (H(2)O(2)) in an age-dependent manner. Left ventricular developed pressure (LVDP) was measured in Langendorff perfused newborn or adult rabbit hearts, exposed to 200μM H(2)O(2), with perfusion of rosiglitazone (RGZ) or pioglitazone (PGZ), PPARγ agonists. We found: (1) H(2)O(2) significantly decreased sarcomere shortening in newborn ventricular cells but not in adult cells. Lactate dehydrogenase (LDH) release occurred earlier in newborn than in adult heart, which may be due, in part, to the lower expression of CAT in newborn heart. (2) RGZ increased CAT mRNA and protein as well as activity in newborn but not in adult heart. GW9662 (PPARγ blocker) eliminated the increased CAT mRNA by RGZ. (3) In newborn heart, RGZ and PGZ treatment inhibited release of LDH in response to H(2)O(2) compared to H(2)O(2) alone. GW9662 decreased this inhibition. (4) LVDP was significantly higher in both RGZ+H(2)O(2) and PGZ+H(2)O(2) groups than in the H(2)O(2) group. Block of PPARγ abolished this effect. In contrast, there was no effect of RGZ in adult. (5) The cardioprotective effects of RGZ were abolished by inhibition of CAT. In conclusion, PPARγ activation is cardioprotective to H(2)O(2)-induced stress in the newborn heart by upregulation of catalase. These data suggest that PPARγ activation may be an effective therapy for the young cardiac patient.     

Ligand entry pathways in the ligand binding domain of PPARγ receptor

To address the question of ligand entry process, we report targeted molecular dynamics simulations of the entry of the flexible ionic ligand GW0072 in the ligand binding domain of the nuclear receptor PPARγ. Starting with the ligand outside the receptor the simulations led to a ligand docked inside the binding pocket resulting in a structure very close to the holo-form of the complex. The results showed that entry process is guided by hydrophobic interactions and that entry pathways are very similar to exit pathways. We suggest that TMD method may help in discriminating between ligands generated by in silico docking.     

α-MSH signalling via melanocortin 5 receptor promotes lipolysis and impairs re-esterification in adipocytes

The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on α-melanocyte-stimulating hormone (α-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the α-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of α-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in α-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed.     

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation

Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPβ, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.     

Deciphering PPARγ activation in cardiometabolic syndrome: studies by in silico and in vivo experimental assessment

Cardiometabolic syndrome (CMetS) is a consolidation of metabolic disorders characterized by insulin resistance, dyslipidemia and hypertension. Curcumin, a natural bioactive compound, has been shown to possess notable anti-oxidant activity and it has also been included as a super natural herb in the super natural herbs database. Most of the beneficial effects of Curcumin are possibly due to activation of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ). The present study investigates molecular interactions of curcumin with PPARγ protein through molecular docking and molecular dynamics (MD) simulation studies. Further, effect of curcumin on high fat diet induced CMetS was studied in rats along with western blot for PPARγ and nuclear factor-κB (NF-κB) expressions and histopathological studies. Computational studies presented several significant molecular interactions of curcumin including Ser289, His323, His449 and Tyr473 of PPARγ. The in vivo results further confirmed that curcumin was able to ameliorate the abnormal changes and also, increased PPARγ expressions. The results confirm our hypothesis that activation of PPARγ by curcumin possesses the therapeutic potential to ameliorate the altered levels of metabolic changes in rats in the treatment of CMetS. This is the first report of CMetS treatment by curcumin and study of its underlying mechanism through in silico as well as in vivo experiments.     

Vanadyl acetylacetonate upregulates PPARγ and adiponectin expression in differentiated rat adipocytes

Vanadium compounds are promising agents in the therapeutic treatment of diabetes mellitus, but their mechanism of action has not been fully elucidated. The current work investigated the effects of vanadyl acetylacetonate, VO(acac)₂, on peroxisome-proliferator-activated receptor γ (PPARγ) and adiponectin, which are important targets of antidiabetic drugs. The experimental results revealed that vanadyl complexes increased the expression and multimerization of adiponectin in differentiated rat adipocytes. VO(acac)₂ caused activation of p38 mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) and elevation of PPARγ levels. The specific inhibitors SB203580 (p38 MAPK inhibitor) and T0070907 (PPARγ inhibitor) decreased the expression of adiponectin; however, compound C (AMPK inhibitor) did not significantly reduce the expression of adiponectin. In addition, vanadyl complexes induced protein–protein interaction between PPARγ and a vanadium-binding chaperone, heat shock protein 60 kDa. Overall, our results suggest that vanadyl complexes may upregulate PPARγ by suppressing PPARγ degradation, and thus stimulate adiponectin expression and multimerization. The present work has provided new insights into the mechanism of the antidiabetic actions of vanadium compounds.     

Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes

Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH(2)-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.     

Inhibition of protein phosphatase 2A activity by PI3Kγ regulates β-adrenergic receptor function

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Highlights? PI3Kγ inhibits βAR resensitization by regulating PP2A activity ? PI3Kγ inhibits PP2A activity by phosphorylating I2PP2A on Serine 9 and 93 ? Phosphorylation of I2PP2A leads to its agonist-mediated interaction with PP2A ? siRNA-targeted depletion of I2PP2A results in βAR resensitization