PI3K/Akt信号通路与胰岛素抵抗相关疾病的关系

磷脂酰肌醇3-激酶（PI3K）/Akt信号通路是参与多重生命活动的关键的信号通路,参与调节细胞的分裂、分化、凋亡等活动.PI3K/Akt信号通路与胰岛素抵抗（insulin resistance,IR）相关疾病如Ⅱ型糖尿病、心血管疾病、肥胖等关系密切.对PI3K/Akt通路的深入研究将为胰岛素抵抗相关疾病的防治提供新思路.     

Ginsenoside Rb1 promotes PC12 cell cycle kinetics through an adenylate cyclase–dependent protein kinase A pathway

Ginsenoside Rb1 (G-Rb1), a constituent of ginseng, bears various beneficial effects on neuroendocrine cells. Previous studies have indicated that G-Rb1 can enhance glutamate release in undifferentiated and differentiated PC12 cells via the protein kinase A (PKA)–dependent signaling pathway. We hypothesized that G-Rb1 stimulates rat adrenomedullary chromaffin cell line PC12 (PC12 cells) proliferation and mitosis by promoting the cell cycle at all regulatory points. This mechanism is partly mediated via the adenylate cyclase–dependent PKA signaling pathway. In the present study, we investigated the mechanism by which G-Rb1 promotes cell cycle kinetics from the PC12 cells. The cell cycle kinetics of these cells were determined using flow cytometric DNA analysis. Analysis of the PC12 cell cycle revealed that G-Rb1 may affect all phases of the cell cycle and accelerate cell cycle kinetics by stimulating G0G1 phase transiting to S and G2M phases. The cell cycle kinetics were decreased by coincubating with the adenylate cyclase inhibitor SQ22536. Compared with the G-Rb1–treated group, the PKA inhibitor H89 produced a marked decrease in the G-Rb1–stimulated cell cycle kinetics by inhibiting G0G1 phase from transiting to the S phase. These results support the position that G-Rb1 exerts a stimulatory effect on cell cycle kinetics to promote PC12 cell proliferation. The result also suggests that the division rate is mediated via the adenylate cyclase–dependent PKA signaling pathway.     

长期酒精摄入对大鼠骨骼肌组织IR、IRS-1和PI-3K基因表达的影响

目的研究酒精长期作用下对大鼠骨骼肌胰岛素受体(IR)、胰岛素受体底物-1(IRS-1)、磷脂酰肌醇3-激酶p85亚单位(PI-3K)mRNA表达的影响,探讨酒精与胰岛素抵抗的关系及相关分子机制。方法清洁级Wistar大鼠80只(雌雄各半),按体重随机分为对照组和低、中、高剂量组,分别给予蒸馏水以及10%、20%和33%酒精溶液,灌胃剂量为每天10ml/kgbw。第19周末,断头处死大鼠,测定空腹血糖和血胰岛素,计算HOMA胰岛素抵抗指数(HOMA-IR)。提取骨骼肌总RNA,通过RT-PCR测定IR、IRS-1、PI-3K(p85)mRNA表达水平。结果雄性大鼠,与对照组比较高剂量组空腹血糖,低、中剂量组空腹胰岛素水平升高,各酒精剂量组HOMA-IR指数均升高。IRmRNA的表达在中、高剂量组降低,而IRS-1、PI-3K(p85)mRNA表达水平表现为随着酒精剂量的增加先升高后降低,与对照组比较差异有显著性(P<0.05);与对照组比较,雌性大鼠高剂量组空腹血糖升高、空腹血胰岛素下降,IR、IRS-1、PI-3K(p85)mRNA的表达在中、高剂量组降低(P<0.05)。各剂量组HOMA-IR指数与对照组比较差异无显著性。结论长期摄入过量酒精可以造成骨骼肌组织IR、IRS-1、PI-3K(p85)mRNA表达的降低,这可能是酒精降低胰岛素敏感性,引起胰岛素抵抗的分子机制。     

Activation of PI3K/Akt Signaling Pathway in Rat Hypothalamus Induced by an Acute Oral Administration of D-Pinitol

D-Pinitol (DPIN) is a natural occurring inositol capable of activating the insulin pathway in peripheral tissues, whereas this has not been thoroughly studied in the central nervous system. The present study assessed the potential regulatory effects of DPIN on the hypothalamic insulin signaling pathway. To this end we investigated the Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (Akt) signaling cascade in a rat model following oral administration of DPIN. The PI3K/Akt-associated proteins were quantified by Western blot in terms of phosphorylation and total expression. Results indicate that the acute administration of DPIN induced time-dependent phosphorylation of PI3K/Akt and its related substrates within the hypothalamus, indicating an activation of the insulin signaling pathway. This profile is consistent with DPIN as an insulin sensitizer since we also found a decrease in the circulating concentration of this hormone. Overall, the present study shows the pharmacological action of DPIN in the hypothalamus through the PI3K/Akt pathway when giving in fasted animals. These findings suggest that DPIN might be a candidate to treat brain insulin-resistance associated disorders by activating insulin response beyond the insulin receptor.     

长期酒精摄入对雄性大鼠胰岛素敏感性及肝脏IR、IRS-1、IRS-2 mRNA表达的影响

目的从基因表达水平探讨长期酒精摄入影响肝脏胰岛素敏感性的分子机制。方法清洁级Wistar雄性大鼠40只,随机分为对照组和低、中、高剂量酒精组,每天摄入酒精剂量分别为0、0·8、1·6和2·4g/kg bw。给予酒精19周后,测定空腹血糖、血胰岛素,计算胰岛素抵抗指数(HOMA-IR)。提取肝脏总RNA,通过RT-PCR测定胰岛素受体(IR)、胰岛素受体底物1(IRS-1)、胰岛素受体底物2(IRS-2)的mRNA表达水平。结果与对照组相比,高剂量组血糖升高(P<0·05);各剂量组血胰岛素浓度均升高,低、中剂量组升高有显著性差异(P<0·05);各剂量组HOMA-IR均显著高于对照组(P<0·05)。各剂量组IR mRNA表达均降低;IRS-1及IRS-2mRNA表达在低、中剂量组升高,高剂量组降低。结论长期过量酒精摄入可以引起雄性大鼠胰岛素抵抗,肝脏IR、IRS-1、IRS-2mRNA表达降低是酒精影响胰岛素敏感性的分子机制之一。     

Antioxidants Supplementation Reduces Ceramide Synthesis Improving the Cardiac Insulin Transduction Pathway in a Rodent Model of Obesity

Obesity-related disruption in lipid metabolism contributes to cardiovascular dysfunction. Despite numerous studies on lipid metabolism in the left ventricle, there is no data describing the influence of n-acetylcysteine (NAC) and α-lipoic acid (ALA), as glutathione precursors, on sphingolipid metabolism, and insulin resistance (IR) occurrence. The aim of our experiment was to evaluate the influence of chronic antioxidants administration on myocardial sphingolipid state and intracellular insulin signaling as a potential therapeutic strategy for obesity-related cardiovascular IR. The experiment was conducted on male Wistar rats fed a standard rodent chow or a high-fat diet with intragastric administration of NAC or ALA for eight weeks. Cardiac and plasma sphingolipid species were assessed by high-performance liquid chromatography (HPLC). The proteins expressed from sphingolipid and insulin signaling pathways were determined by Western blot. Antioxidant supplementation markedly reduced ceramide accumulation by lowering the expression of selected proteins from the sphingolipid pathway and simultaneously increased the myocardial sphingosine-1-phosphate level. Moreover, NAC and ALA augmented the expression of GLUT4 and the phosphorylation state of Akt (Ser473) and GSK3β (Ser9), which improved the intracellular insulin transduction pathway. Based on our results, we may postulate that NAC and ALA have a beneficial influence on the cardiac ceramidose under IR conditions.     

Implications of Resveratrol in Obesity and Insulin Resistance: A State-of-the-Art Review

Background: Resveratrol is a polyphenol chemical that naturally occurs in many plant-based dietary products, most notably, red wine. Discovered in 1939, widespread interest in the potential health benefits of resveratrol emerged in the 1970s in response to epidemiological data on the cardioprotective effects of wine. Objective: To explore the background of resveratrol (including its origins, stability, and metabolism), the metabolic effects of resveratrol and its mechanisms of action, and a potential future role of dietary resveratrol in the lifestyle management of obesity. Data sources: We performed a narrative review, based on relevant articles written in English from a Pubmed search, using the following search terms: “resveratrol”, “obesity”, “Diabetes Mellitus”, and “insulin sensitivity”. Results: Following its ingestion, resveratrol undergoes extensive metabolism. This includes conjugation (with sulfate and glucuronate) within enterocytes, hydrolyzation and reduction within the gut through the action of the microbiota (with the formation of metabolites such as dihydroresveratrol), and enterohepatic circulation via the bile. Ex vivo studies on adipose tissue reveal that resveratrol inhibits adipogenesis and prevents the accumulation of triglycerides through effects on the expression of Peroxisome Proliferator-activated Receptor γ (PPARγ) and sirtuin 1, respectively. Furthermore, resveratrol induces anti-inflammatory effects, supported by data from animal-based studies. Limited data from human-based studies reveal that resveratrol improves insulin sensitivity and fasting glucose levels in patients with Type 2 Diabetes Mellitus and may improve inflammatory status in human obesity. Although numerous mechanisms may underlie the metabolic benefits of resveratrol, evidence supports a role in its interaction with the gut microbiota and modulation of protein targets, including sirtuins and proteins related to nitric oxide, insulin, and nuclear hormone receptors (such as PPARγ). Conclusions: Despite much interest, there remain important unanswered questions regarding its optimal dosage (and how this may differ between and within individuals), and possible benefits within the general population, including the potential for weight-loss and improved metabolic function. Future studies should properly address these important questions before we can advocate the widespread adoption of dietary resveratrol supplementation.     

1 - 硝基芘对GC - 2细胞的损伤效应及AhR信号通路的活化

目的 研究1 - 硝基芘（1 - nitropyrene, 1 - NP）对小鼠精母细胞株GC - 2的损伤效应及芳香烃受体（aryl hydrocarbon receptor, AhR）信号通路的影响。方法 建立不同剂量1 - NP染毒的GC - 2细胞模型，采用CCK - 8法检测细胞活性，流式细胞术检测细胞周期，Western blot分析各组细胞DNA双链断裂损伤标志物γ - H2AX、DNA损伤反应通路蛋白以及AhR通路蛋白的表达。实时荧光定量PCR检测细胞色素450 (CYP)1a1和1b1 mRNA表达水平。结果 1 - NP处理可导致GC - 2细胞活性降低及G2/M期细胞比例升高，同时诱导γ - H2AX蛋白表达升高，DNA损伤反应及细胞周期G2/M期检测点蛋白ATM、Chk1、Cdc25c、Cdc2的磷酸化表达水平升高，Cyclin B1表达降低。1 - NP染毒也提高了GC - 2细胞中AhR核蛋白及下游靶基因CYP1A1的蛋白表达，提高了Cyp1a1和Cyp1b1的转录表达水平。结论 1 - NP暴露造成小鼠生精细胞的DNA损伤、细胞周期阻滞和细胞活性抑制，其效应可能与AhR信号通路的活化及对化学物的代谢活化有关。     

Elimination of Vitamin D Signaling Causes Increased Mortality in a Model of Overactivation of the Insulin Receptor: Role of Lipid Metabolism

Vitamin D (VD) deficiency has been associated with cancer and diabetes. Insulin signaling through the insulin receptor (IR) stimulates cellular responses by activating the PI3K/AKT pathway. PTEN is a tumor suppressor and a negative regulator of the pathway. Its absence enhances insulin signaling leading to hypoglycemia, a dangerous complication found after insulin overdose. We analyzed the effect of VD signaling in a model of overactivation of the IR. We generated inducible double KO (DKO) mice for the VD receptor (VDR) and PTEN. DKO mice showed severe hypoglycemia, lower total cholesterol and increased mortality. No macroscopic tumors were detected. Analysis of the glucose metabolism did not show clear differences that would explain the increased mortality. Glucose supplementation, either systemically or directly into the brain, did not enhance DKO survival. Lipidic liver metabolism was altered as there was a delay in the activation of genes related to β-oxidation and a decrease in lipogenesis in DKO mice. High-fat diet administration in DKO significantly improved its life span. Lack of vitamin D signaling increases mortality in a model of overactivation of the IR by impairing lipid metabolism. Clinically, these results reveal the importance of adequate Vitamin D levels in T1D patients.     

Downregulation of the cyclin D1/Cdk4 complex occurs during resveratrol-induced cell cycle arrest in colon cancer cell lines

Resveratrol is a naturally occurring polyphenol with cancer chemopreventive properties. The objective of the current study was to investigate the effect of resveratrol on the human colonic adenocarcinoma cell line Caco-2. The compound inhibited cell growth and proliferation of Caco-2 cells in a dose-dependent manner (12.5-200 micromol/L) as assessed by crystal violet assay, [(3)H]thymidine and [(14)C]leucine incorporation. Furthermore, apoptosis was determined by measuring caspase-3 activity, which increased significantly after 24 and 48 h of treatment with 200 micromol/L resveratrol. Perturbed cell cycle progression from the S to G2 phase was observed for concentrations up to 50 micromol/L, whereas higher concentrations led to reversal of the S phase arrest. These effects were specific for resveratrol; they were not observed after incubation with the stilbene analogs stilbenemethanol and rhapontin. Levels of cyclin D1 and cyclin-dependent kinase (cdk) 4 proteins were decreased, as revealed by immunoblotting. In addition, resveratrol enhanced the expression of cyclin E and cyclin A. The protein levels of cdk2, cdk6 and proliferating cell nuclear antigen were unaffected. Similar results were obtained for the colon carcinoma cell line HCT-116, indicating that cell cycle inhibition by resveratrol is independent of cyclooxygenase inhibition. The phosphorylation state of the retinoblastoma protein in Caco-2 cells was shifted from hyperphosphorylated to hypophosphorylated at 200 micromol/L, which may account for reversal of the S phase block at concentrations exceeding 50 micromol/L. These findings suggest that resveratrol exerts chemopreventive effects on colonic cancer cells by inhibition of the cell cycle.     

Vitamin D Receptor Protects against Radiation-Induced Intestinal Injury in Mice via Inhibition of Intestinal Crypt Stem/Progenitor Cell Apoptosis

It is urgent to seek new potential targets for the prevention or relief of gastrointestinal syndrome in clinical radiation therapy for cancers. Vitamin D, mediated through the vitamin D receptor (VDR), has been identified as a protective nutrient against ionizing radiation (IR)-induced damage. This study investigated whether VDR could inhibit IR-induced intestinal injury and explored underlying mechanism. We first found that vitamin D induced VDR expression and inhibited IR-induced DNA damage and apoptosis in vitro. VDR was highly expressed in intestinal crypts and was critical for crypt stem/progenitor cell proliferation under physiological conditions. Next, VDR-deficient mice exposed to IR significantly increased DNA damage and crypt stem/progenitor cell apoptosis, leading to impaired intestinal regeneration as well as shorter survival time. Furthermore, VDR deficiency activated the Pmaip1-mediated apoptotic pathway of intestinal crypt stem/progenitor cells in IR-treated mice, whereas inhibition of Pmaip1 expression by siRNA transfection protected against IR-induced cell apoptosis. Therefore, VDR protects against IR-induced intestinal injury through inhibition of crypt stem/progenitor cell apoptosis via the Pmaip1-mediated pathway. Our results reveal the importance of VDR level in clinical radiation therapy, and targeting VDR may be a useful strategy for treatment of gastrointestinal syndrome.     

Foxtail Millet Improves Blood Glucose Metabolism in Diabetic Rats through PI3K/AKT and NF-κB Signaling Pathways Mediated by Gut Microbiota

Foxtail millet (FM) is receiving ongoing increased attention due to its beneficial health effects, including the hypoglycemic effect. However, the underlying mechanisms of the hypoglycemic effect have been underexplored. In the present study, the hypoglycemic effect of FM supplementation was confirmed again in high-fat diet and streptozotocin-induced diabetic rats with significantly decreased fasting glucose (FG), glycated serum protein, and areas under the glucose tolerance test (p < 0.05). We employed 16S rRNA and liver RNA sequencing technologies to identify the target gut microbes and signaling pathways involved in the hypoglycemic effect of FM supplementation. The results showed that FM supplementation significantly increased the relative abundance of Lactobacillus and Ruminococcus_2, which were significantly negatively correlated with FG and 2-h glucose. FM supplementation significantly reversed the trends of gene expression in diabetic rats. Specifically, FM supplementation inhibited gluconeogenesis, stimulated glycolysis, and restored fatty acid synthesis through activation of the PI3K/AKT signaling pathway. FM also reduced inflammation through inhibition of the NF-κB signaling pathway. Spearman’s correlation analysis indicated a complicated set of interdependencies among the gut microbiota, signaling pathways, and metabolic parameters. Collectively, the above results suggest that the hypoglycemic effect of FM was at least partially mediated by the increased relative abundance of Lactobacillus, activation of the PI3K/AKT signaling pathway, and inhibition of the NF-κB signaling pathway.     

重组人生长激素体外激活人结肠癌细胞JAK2-STAT3通路

目的探讨重组人生长激素（rhGH）对不同生长激素受体（GHR）表达状态的人结肠癌细胞生长及JAK2-STAT3通路的影响。方法采用流式细胞术根据GHR表达状态不同筛选结肠癌细胞株,选择LOVO细胞株和HCT-8细胞株进入实验,采用MTT法分析rhGH对人结肠癌细胞生长的作用,采用流式细胞术进行细胞增殖指数（PI）、细胞周期分析及凋亡检测,采用蛋白质印迹法分析JAK2-STAT3通路相关分子的蛋白水平变化。结果HCT-8细胞株GHR呈阳性表达（59．6％）,LOVO细胞株GHR呈阴性表达（3．5％）。rhGH显著促进HCT-8细胞生长,G2／M期比例明显高于未处理组（P=0．0073）,PI升高,凋亡率降低,且pJAK2、pSTAT3、VEGF、CyelinD1、Bcl-xL蛋白表达增加。经rhGH处理后,LOVO细胞的上述各项检测指标未出现明显变化。结论rhGH促进GHR表达较高的HCT-8细胞生长,上调JAK2-STAT3信号转导通路多个关键节点的基因表达;不促进GHR低表达的LOVO细胞生长及JAK2-STAT3通路因子表达变化。