FGF19 protects skeletal muscle against obesity-induced muscle atrophy,metabolic derangement and abnormal irisin levels via the AMPK/SIRT-1/PGC-α pathway

Obesity is associated with biological dysfunction in skeletal muscle. As a condition of obesity accompanied by muscle mass loss and physical dysfunction, sarcopenic obesity (SO) has become a novel public health problem. Human fibroblast growth factor 19 (FGF19) plays a therapeutic role in metabolic diseases. However, the protective effects of FGF19 on skeletal muscle in obesity and SO are still not completely understood. Our results showed that FGF19 administration improved muscle loss and grip strength in young and aged mice fed a high-fat diet (HFD). Increases in muscle atrophy markers (FOXO-3, Atrogin-1, MuRF-1) were abrogated by FGF19 in palmitic acid (PA)-treated C2C12 myotubes and in the skeletal muscle of HFD-fed mice. FGF19 not only reduced HFD-induced body weight gain, excessive lipid accumulation and hyperlipidaemia but also promoted energy expenditure (PGC-1α, UCP-1, PPAR-γ) in brown adipose tissue (BAT). FGF19 treatment restored PA- and HFD-induced hyperglycaemia, impaired glucose tolerance and insulin resistance (IRS-1, GLUT-4) and mitigated the PA- and HFD-induced decrease in FNDC-5/irisin expression. However, these beneficial effects of FGF19 on skeletal muscle were abolished by inhibiting AMPK, SIRT-1 and PGC-1α expression. Taken together, this study suggests that FGF19 protects skeletal muscle against obesity-induced muscle atrophy, metabolic derangement and abnormal irisin secretion partially through the AMPK/SIRT-1/PGC-α signalling pathway, which might be a potential therapeutic target for obesity and SO.     

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A)-AR. α(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited α(1B)-AR- but not α(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.     

PKCδ stimulates macropinocytosis via activation of SSH1-cofilin pathway

Macropinocytosis is an actin-dependent endocytic mechanism mediating internalization of extracellular fluid and associated solutes into cells. The present study was designed to identify the specific protein kinase C (PKC) isoform(s) and downstream effectors regulating actin dynamics during macropinocytosis. We utilized various cellular and molecular biology techniques, pharmacological inhibitors and genetically modified mice to study the signaling mechanisms mediating macropinocytosis in macrophages. The qRT-PCR experiments identified PKCδ as the predominant PKC isoform in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated the functional role of PKCδ in phorbol ester- and hepatocyte growth factor (HGF)-induced macropinocytosis. Western blot analysis demonstrated that phorbol ester and HGF stimulate activation of slingshot phosphatase homolog 1 (SSH1) and induce cofilin Ser-3 dephosphorylation via PKCδ in macrophages. Silencing of SSH1 inhibited cofilin dephosphorylation and macropinocytosis stimulation. Interestingly, we also found that incubation of macrophages with BMS-5, a potent inhibitor of LIM kinase, does not stimulate macropinocytosis. In conclusion, the findings of the present study demonstrate a previously unidentified mechanism by which PKCδ via activation of SSH1 and cofilin dephosphorylation stimulates membrane ruffle formation and macropinocytosis. The results of the present study may contribute to a better understanding of the regulatory mechanisms during macrophage macropinocytosis.     

Resveratrol regulates muscle fiber type conversion via miR-22-3p and AMPK/SIRT1/PGC-1α pathway

This study investigated the effects of resveratrol and miR-22-3p on muscle fiber type conversion in mouse C2C12 myotubes. Here we showed that resveratrol significantly increased the protein level of slow myosin heavy chain (MyHC) and the activities of succinic dehydrogenase and malate dehydrogenase, as well as markedly decreased the protein level of fast MyHC and the activity of lactate dehydrogenase. Immunofluorescence staining showed that resveratrol remarkably upregulated the number of slow MyHC-positive myotubes and downregulated the number of fast MyHC-positive myotubes, suggesting that resveratrol promoted muscle fiber type conversion from fast-twitch to slow-twitch in C2C12 myotubes. We also showed that miR-22-3p had an opposite function on muscle fiber type conversion and resveratrol was able to repress the expression of miR-22-3p. Furthermore, AMP-activated protein kinase (AMPK) inhibitor Compound C and miR-22-3p mimics could attenuate and eliminate muscle fiber type conversion from fast-twitch to slow-twitch cause by resveratrol, respectively. Together, we provided the first evidence that resveratrol promotes muscle fiber type conversion from fast-twitch to slow-twitch via miR-22-3p and AMPK/SIRT1/PGC-1α pathway in C2C12 myotubes.     

PGC-1α-mediated changes in phospholipid profiles of exercise-trained skeletal muscle

Exercise training influences phospholipid fatty acid composition in skeletal muscle and these changes are associated with physiological phenotypes; however, the molecular mechanism of this influence on compositional changes is poorly understood. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, the fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training induces these adaptations, together with increased PGC-1α, PGC-1α may contribute to the exercise-mediated change in phospholipid fatty acid composition. To determine the role of PGC-1α, we performed lipidomic analyses of skeletal muscle from genetically modified mice that overexpress PGC-1α in skeletal muscle or that carry KO alleles of PGC-1α. We found that PGC-1α affected lipid profiles in skeletal muscle and increased several phospholipid species in glycolytic muscle, namely phosphatidylcholine (PC) (18:0/22:6) and phosphatidylethanolamine (PE) (18:0/22:6). We also found that exercise training increased PC (18:0/22:6) and PE (18:0/22:6) in glycolytic muscle and that PGC-1α was required for these alterations. Because phospholipid fatty acid composition influences cell permeability and receptor stability at the cell membrane, these phospholipids may contribute to exercise training-mediated functional changes in the skeletal muscle.     

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A₂ via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca²⁺]_i) in MHS macrophage cells. Ionomycin increased [Ca²⁺]_i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca²⁺→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca²⁺ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca²⁺→ ERK pathway, which plays a key role in suppression of inflammatory reactions.     

Laminin regulates the osteogenic differentiation of dental follicle cells via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway

Dental follicle cells (DFCs) are ideal for studies concerning the differentiation of dental precursor cells into alveolar osteoblasts and cementoblasts. Previous investigations have suggested that the extracellular matrix (ECM) protein laminin and the ECM receptor integrin-α2/-β1 play regulatory roles during the osteogenic differentiation of DFCs. Our present data indicate that laminin impairs alkaline phosphatase (ALP) activity following osteogenic induction while inducing integrin-α2/-β1 expression, osteogenic differentiation marker elevation, and DFC biomineralization. Integrin-α2/-β1 facilitates the laminin-dependent expression of osteogenic differentiation markers and the laminin-dependent inhibition of ALP activity. Moreover, these laminin-dependent effects on the osteogenic differentiation of DFCs can be reversed by the inhibition of the FAK/ERK signaling pathway. Thus, laminin regulates the inhibition of early osteogenic differentiation markers and the induction of late osteogenic differentiation markers via integrin-α2/-β1 and the activation of the FAK/ERK signaling pathway.     

PPAR-γ/PGC-1α对支气管哮喘豚鼠肺组织Nrf2/γ-GCS-h作用

目的：观察PPAR-γ、PGC-1α、Nrf2和γ-GCS-h在豚鼠支气管哮喘肺组织中表达而探索PPAR-γ/PGC-1α对Nrf2/γ-GCS-h作用。方法：40只健康雄性豚鼠随机化原则分成对照组（A组）、哮喘组（B组）、地塞米松（C组）和罗格列酮治疗组（D组）,每组10只豚鼠,卵蛋白致敏法复制哮喘模型。原位杂交检测PPAR-γ、PGC-1α、Nrf2和γ-GCS-hmRNA表达,免疫组化和Western blot检测四种蛋白表达。结果：PPAR-γ、PGC-1α、Nrf2和γ-GCS-h的mRNA哮喘组表达最低,四组表达差异有统计学意义（P均〈0.01）;免疫组化和Western blot显示PPAR-γ、PGC-1α、Nrf2和γ-GCS-h的蛋白哮喘组表达几乎都呈阴性而且以核内表达为主,四组差异均有统计学意义（P均〈0.01）。PPAR-γ表达与PGC-1α表达呈正相关,γ-GCS-h mRNA表达与PPAR-γ、PGC-1α、Nrf2核内表达均呈正相关,Nrf2表达与PPAR-γ和PGC-1α表达均呈正相关。结论：PPAR-γ、PGC-1α、Nrf2和γ-GCS-h在卵蛋白致敏急性支气管哮喘模型中表达下降;PPAR-γ/PGC-1α可通过上调Nrf2/γ-GCS-h表达提高组织的抗氧化能力,因而PPAR-γ/PGC-1α在哮喘的发病和防治可能起重要的作用。     

Sufentanil protects the rat myocardium against ischemia–reperfusion injury via activation of the ERK1/2 pathway

Sufentanil, a lipophilic opioid, is the most frequently used clinical drug for ischemic heart disease. The effects of sufentanil on MAPK signaling in ischemic heart disease were explored. The effects of sufentanil on ischemia–reperfusion (IR)-induced myocardial injury in a rat model were examined. The serum levels of CK, LDH, MDA and SOD, and the activities of Na⁺–K⁺-ATPase and Ca²⁺–Mg²⁺-ATPase were measured. The levels of total and phosphorylated ERK1/2, JNK, and p38 were measured by western blotting in the heart, and the myocardial H9C2 cell line was studied. Using the Cell Counting Kit-8, the growth rate of H9C2 cells affected by sufentanil was studied. The serum levels of CK, LDH and MDA were higher in the IR group than in the SO and SUF groups. The SOD level, as well as the activities of Na⁺–K⁺-ATPase and Ca²⁺–Mg²⁺-ATPase, were lower in the SO and SUF groups than in the IR group. The phosphorylated ERK1/2 level was lower in the IR group than in the SO and SUF groups. The growth rate of H9C2 cells increased with the concentration of sufentanil and the exposure time. The phosphorylated ERK level was upregulated by 4–12 h of sufentanil exposure, indicating that the effects were time-dependent. Furthermore, an inhibition of ERK signaling by chemical inhibition suppressed the sufentanil-mediated increase in the growth rate of H9C2 cells. Sufentanil appears to be beneficial for cases of worsening ischemic heart disease. Further studies are necessary before a clinical application is considered.     

α-Lipoic acid increases tolerance of cardiomyoblasts to glucose/glucose oxidase-induced injury via ROS-dependent ERK1/2 activation

α-Lipoic acid (LA) has been shown to improve the diabetic cardiac symptoms. However, the underlying mechanisms have not been elucidated precisely. We have reported recently that LA potentially protected neurons from substance-induced apoptosis. We hypothesized that LA could attenuate cardiac cells death induced by oxidative stress derived from high glucose. To test this possibility, we examined the effects of LA on d-glucose/glucose oxidase (DG/GO, 30mM/5mU)-induced injury in rat cardiomyoblast H9c2 cells. We observed that LA pretreatment significantly increased cell viability in DG/GO-challenged cells. LA pretreatment also attenuated DG/GO-induced apoptosis as evidenced by decreases in both nuclear condensation and loss of mitochondrial potential. In addition, LA activated ERK1/2 and moderately increased ROS production. Blockade of ERK1/2 activation by PD98059 completely abolished LA-induced protection against DG/GO challenge. Inhibition of ROS by N-acetylcysteine abrogated LA-induced ERK1/2 activation and cytoprotection. Furthermore, we observed that the ROS production induced by LA was significantly slower and milder than that by DG/GO. Our results suggest that pretreatment with LA moderately increased ROS production to induce a preconditioning-like effect by ERK1/2 activation thereby increased tolerance of H9c2 cells to DG/GO challenge.     

Scutellarin ameliorates nonalcoholic fatty liver disease through the PPARγ/PGC-1α-Nrf2 pathway

Nonalcoholic fatty liver disease (NAFLD) is characterised by excessive accumulation of hepatic lipids and oxidative injury of hepatocytes. Scutellarin is a flavonoid glycoside having antioxidative stress activity. Our current study aims to investigate the molecular mechanism of scutellarin ameliorating NAFLD. Scutellarin treatment was applied to male C57BL/6 mice maintained on a high-fat diet (HFD) and HepG2 cells challenged with oleic acid. The antioxidation biochemical indicators and lipid levels in the liver and cells were detected by kits. Liver pathology was observed by light microscope, Oil Red O staining, and transmission electron microscope (TEM). In addition, quantitative real-time polymerase chain reactions (qRT-PCR) and western blot assays were employed to detect the mRNA and protein levels of various antioxidative-related genes in the presence or absence of peroxisome proliferator-activated receptor gamma (PPARγ); inhibitor GW9662. Our results showed that scutellarin could significantly reduce blood lipid levels and enhance antioxidative capacities in both the models. In addition, scutellarin treatment conspicuously activated PPARγ, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear factor erythroid-2-related factor (Nrf2), haem oxygenase-1 (HO-1), glutathione S-transferase (GST), and NAD(P)H quinone dehydrogenase one (NQO1), while it significantly inhibited nuclear factor kappa B (NF-κB), Kelch-like ECH-associated protein 1 (Keap1) at both the mRNA and protein levels. However, after interfered by GW9662, scutellarin effect was significantly decreased. The experimental data demonstrated that scutellarin showed strong hypolipidaemic, antioxidative, and liver protective activity which could be attributed to its regulating activity in the PPARγ/PGC-1α-Nrf2 signaling pathway.     

Treatment with celastrol protects against obesity through suppression of galanin-induced fat intake and activation of PGC-1α/GLUT4 axis-mediated glucose consumption

Overweight and obesity may cause several metabolic complications, including type 2 diabetes mellitus and hyperlipidemia. Despite years of progress in medicine, there are no highly effective pharmacological treatments for obesity. The natural compound celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium Wilfordi (thunder god vine) plant, exerts various bioactivities including anti-diabetic and anti-obese effects. Although celastrol could decrease food intake and obesity, the detailed mechanism for celastrol is still unclear as yet. Herein, we intended to determine the effect of celastrol on obesity and the underlying mechanisms. In the present study, diet-induced obese mice were treated with 100 μg/kg/d celastrol for the last 21 days, and 3T3-L1 cells were treated with celastrol for 6 h. The present findings showed that celastrol suppresses fat intake, and leads to weight loss by inhibiting galanin and its receptor expression in the hypothalamus of mice fed a high-fat diet. More importantly, in addition to these direct anti-obesity activities, celastrol augmented the PGC-1α and GLUT4 expression in adipocytes and skeletal muscles to increase glucose uptake through AKT and P38 MAPK activation. Celastrol also inhibited gluconeogenic activity through a CREB/PGC-1α pathway. In conclusion, the weight-lowering effects of celastrol are driven by decreased galanin-induced food consumption. Thus, this study contributes to our understanding of the anti-obese role of celastrol, and provides a possibility of using celastrol to treat obesity in clinic.