Trans‐repression of NFκB pathway mediated by PPARγ improves vascular endothelium insulin resistance

Previous study has shown that thiazolidinediones (TZDs) improved endothelium insulin resistance (IR) induced by high glucose concentration (HG)/hyperglycaemia through a PPARγ‐dependent‐NFκB trans‐repression mechanism. However, it is unclear, whether changes in PPARγ expression affect the endothelium IR and what the underlying mechanism is. In the present study, we aimed to address this issue. HG‐treated human umbilical vascular endothelial cells (HUVEC) were transfected by either PPARγ‐overexpressing (Ad‐PPARγ) or PPARγ‐shRNA‐containing (Ad‐PPARγ‐shRNA) adenoviral vectors. Likewise, the rats fed by high‐fat diet (HFD) were infected by intravenous administration of Ad‐PPARγ or Ad‐PPARγ‐shRNA. The levels of nitric oxide (NO), endothelin‐1 (ET‐1) and cytokines (TNFα, IL‐6, sICAM‐1 and sVCAM‐1) and the expression levels of PPARγ, eNOS, AKT, p‐AKT, IKKα/β and p‐IKKα/β and IκBα were examined; and the interaction between PPARγ and NFκB‐P65 as well as vascular function were evaluated. Our present results showed that overexpression of PPARγ notably increased the levels of NO, eNOS, p‐AKT and IκBα as well as the interaction of PPARγ and NFκB‐P65, and decreased the levels of ET‐1, p‐IKKα/β, TNFα, IL‐6, sICAM‐1 and sVCAM‐1. In contrast, down‐expression of PPARγ displayed the opposite effects. The results demonstrate that the overexpression of PPARγ improves while the down‐expression worsens the endothelium IR via a PPARγ‐mediated NFκB trans‐repression dependent manner. The findings suggest PPARγ is a potential therapeutic target for diabetic vascular complications.     

Adipocyte NCoR knockout decreases PPARγ phosphorylation and enhances PPARγ activity and insulin sensitivity

Insulin resistance, tissue inflammation, and adipose tissue dysfunction are features of obesity and Type 2 diabetes. We generated adipocyte-specific Nuclear Receptor Corepressor (NCoR) knockout (AKO) mice to investigate the function of NCoR in adipocyte biology, glucose and insulin homeostasis. Despite increased obesity, glucose tolerance was improved in AKO mice, and clamp studies demonstrated enhanced insulin sensitivity in liver, muscle, and fat. Adipose tissue macrophage infiltration and inflammation were also decreased. PPARγ response genes were upregulated in adipose tissue from AKO mice and CDK5-mediated PPARγ ser-273 phosphorylation was reduced, creating a constitutively active PPARγ state. This identifies NCoR as an adaptor protein that enhances the ability of CDK5 to associate with and phosphorylate PPARγ. The dominant function of adipocyte NCoR is to transrepress PPARγ and promote PPARγ ser-273 phosphorylation, such that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity, phenocopying the TZD-treated state.     

Peroxisome proliferator-activated receptors gamma reverses hepatic nutritional fibrosis in mice and suppresses activation of hepatic stellate cells in vitro

Nonalcoholic steatohepatitis with fibrosis is a more severe form of nonalcoholic fatty liver disease, one of the most common liver diseases. We have previously shown that peroxisome proliferator-activated receptors gamma (PPARγ) ligand, rosiglitazone, prevented the development of the methionine choline deficient (MCD) diet-induced fibrosing steatohepatitis. We have now tested whether overexpression of PPARγ ameliorates established steatohepatitis and fibrosis. Male C57BL6 mice fed with MCD diet for 8 weeks developed hepatic fibrosis with increased hepatic expression of collagen1α(I), inhibitors of fibrosis reversal-1, regulator involved in matrix degradation-9 and connective tissue growth factor. After 2 weeks of transduction of PPARγ through an adenovirus-expressing PPARγ (Ad-PPARγ), expression of these genes was reduced in a manner that paralleled the reduction in activated hepatic stellate cells (HSCs) and resolution of liver fibrosis. On the in vitro study, PPARγ is expressed in primary quiescent HSC, but depleted in culture activated HSC. Conversely, ectopic expression of PPARγ in activated HSC achieved the phenotypic reversal to the quiescent cell. Such induction markedly suppressed cell viability and cell proliferation, downregulated proliferating cell nuclear antigen, and caused cell cycle arrest at G0/G1 phase. Further, introduction of PPARγ in HSC increased cell apoptosis, this was confirmed by enhanced expression of FasL, cleaved caspase-3, cleaved caspase-7 and poly ADP-ribose polymerase, indicating an extrinsic apoptosis pathway. In conclusion, the present study shows that MCD diet-induced fibrosing steatohepatitis can be reversed by overexpression of PPARγ. It is likely that PPARγ reverses fibrosis by reducing HSCs proliferation, inducing cell cycle arrest and apoptosis.     

Pioglitazone inhibits advanced glycation end product-induced matrix metalloproteinases and apoptosis by suppressing the activation of MAPK and NF-κB

Apoptosis and degeneration coming mainly from chondrocytes are important mechanisms in the onset and progression of osteoarthritis. Specifically, advanced glycation end products (AGEs) play an important role in the pathogenesis of osteoarthritis. Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist has a protective effect on cartilage. This study aims to evaluate the effect of pioglitazone on AGEs-induced chondrocyte apoptosis and degeneration and their underlying mechanism. The in vitro study shows that AGEs induce cleavage of caspase-3 and PARP, up-regulate MMP-13 expression, enhance chondrocyte apoptosis and down-regulate PPARγ expression in human primary chondrocytes, which is reversed by pioglitazone. Furthermore, AGEs activate phosphorylation of Erk, JNK, and p38, and pioglitazone reverses AGEs-induced phosphorylation of Erk and p38. AGEs-induced degradation of IκBα and translocation of nuclear NF-κB p65 is reversed by pioglitazone. Pretreatment of chondrocytes with SB202190 (p38 inhibitor), SP600125 (JNK inhibitor) and BAY-11-7082 (NF-κB inhibitor) inhibit AGEs-induced apoptosis and degeneration. In vivo experiments suggest that pioglitazone reverses AGEs-induced cartilage degeneration and apoptosis in a mouse model, as demonstrated by HE and Safranin O staining, immunohistochemical analyses of Type II collagen (Col II), metalloproteinases (MMPs) and caspase-3. These findings suggest that pioglitazone, a PPARγ agonist, inhibits AGEs-induced chondrocytes apoptosis and degeneration via suppressing the activation of MAPK and NF-κB.     

Nitrated oleic acid up-regulates PPARγ and attenuates experimental inflammatory bowel disease

Nitric oxide and its metabolites undergo nitration reactions with unsaturated fatty acids during oxidative inflammatory conditions, forming electrophilic nitro-fatty acid derivatives. These endogenous electrophilic mediators activate anti-inflammatory signaling reactions, serving as high-affinity ligands for peroxisome proliferator-activated receptor γ (PPARγ). Here we examined the therapeutic effects of 9- or 10-nitro-octadecenoic oleic acid (OA-NO₂) and native oleic acid (OA) in a mouse model of colitis. OA-NO₂ reduced the disease activity index and completely prevented dextran sulfate sodium-induced colon shortening and the increase in colonic p65 expression. Increased PPARγ expression was observed in colon samples as well as in cells after OA-NO₂ administration, whereas no effect was seen with OA. This induction of PPARγ expression was completely abolished by the PPARγ antagonist GW9662. 5-Aminosalicylic acid, an anti-inflammatory drug routinely used in the management of inflammatory bowel disease, also increased PPARγ expression but to a lesser extent. Altogether, these findings demonstrate that administration of OA-NO₂ attenuates colonic inflammation and improves clinical symptoms in experimental inflammatory bowel disease. This protection involves activation of colonic PPARγ.     

25-Hydroxycholesterol-3-sulfate attenuates inflammatory response via PPARγ signaling in human THP-1 macrophages

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ～1 μM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1β. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1β expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.     

Bacterial Peptidoglycan Stimulates Adipocyte Lipolysis via NOD1

Obesity is associated with inflammation that can drive metabolic defects such as hyperlipidemia and insulin resistance. Specific metabolites can contribute to inflammation, but nutrient intake and obesity are also associated with altered bacterial load in metabolic tissues (i.e. metabolic endotoxemia). These bacterial cues can contribute to obesity-induced inflammation. The specific bacterial components and host receptors that underpin altered metabolic responses are emerging. We previously showed that Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) activation with bacterial peptidoglycan (PGN) caused insulin resistance in mice. We now show that PGN induces cell-autonomous lipolysis in adipocytes via NOD1. Specific bacterial PGN motifs stimulated lipolysis in white adipose tissue (WAT) explants from WT, but not NOD1^−/− mice. NOD1-activating PGN stimulated mitogen activated protein kinases (MAPK),protein kinase A (PKA), and NF-κB in 3T3-L1 adipocytes. The NOD1-mediated lipolysis response was partially reduced by inhibition of ERK1/2 or PKA alone, but not c-Jun N-terminal kinase (JNK). NOD1-stimulated lipolysis was partially dependent on NF-κB and was completely suppressed by inhibiting ERK1/2 and PKA simultaneously or hormone sensitive lipase (HSL). Our results demonstrate that bacterial PGN stimulates lipolysis in adipocytes by engaging a stress kinase, PKA, NF-κB-dependent lipolytic program. Bacterial NOD1 activation is positioned as a component of metabolic endotoxemia that can contribute to hyperlipidemia, systemic inflammation and insulin resistance by acting directly on adipocytes.     

Design,synthesis, and evaluation of potent novel peroxisome proliferator-activated receptor γ indole partial agonists

Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a nuclear receptor important for glucose homeostasis and insulin sensitivity. The anti-diabetic drugs thiazolidinediones improve insulin sensitivity by blocking PPARγ phosphorylation at S273; however, their full agonism on PPARγ also causes significant unwanted side effects. The indole derivative UHC1 displays insulin-sensitizing effect by acting as a partial agonist through the inhibition of PPARγ S273 phosphorylation, but without full agonist-associated side effects; however, its potency leaves much to be desired. Herein we report the design and synthesis of potent indole analogs as partial PPARγ agonists via the structure-activity relationship studies. Our studies revealed that vanillylamine and piperonyl benzylamine at Site 1 are favored to bind PPARγ with either biphenyl or 3-trifluoromethyl benzyl group at Site 2. In particular, compound WO91A with vanillylamine at Site 1 displays highly potent PPARγ binding affinity (IC₅₀ = 16.7 nM), over 30-fold more potent than the parental compound UHC1, yet with less side effect-associated transactivation activity.     

Dietary fish oil reduces systemic inflammation and ameliorates sepsis-induced liver injury by up-regulating the peroxisome proliferator-activated receptor gamma-mediated pathway in septic mice

This study investigated the effect of dietary fish oil on systemic inflammation and hepatic injury in mice with polymicrobial sepsis. Male ICR mice were assigned to a control group (C, n=30) and a fish oil group (FO, n=30). Mice in the C group were fed a semi-purified diet with 10% soybean oil, and those in the FO group were fed a fish oil diet (2.5% fish oil+7.5% soybean oil; w/w). Three weeks later, sepsis was induced by cecal ligation and puncture (CLP), and mice were sacrificed at 0, 6 and 24 h after CLP, respectively. Results showed that compared with C group, the FO group had lower plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and nitrite at 6 and 24 h after CLP. Also, peritoneal lavage fluid concentrations of TNF-α and prostaglandin (PG) E2 were significantly lower at 24 h in the FO than in the C group. The FO group had lower myeloperoxidase activities at 6 h after CLP in various organs. Plasma aminotransferase and alanine aminotransferase activities revealed significantly decreased in the FO group. The DNA-binding activity of peroxisome proliferators-activated receptor gamma (PPARγ) and mRNA expression of I kappaB alpha (IκBα) were up-regulated while nuclear factor (NF)-κB p65 DNA-binding activity, inducible nitric oxide synthase protein expression and the concentration of nitrotyrosine were significantly decreased in the FO group in liver after CLP. These results indicate that dietary fish oil administration may attenuate systemic inflammation and up-regulate hepatic PPARγ DNA-binding activity, which may consequently have ameliorated liver injury in these septic mice.     

Differential TLR2 downstream signaling regulates lipid metabolism and cytokine production triggered by Mycobacterium bovis BCG infection

The nuclear receptor PPARγ acts as a key modulator of lipid metabolism, inflammation and pathogenesis in BCG-infected macrophages. However, the molecular mechanisms involved in PPARγ expression and functions during infection are not completely understood. Here, we investigate signaling pathways triggered by TLR2, the involvement of co-receptors and lipid rafts in the mechanism of PPARγ expression, lipid body formation and cytokine synthesis in macrophages during BCG infection. BCG induces NF-κB activation and increased PPARγ expression in a TLR2-dependent manner. Furthermore, BCG-triggered increase of lipid body biogenesis was inhibited by the PPARγ antagonist GW9662, but not by the NF-κB inhibitor JSH-23. In contrast, KC/CXCL1 production was largely dependent on NF-κB but not on PPARγ. BCG infection induced increased expression of CD36 in macrophages in vitro. Moreover, CD36 co-immunoprecipitates with TLR2 in BCG-infected macrophages, suggesting its interaction with TLR2 in BCG signaling. Pretreatment with CD36 neutralizing antibodies significantly inhibited PPARγ expression, lipid body formation and PGE₂ production induced by BCG. Involvement of CD36 in lipid body formation was further confirmed by decreased BCG-induced lipid body formation in CD36 deficient macrophages. Similarly, CD14 and CD11b/CD18 blockage also inhibited BCG-induced lipid body formation, whereas TNF-α synthesis was not affected. Disruption of rafts recapitulates the latter result, inhibiting lipid body formation, but not TNF-α synthesis in BCG-infected macrophages. In conclusion, our results suggest that CD36-TLR2 cooperation and signaling compartmentalization within rafts, divert host response signaling through PPARγ-dependent and NF-κB-independent pathways, leading to increased macrophage lipid accumulation and down-modulation of macrophage response.     

Oleate prevents palmitate-induced mitochondrial dysfunction,insulin resistance and inflammatory signaling in neuronal cells

Elevated circulating levels of saturated free fatty acids (sFFAs; e.g. palmitate) are known to provoke inflammatory responses and cause insulin resistance in peripheral tissue. By contrast, mono- or poly-unsaturated FFAs are protective against sFFAs. An excess of sFFAs in the brain circulation may also trigger neuroinflammation and insulin resistance, however the underlying signaling changes have not been clarified in neuronal cells. In the present study, we examined the effects of palmitate on mitochondrial function and viability as well as on intracellular insulin and nuclear factor-κB (NF-κB) signaling pathways in Neuro-2a and primary rat cortical neurons. We next tested whether oleate preconditioning has a protective effect against palmitate-induced toxicity. Palmitate induced both mitochondrial dysfunction and insulin resistance while promoting the phosphorylation of mitogen-activated protein kinases and nuclear translocation of NF-κB p65. Oleate pre-exposure and then removal was sufficient to completely block subsequent palmitate-induced intracellular signaling and metabolic derangements. Oleate also prevented ceramide-induced insulin resistance. Moreover, oleate stimulated ATP while decreasing mitochondrial superoxide productions. The latter were associated with increased levels of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Inhibition of protein kinase A (PKA) attenuated the protective effect of oleate against palmitate, implicating PKA in the mechanism of oleate action. Oleate increased triglyceride and blocked palmitate-induced diacylglycerol accumulations. Oleate preconditioning was superior to docosahexaenoic acid (DHA) or linoleate in the protection of neuronal cells against palmitate- or ceramide-induced cytotoxicity. We conclude that oleate has beneficial properties against sFFA and ceramide models of insulin resistance-associated damage to neuronal cells.     

PPARα protects against trans-fatty-acid-containing diet-induced steatohepatitis

Consumption of trans-fatty acids (TFA), unsaturated fatty acids (FA) containing trans double bonds, is a risk factor for metabolic syndrome and steatohepatitis. Peroxisome proliferator-activated receptor α (PPARα) is a master regulator of hepatic lipid homeostasis. To examine the contribution of PPARα to changes in liver phenotypes induced by TFA, two diets were used: a purified control diet and an isocaloric diet in which most of the soybean oil, a major source of FA in the diet, was replaced with TFA-rich shortening. The diets were fed to wild-type and Ppara-null mice for 2 months. Ppara-null mice fed a TFA-containing diet showed more severe hepatic steatosis and liver damage compared with similarly treated wild-type mice, as revealed by increased hepatic triglyceride (TG) contents and serum alanine aminotransferase activities. While the TFA-rich diet increased the hepatic expression of enzymes involved in de novo FA synthesis and decreased TG-hydrolyzing enzymes in both genotypes, the expression of FA-catabolizing enzymes was decreased in Ppara-null mice, resulting in more severe hepatosteatosis. Additionally, the expression levels of key contributors to inflammation, such as osteopontin, were increased, and nuclear factor-kappa B was activated in TFA-containing diet-fed Ppara-null mice. Enhanced inflammatory signaling in these mice was presumably mediated by toll-like receptor 2, with no accompanying inflammasome activation. Collectively, these results suggest a protective role for PPARα in the pathological changes in the liver following TFA consumption. PPARα might prevent TFA-containing diet-induced steatohepatitis.