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.     

Oridonin attenuates atherosclerosis by inhibiting foam macrophage formation and inflammation through FABP4/PPARγ signalling

Both lipid accumulation and inflammatory response in lesion macrophages fuel the progression of atherosclerosis, leading to high mortality of cardiovascular disease. A therapeutic strategy concurrently targeting these two risk factors is promising, but still scarce. Oridonin, the bioactive medicinal compound, is known to protect against inflammatory response and lipid dysfunction. However, its effect on atherosclerosis and the underlying molecular mechanism remain elusive. Here, we showed that oridonin attenuated atherosclerosis in hyperlipidemic ApoE knockout mice. Meanwhile, we confirmed the protective effect of oridonin on the oxidized low-density lipoprotein (oxLDL)-induced foam macrophage formation, resulting from increased cholesterol efflux, as well as reduced inflammatory response. Mechanistically, the network pharmacology prediction and further experiments revealed that oridonin dramatically facilitated the expression of peroxisome proliferator-activated receptor gamma (PPARγ), thereby regulating liver X receptor-alpha (LXRα)-induced ATP-binding cassette transporter A1 (ABCA1) expression and nuclear factor NF-kappa-B (NF-κB) translocation. Antagonist of PPARγ reversed the cholesterol accumulation and inflammatory response mediated by oridonin. Besides, RNA sequencing analysis revealed that fatty acid binding protein 4 (FABP4) was altered responding to lipid modulation effect of oridonin. Overexpression of FABP4 inhibited PPARγ activation and blunted the benefit effect of oridonin on foam macrophages. Taken together, oridonin might have potential to protect against atherosclerosis by modulating the formation and inflammatory response in foam macrophages through FABP4/PPARγ signalling.     

Selective inhibition of PKCβ2 improves Caveolin-3/eNOS signaling and attenuates lipopolysaccharide-induced injury by inhibiting autophagy in H9C2 cardiomyocytes

Journal of Molecular Histology - Lipopolysaccharide (LPS)-induced autophagy is involved in sepsis-associated myocardial injury with increased PKCβ2 activation. We previously found...     

Oral treatment with γ-aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in high fat diet-fed mice

Adipocyte and β-cell dysfunction and macrophage-related chronic inflammation are critical for the development of obesity-related insulin resistance and type 2 diabetes mellitus (T2DM), which can be negatively regulated by Tregs. Our previous studies and those of others have shown that activation of γ-aminobutyric acid (GABA) receptors inhibits inflammation in mice. However, whether GABA could modulate high fat diet (HFD)-induced obesity, glucose intolerance and insulin resistance has not been explored. Here, we show that although oral treatment with GABA does not affect water and food consumption it inhibits the HFD-induced gain in body weights in C57BL/6 mice. Furthermore, oral treatment with GABA significantly reduced the concentrations of fasting blood glucose, and improved glucose tolerance and insulin sensitivity in the HFD-fed mice. More importantly, after the onset of obesity and T2DM, oral treatment with GABA inhibited the continual HFD-induced gain in body weights, reduced the concentrations of fasting blood glucose and improved glucose tolerance and insulin sensitivity in mice. In addition, oral treatment with GABA reduced the epididymal fat mass, adipocyte size, and the frequency of macrophage infiltrates in the adipose tissues of HFD-fed mice. Notably, oral treatment with GABA significantly increased the frequency of CD4(+)Foxp3(+) Tregs in mice. Collectively, our data indicated that activation of peripheral GABA receptors inhibited the HFD-induced glucose intolerance, insulin resistance, and obesity by inhibiting obesity-related inflammation and up-regulating Treg responses in vivo. Given that GABA is safe for human consumption, activators of GABA receptors may be valuable for the prevention of obesity and intervention of T2DM in the clinic.     

D2 Thr92Ala and PPARγ2 Pro12Ala polymorphisms interact in the modulation of insulin resistance in type 2 diabetic patients

Type 2 deiodinase (D2) converts T4 into its active metabolite T3, an essential step in thyroid metabolism. A Thr92Ala polymorphism in the gene encoding D2 has been inconsistently associated with insulin resistance (IR). Recently, it was reported that the D2 Thr92Ala (rs225014) and the peroxisome proliferator-activated receptor (PPAR) γ2 Pro12Ala (rs1801282) polymorphisms interact in the modulation of metabolic syndrome in nondiabetic subjects. Here, we investigated the effect of both polymorphisms, isolated or in combination, on IR in patients with type 2 diabetes mellitus (DM2). The D2 Thr92Ala and PPARγ2 Pro12Ala polymorphisms were genotyped in 721 DM2 patients. IR was evaluated using the homeostasis model assessment-IR (HOMA(IR)) index in a subgroup of 246 DM2 subjects. The frequencies of D2 Ala92 and PPARγ2 Ala12 variants were 0.390 and 0.074, respectively. Patients carrying D2 Ala/Ala genotype had a higher fasting plasma insulin and HOMA(IR) index as compared to patients carrying Thr/Ala or Thr/Thr genotypes (P = 0.022 and P = 0.001, respectively). A significant synergistic effect was observed between D2 Thr92Ala and PPARγ2 Pro12Ala polymorphisms on HOMA(IR) index, with carriers of both D2 Ala/Ala genotype and PPARγ2 Ala12 allele showing the highest HOMA(IR) values, after adjusting for age, gender, BMI, and use of medication for DM2 (P = 0.010). In conclusion, DM2 patients harboring both D2 Ala/Ala genotype and PPARγ2 Ala12 allele seem to present more severe IR than those with other D2/PPARγ2 genotype combinations. These findings suggest that these polymorphisms interact in the IR modulation, which may constitute a potential therapeutic target.     

Influence of different CLA isomers on insulin resistance and adipocytokines in pre-diabetic, middle-aged men with PPARγ2 Pro12Ala polymorphism

Conjugated linoleic acids (CLAs) are natural PPARγ ligands, which showed conflicting effects on metabolism in humans. We examined metabolic effects of different isomers of CLA in subjects with PPARγ2 Pro12Ala polymorphisms. A total of 35 men underwent four intervention periods in a crossover study design: subjects with either genotypes received c9, t11 CLA or t10, c12 CLA, a commercially available 1:1 mix of both isomers or reference oil (linoleic acid (LA)). Adipocytokines, insulin, glucose and triglycerides were assessed in the fasting state and after a standardized mixed meal. Across all genotypes, there was a significant (p = 0.025) CLA treatment effect upon postprandial (pp) HOMA-IR values, with c9, t11 CLA and CLA isomer mix improving, but t10, c12 CLA isomer worsening. In Ala12Ala subjects, the t10, c12 isomer caused weight gain (p = 0.03) and tended to increase postprandial insulin levels (p = 0.05). In Pro12Pro subjects, t10, c12 resulted in reduction in waist circumference (p = 0.03). The comparison of the different genotype groups revealed statistically different changes in fasting and postprandial insulin, HOMA-IR and leptin after intervention. c9, t11 CLA and the commercial CLA mix showed beneficial effects on insulin sensitivity compared with LA, while t10, c12 CLA adversely affects body weight and insulin sensitivity in different PPAR genotypes. CLA isomers have different effects on metabolism in Ala and Pro carriers.     

Osthole improves an accelerated focal segmental glomerulosclerosis model in the early stage by activating the Nrf2 antioxidant pathway and subsequently inhibiting NF-κB-mediated COX-2 expression and apoptosis

Inflammatory reactions and oxidative stress are implicated in the pathogenesis of focal segmental glomerulosclerosis (FSGS), a common chronic kidney disease with relatively poor prognosis and unsatisfactory treatment regimens. Previously, we showed that osthole, a coumarin compound isolated from the seeds of Cnidium monnieri, can inhibit reactive oxygen species generation, NF-κB activation, and cyclooxygenase-2 expression in lipopolysaccharide-activated macrophages. In this study, we further evaluated its renoprotective effect in a mouse model of accelerated FSGS (acFSGS), featuring early development of proteinuria, followed by impaired renal function, glomerular epithelial cell hyperplasia lesions (a sensitive sign that precedes the development of glomerular sclerosis), periglomerular inflammation, and glomerular hyalinosis/sclerosis. The results show that osthole significantly prevented the development of the acFSGS model in the treated group of mice. The mechanisms involved in the renoprotective effects of osthole on the acFSGS model were mainly a result of an activated Nrf2-mediated antioxidant pathway in the early stage (proteinuria and ischemic collapse of the glomeruli) of acFSGS, followed by a decrease in: (1) NF-κB activation and COX-2 expression as well as PGE₂ production, (2) podocyte injury, and (3) apoptosis. Our data support that targeting the Nrf2 antioxidant pathway may justify osthole being established as a candidate renoprotective compound for FSGS.     

Curcumin ameliorates IL-1β-induced apoptosis by activating autophagy and inhibiting the NF-κB signaling pathway in rat primary articular chondrocytes

Articular cartilage damage and chondrocyte apoptosis are common features of rheumatoid arthritis and osteoarthritis. Recently, curcumin has been reported to exhibit protective effects on degeneration in articular cartilage diseases. However, the effects and mechanisms of curcumin on articular chondrocyte injury remain to be elucidated. The aim of the present study is to investigate the chondroprotective mechanisms of curcumin on interleukin-1β (IL-1β)-induced chondrocyte apoptosis in vitro. The results revealed that IL-1β decreased cell viability and induced apoptosis in primary articular chondrocytes. Curcumin pretreatment reduced IL-1β-induced articular chondrocyte apoptosis. In addition, treatment with curcumin increased autophagy in articular chondrocytes and protected against IL-1β-induced apoptosis. The curcumin-mediated protection against IL-1β induced apoptosis was abolished when cells were treated with the autophagy inhibitor 3-methyladenine or transfected with Beclin-1 small interfering RNA. Furthermore, IL-1β stimulation significantly increased the phosphorylation levels of nuclear factor (NF)-κB p65 and glycogen synthase kinase-3β, and decreased the phosphorylation levels of β-catenin in articular chondrocytes, and these alterations to the phosphorylation levels were partly reversed by treatment with curcumin. Dual-luciferase and electrophoretic mobility shift assays demonstrated that IL-1β increased NF-κB p65 promoter activity in chondrocytes, and this was also reversed by curcumin. Pretreatment with the NF-κB inhibitor pyrrolidine dithiocarbamate enhanced the protective effects of curcumin on chondrocyte apoptosis, but Wnt/β-catenin inhibitor, XAV-939, did not exhibit this effect. Molecular docking and dynamic simulation studies results showed that curcumin could bound to RelA (p65) protein. These results indicate that curcumin may suppress IL-1β-induced chondrocyte apoptosis through activating autophagy and restraining NF-κB signaling pathway.     

PPARδ activation attenuates hepatic steatosis in Ldlr?/? mice by enhanced fat oxidation,reduced lipogenesis,and improved insulin sensitivity

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr^−/− mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA β-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKβ1^−/− hepatocytes. However, FA oxidation was only partially reduced in AMPKβ1^−/− hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKβ1^−/− hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.     

Xanthine-based KMUP-1 improves HDL via PPARγ/SR-B1, LDL via LDLRs,and HSL via PKA/PKG for hepatic fat loss

The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1–14 or 8–14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10⁻⁷∼10⁻⁵ M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3′,5′-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit ¹⁴C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight.     

An increase in liver PPARγ2 is an initial event to induce fatty liver in response to a diet high in butter: PPARγ2 knockdown improves fatty liver induced by high-saturated fat

The effects of a diet rich in saturated fat on fatty liver formation and the related mechanisms that induce fatty liver were examined. C57BL/6J mice were fed butter or safflower oil as a high-fat (HF) diet (40% fat calories) for 2, 4, 10, or 17 weeks. Although both HF diets induced similar levels of obesity, HF butter-fed mice showed a two to threefold increase in liver triacylglycerol (TG) concentration compared to HF safflower oil-fed mice at 4 or 10 weeks without hyperinsulinemia. At 4 weeks, increases in peroxisome proliferator-activated receptor γ2 (PPARγ2), CD36, and adipose differentiation-related protein (ADRP) mRNAs were observed in HF butter-fed mice; at 10 weeks, an increase in sterol regulatory element-binding protein-1c (SREBP-1c) was observed; at 17 weeks, these increases were attenuated. At 4 weeks, a single injection of adenoviral vector-based short hairpin interfering RNA against PPARγ2 in HF butter-fed mice reduced PPARγ protein and mRNA of its target genes (CD36 and ADRP) by 43%, 43%, and 39%, respectively, with a reduction in liver TG concentration by 38% in 5 days. PPARγ2 knockdown also reduced mRNAs in lipogenic genes (fatty-acid-synthase, stearoyl-CoA desaturase 1, acetyl-CoA carboxylase 1) without alteration of SREBP-1c mRNA. PPARγ2 knockdown reduced mRNAs in genes related to inflammation (CD68, interleukin-1β, tumor necrosis factor-α, and monocyte chemoattractant protein-1). In conclusion, saturated fatty acid-rich oil induced fatty liver in mice, and this was triggered initially by an increase in PPARγ2 protein in the liver, which led to increased expression of lipogenic genes. Inactivation of PPARγ2 may improve fatty liver induced by HF saturated fat.     

Role of inhibitory κB kinase and c-Jun NH2-terminal kinase in the development of hepatic insulin resistance in critical illness diabetes

Hyperglycemia and insulin resistance induced by acute injuries or critical illness are associated with increased mortality and morbidity, as well as later development of type 2 diabetes. The molecular mechanisms underlying the acute onset of insulin resistance following critical illness remain poorly understood. In the present studies, the roles of serine kinases, inhibitory κB kinase (IKK) and c-Jun NH(2)-terminal kinase (JNK), in the acute development of hepatic insulin resistance were investigated. In our animal model of critical illness diabetes, activation of hepatic IKK and JNK was observed as early as 15 min, concomitant with the rapid impairment of hepatic insulin signaling and increased serine phosphorylation of insulin receptor substrate 1. Inhibition of IKKα or IKKβ, or both, by adenovirus vector-mediated expression of dominant-negative IKKα or IKKβ in liver partially restored insulin signaling. Similarly, inhibition of JNK1 kinase by expression of dominant-negative JNK1 also resulted in improved hepatic insulin signaling, indicating that IKK and JNK1 kinases contribute to critical illness-induced insulin resistance in liver.     

Knockdown of TRPV4 suppresses osteoclast differentiation and osteoporosis by inhibiting autophagy through Ca2+–calcineurin–NFATc1 pathway

The aim of this study is to evaluate the effect of transient receptor potential vanilloid 4 (TRPV4) on osteoclast differentiation and osteoporosis, and to investigate the underlying mechanism. The results showed that TRPV4 expression and intracellular Ca²⁺ concentration were significantly upregulated in macrophage colony-stimulating factor (M-CSF)-stimulated and receptor activator of nuclear factor κΒ ligand (RANKL)-stimulated RAW264.7 cells. Furthermore, TRPV4 overexpression further increased the M-CSF- and RANKL-induced number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and expression of osteoclastogenesis-related genes (TRAP, c-Fos, and nuclear factor of activated T cells [NFATc1]), activated the Ca ²⁺–calcineurin–NFATc1 signaling and increased autophagy-related proteins (light chain [LC] 3II and Beclin-1) during osteoclast differentiation. In contrast, TRPV4 knockdown exerted the opposite effects. Mechanically, inhibition of Ca ²⁺–calcineurin–NFATc1 signaling by FK506 or 11R-VIVIT abrogated the TRPV4 overexpression-induced osteoclast differentiation and autophagy induction. Moreover, suppression of autophagy by 3-methyladenine attenuated the TRPV4-induced osteoclast differentiation. In addition, short hairpin RNA TRPV4-lentivirus administration significantly diminished the increased levels of several osteoclastogenesis-related genes (RANKL, TRAP, and tumor necrosis factor-α), alleviated the disturbed microarchitecture of lumbar vertebrae, restored the decreased bone mineral density, ratio of bone volume to total tissue volume, trabecular thickness, and trabecular number, and diminished the increased trabecular separation, in ovariectomy (OVX)-induced osteoporosis mice. Consistent with the in vitro data, TRPV4 knockdown significantly decreased the induced number of TRAP-positive osteoclasts, the increased LC3 and NFATc1 expression in the lumbar vertebrae of OVX mice. In conclusion, TRPV4 knockdown suppresses osteoclast differentiation and osteoporosis by inhibiting autophagy through Ca ²⁺–calcineurin–NFATc1 pathway.     

Wnt5b stimulates adipogenesis by activating PPARγ, and inhibiting the β-catenin dependent Wnt signaling pathway together with Wnt5a

Correct Wnt signaling is required for adipogenesis and alterations occur in Type 2 diabetes mellitus (T2DM). Gene expression studies showed that β-catenin independent Wnt5b was down-regulated in T2DM preadipocytes, while its paralog Wnt5a was unchanged. Our study aimed at defining the expression profile and function of Wnt5a and Wnt5b during adipogenesis by determining their effect on aP2 and PPARγ expression and assessing the level of β-catenin translocation in mouse 3T3-L1 preadipocytes. Additionally, we explored the effect on adipogenic capacity by Wnt5b overexpression in combination with stimulation of the β-catenin dependent or β-catenin independent Wnt signaling. Expression of Wnt5b was, like Wnt5a, down-regulated upon induction of differentiation and both inhibit β-catenin dependent Wnt signaling at the initiation of adipogenesis. Wnt5b additionally appears to be a potent enhancer of adipogenic capacity by stimulation of PPARγ and aP2. Down-regulation of Wnt5b could therefore contribute to decreased adipogenesis observed in T2DM diabetic subjects.