12b-hydroxy-des-D-garcigerin A enhances glucose metabolism in insulin-resistant HepG2 cells via the IRS-1/PI3-K/Akt cell signaling pathway

HepG2 cells were induced with a high concentration of insulin to establish an insulin-resistant cell model (HepG2/IR). The effect of 12b-hydroxy-des-D-garcigerin A (DGA) on the glucose consumption (GC) of HepG2/IR cells was analyzed with the glucose oxidase/peroxidase assay. The results showed that DGA significantly stimulated GC by enhancing the activity of hexokinase (HK) and pyruvate kinase (PK) in HepG2/IR cells. The cell signaling pathway by which DGA enhances the GC of HepG2/IR cells was explored. The results showed that DGA promoted the expression of insulin receptor (InsR) protein, and stimulated the expression of insulin receptor substrate 1 (IRS-1), phosphatidylinositol-3 kinase (p-PI3-K), and phospho-protein kinase B Serine⁴⁷³ (p-AKT ser⁴⁷³). Therefore, we concluded that DGA improved the insulin-resistance of HepG2/IR cells by inducing the IRS-1/PI3-K/Akt cell signaling pathway. Interestingly, DGA had no effect on the phosphorylation of threonine¹⁷² (Thr¹⁷²) in AMP-activated protein kinase (AMPK).     

吡格列酮对胰岛素抵抗HepG2细胞模型的药理学评价

目的应用高胰岛素诱导培养HepG2细胞,建立胰岛素抵抗的细胞模型。并探讨吡格列酮对该模型胰岛素敏感性和糖代谢的影响。方法将HepG2细胞置于5×10-7mol.L-1胰岛素培养液中16 h,采用3H-D-葡萄糖参入试验观察高胰岛素对HepG2细胞葡萄糖摄取率的影响。模型建立后,培养液中加入吡格列酮共同孵育,观察吡格列酮对胰岛素抵抗细胞模型葡萄糖摄取率和葡萄糖消耗量的影响。结果高胰岛素诱导培养的HepG2细胞葡萄糖参入率明显低于未用高胰岛素诱导的HepG2细胞(对照细胞)。将高胰岛素诱导培养的HepG2细胞置于不含胰岛素的培养液中60 h,其细胞葡萄糖摄取率仍明显低于对照细胞。含有吡格列酮(浓度为1×10-6~1×10-4mol.L-1)的胰岛素抵抗HepG2细胞的葡萄糖参入率与葡萄糖消耗量明显高于不含吡格列酮的胰岛素抵抗HepG2细胞(P<0.01)。结论将HepG2置于5×10-7mol.L-1胰岛素环境中16 h,该细胞对胰岛素的生物学效应产生抵抗,其胰岛素抵抗状态可维持60 h。该方法较为简便、易行、重复性好、成功率高,可广泛用于胰岛素抵抗的研究。吡格列酮能增加胰岛素抵抗模型细胞的胰岛素敏感性,并能明显改善糖代谢。     

芒果苷对胰岛素抵抗HepG2细胞糖脂代谢的影响

目的:分析芒果苷(MGF)对胰岛素抵抗HepG2细胞(IR-HepG2细胞)糖脂代谢的影响,并探讨潜在机制.方法:以人肝癌HepG2细胞为对象,以1 mmol/L棕榈酸+2 mmol/L油酸联合培养建立IR-HepG2细胞模型.以盐酸二甲双胍为阳性对照,分别检测低、中、高浓度MGF(125、250、500μmol/L)...     

Bis(alpha-furancarboxylato)oxovanadium(IV) prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes

Previous studies showed that bis(alpha-furancarboxylato)oxovanadium(IV) (BFOV), an orally active anti-diabetic organic vanadium complex, could improve insulin resistance in animals with type 2 diabetes. The present study has been carried out to evaluate the effects of BFOV on insulin-resistant glucose metabolism using dexamethasone-treated 3T3-L1 adipocytes as an in-vitro model of insulin resistance. The results showed that BFOV, similar to vanadyl sulfate and rosiglitazone, caused a concentration-dependent increase in glucose consumption by insulin-resistant adipocytes. Moreover, BFOV enhanced the action of insulin and completely prevented the development of insulin resistance induced by dexamethasone, leading to glucose consumption equal to that by normal cells. In addition, dexamethasone reduced the mRNA expression of insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT4) in 3T3-L1 adipocytes, while BFOV normalized the expression of IRS-1 and GLUT4. These findings suggest that BFOV prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes by enhancing expression of IRS-1 and GLUT4 mRNA.     

Flavonoids from Camellia sinensis (L.) O. Kuntze seed ameliorates TNF-α induced insulin resistance in HepG2 cells

The aim of this study is to discuss the non-catechin flavonoids (NCF) from Camellia sinensis (L.) O. Kuntze seed improving TNF-α impaired insulin stimulated glucose uptake and insulin signaling. Flavonoids had anti-metabolic syndrome and anti-inflammatory properties. It had widely been known for biological activity of catechins in tea, but very few research reports discussed the biological activity of non-catechin flavonoids in tea seed. We used HepG2 cell to treat with 5 μM insulin or with 5 μM insulin + 30 ng/ml TNF-α. Detecting the glucose concentration of medium, insulin decreased the glucose levels of medium meant that insulin promoted glucose uptake into cells, but TNF-α inhibited the glucose uptake effect of insulin. Furthermore, insulin increased the protein expressions of IR, IRS-1, IRS-2, PI3K-α, Akt/PKB, GLUT-2, AMPK, GCK, pyruvate kinase, and PPAR-γ. TNF-α activated p65 and MAPKs (p38, JNK1/2 and ERK1/2), iNOS and COX-2 which worsened the insulin signaling expressions of IR, IRS-1, IRS-2, PI3K-α, Akt/PKB, GLUT-2, AMPK, GCK, pyruvate kinase, and PPAR-γ. We added NCF (500, 1000, 2000 ppm) to cell with insulin and TNF-α. Not only glucose levels of medium were lowered, and the protein expressions of insulin signaling were increased, but p38, JNK1/2, iNOS and COX-2 were also reduced. NCF could ameliorate TNF-α induced insulin resistance through inhibiting p38, JNK1/2, iNOS and COX-2, and suggested that it might be used in the future to help control insulin resistance. This finding is the first report to present the discovery.     

橙皮苷对胰岛素抵抗HepG2细胞体外糖代谢的影响

目的探讨橙皮苷(hesperidin)对胰岛素抵抗HepG2细胞体外糖代谢的影响。方法采用高浓度胰岛素(INS)持续作用HepG2 12h建立胰岛素抵抗(IR-HepG2)模型,同时培养液中给予不同质量浓度橙皮苷(10,40和60μg.mL-1)或盐酸二甲双胍(30μg.mL-1)干预。药物作用12h后,换含低浓度INS的培养液培养细胞12h,使细胞同步化。然后,测定各组细胞葡萄糖消耗量、细胞内肝糖原含量、己糖激酶(hexokinase,HK)和丙酮酸激酶(pyruvate kinase,PK)的活力。结果与正常组相比,模型组细胞葡萄糖消耗量、肝糖原含量及HK和PK酶活力显著下降(P<0.01);与模型组相比,40和60μg.mL-1的橙皮苷可极显著增加IR-HepG2细胞葡萄糖消耗量、肝糖原合成量及HK酶活力(P<0.01),显著增加PK酶活力(P<0.05)。结论橙皮苷可增加IR-HepG2细胞对葡萄糖的利用,增加肝糖原合成量,提高HK、PK活力,从而促进IR-HepG2细胞糖代谢,改善胰岛素抵抗能力。     

Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells differentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of differentiation of the C2C12 myotubes. Insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to significant levels (P<0.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were unaffected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin- and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the effect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.     

Fucosterol activates the insulin signaling pathway in insulin resistant HepG2 cells via inhibiting PTP1B

Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. This study investigated the modulatory effects of fucosterol on the insulin signaling pathway in insulin-resistant HepG2 cells by inhibiting protein tyrosine phosphatase 1B (PTP1B). In addition, molecular docking simulation studies were performed to predict binding energies, the specific binding site of fucosterol to PTP1B, and to identify interacting residues using Autodock 4.2 software. Glucose uptake was determined using a fluorescent d-glucose analogue and the glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose, and the signaling pathway was detected by Western blot analysis. We found that fucosterol enhanced insulin-provoked glucose uptake and conjointly decreased PTP1B expression level in insulin-resistant HepG2 cells. Moreover, fucosterol significantly reduced insulin-stimulated serine (Ser307) phosphorylation of insulin receptor substrate 1 (IRS1) and increased phosphorylation of Akt, phosphatidylinositol-3-kinase, and extracellular signal- regulated kinase 1 at concentrations of 12.5, 25, and 50 µM in insulin-resistant HepG2 cells. Fucosterol inhibited caspase-3 activation and nuclear factor kappa B in insulin-resistant hepatocytes. These results suggest that fucosterol stimulates glucose uptake and improves insulin resistance by downregulating expression of PTP1B and activating the insulin signaling pathway. Thus, fucosterol has potential for development as an anti-diabetic agent.     

Stimulation of glucose uptake and improvement of insulin resistance by aromadendrin

Agents that stimulate glucose uptake and improve insulin resistance may be useful in the management of type 2 diabetes mellitus (DM). Thus, the aims of this study were to assess the effects of aromadendrin, a flavonoid from Gleditsia sinensis Lam., on stimulation of glucose uptake and improvement of insulin resistance and to characterize the molecular mechanisms underlying these activities. Insulin-stimulated glucose uptake was measured in HepG2 cells and in differentiated 3T3-L1 adipocytes using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a fluorescent D-glucose analog. Expression of the peroxisome proliferator-activated receptor-γ2 (PPARγ2) and adipocyte-specific fatty acid binding protein (aP2) mRNAs and the PPARγ2 protein was analyzed in adipocytes using RT-PCR and immunoblotting, respectively. Insulin-stimulated protein kinase B (Akt/PKB) phosphorylation was measured in high glucose-induced, insulin-resistant HepG2 cells. Similar to 30 μmol/l rosiglitazone, treatment with 30 μmol/l aromadendrin significantly stimulated insulin-sensitive glucose uptake in both HepG2 cells and 3T3-L1 adipocytes. Aromadendrin treatment also enhanced adipogenesis and caused increases in the expression of PPARγ2 and aP2 mRNAs and the PPARγ2 protein in differentiated 3T3-L1 adipocytes. In high glucose-induced, insulin-resistant HepG2 cells, aromadendrin reversed the inhibition of Akt/PKB phosphorylation in response to insulin, which could be abrogated by pretreatment with LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Aromadendrin treatment induced adipogenesis by increases in PPARγ2 expression, resulting in stimulation of glucose uptake and ameliorated insulin resistance. These findings suggest that aromadendrin may represent a potential therapeutic candidate for the management of type 2 DM.     

小檗碱与梓醇及其配伍对胰岛素抵抗3T3-L1脂肪细胞Glut-4、IRS-1、IRS-1 Ser307磷酸化蛋白表达的影响

目的：观察梓醇与小檗碱及其配伍对胰岛素抵抗3T3-L1脂肪细胞葡萄糖消耗及这一过程中葡萄糖转运子-4（Glut-4）、胰岛素受体底物-1（IRS-1）和胰岛素受体底物-1丝氨酸307（IRS-1Ser307）磷酸化蛋白表达的影响。方法：采用高糖联合高胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗，分别给予小檗碱、梓醇、小檗碱＋梓醇、盐酸罗格列酮进行干预，以葡萄糖氧化酶法检测培养液中葡萄糖消耗量，以WesternBlot法检测蛋白的表达。结果：与模型组相比，小檗碱能增加培养液中葡萄糖的消耗（P〈0．01），但对Glut，4蛋白的表达无影响；梓醇、小檗碱＋梓醇均能显著增加培养液中葡萄糖的消耗（P〈0．01），并使细胞中Glut-4蛋白的表达增强（P〈0．05），且小檗碱＋梓醇组的效应优于梓醇组及小檗碱组；与模型组相比，小檗碱与梓醇及其配伍对IRS-1的表达没有显著性影响，但能降低IRS-1 Ser307磷酸化蛋白表达。结论：小檗碱、梓醇及其配伍能改善胰岛素抵抗3T3-L1脂肪细胞的胰岛素敏感性，其作用机制与罗格列酮不同。     

虫草多糖对2型糖尿病小鼠InsR／IRS-1通路及糖代谢的影响

目的：观察虫草多糖对2型糖尿病小鼠InsR／IRS—1通路及糖代谢的影响。方法：采用高脂饲料联合腹腔注射小剂量链脲佐菌素（STZ,25mg·kg-1）建立胰岛素抵抗2型糖尿病模型,每日给予不同剂量虫草多糖（200,400mg·kg-1）治疗,28d后．免疫印迹法检测实验小鼠骨骼肌内胰岛素受体（1nsR）、胰岛素受体底物（IRS-1）及葡萄糖转运蛋白（GLUT4）的表达水平。检测肝脏葡萄糖激酶（GK）、磷酸果糖激酶（PFK）活性水平。结果：虫草多糖治疗组可显著改善糖尿病小鼠InsR／IRS一1及GLUT4蛋白的畀常表达（P〈0．05）;并呈剂量依赖性地增强小鼠肝脏GK及PFK酶活性,与糖尿病模型组比较差畀有统计学意义（P〈0．05或0．01）。结论：虫草多糖可增强胰岛素信号通路敏感性,改善葡萄糖代谢,从而缓解糖尿病小鼠胰岛素抵抗及高血糖症状,为胰岛素抵抗2型糖尿病的临床治疗提供新的依据。     

二甲双胍在慢性高胰岛素处理的Hep G2细胞对胰岛素受体后信号转导的调节作用(英文)

目的:研究慢性高胰岛素对胰岛素受体后信号转导的影响及其与胰岛素抵抗的关系,并探讨二甲双胍治疗胰岛素抵抗是否通过胰岛素信号转导途径的介导。方法:人类肝癌细胞系(Hep G2)在无血清条件下首先与100nmol/L高浓度胰岛素预温育16h以产生胰岛素抵抗细胞模型,并观察不同浓度(0.01-10mmol/L)的二甲双胍对胰岛素受体后磷酯酰肌醇3激酶(PI3K)途径信号转导的影响。结果:高浓度胰岛素100nmol/L慢性处理引起了IRβ、IRS1和IRS2的酪氨酸磷酸化和蛋白表达水平的下调,p85与IRS的相互作用也有显著降低。生理治疗浓度的(0.01-0.1mmol/L)二甲双胍逆转了这种慢性高胰岛素引起的信号下调,细胞用0.1mmol/L二甲双胍预温育后,IRB、IRS1和IRS2的磷酸化反应水平分别增加了2.7倍(P<0.01),6.8倍(P<0.01)和2.3倍(P<0.01),p85与IRS1的相互作用从34％增加至86％(P<0.01),与IRS2的相互作用从30％增加至92％(P<0.01)。相反,药物浓度(1-10mmol/L)的二甲双胍进一步抑制了IRB、IRS的磷酸化及IRS与p85的相互作用。结论:高浓度胰岛素慢性处理可引起胰岛素受体后PI3K途径信号转导的下调,二甲双胍对胰岛素信号转导的作用可能是其治疗胰岛素抵抗的主要分子机制。     

Oligonol promotes glucose uptake by modulating the insulin signaling pathway in insulin-resistant HepG2 cells <Emphasis Type="Italic">via</Emphasis> inhibiting protein tyrosine phosphatase 1B

Insulin resistance and protein tyrosine phosphatase 1B (PTP1B) overexpression are strongly associated with type 2 diabetes mellitus (T2DM), which is characterized by defects in insulin signaling and glucose intolerance. In a previous study, we demonstrated oligonol inhibits PTP1B and α-glucosidase related to T2DM. In this study, we examined the molecular mechanisms underlying the anti-diabetic effects of oligonol in insulin-resistant HepG2 cells. Glucose uptake was assessed using a fluorescent glucose tracer, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose, and the signaling pathway was investigated by western blotting. Oligonol significantly increased insulin-provoked glucose uptake and decreased PTP1B expression, followed by modulation of ERK phosphorylation. In addition, oligonol activated insulin receptor substrate 1 by reducing phosphorylation at serine 307 and increasing that at tyrosine 895, and enhanced the phosphorylations of Akt and phosphatidylinositol 3-kinase. Interestingly, it also reduced the expression of two key enzymes of gluconeogenesis (glucose 6-phosphatase and phosphoenolpyruvate carboxykinase), attenuated oxidative stress by scavenging/inhibiting peroxynitrite, and reactive oxygen species (ROS) generation, and augmented the expression of nuclear factor kappa B. These findings suggest oligonol improved the insulin sensitivity of insulin-resistant HepG2 cells by attenuating the insulin signaling blockade and modulating glucose uptake and production. Furthermore, oligonol attenuated ROS-related inflammation and prevented oxidative damage in our in vitro model of type 2 diabetes. These result indicate oligonol has promising potential as a treatment for T2DM.     

Des-aspartate-angiotensin-I and angiotensin IV improve glucose tolerance and insulin signalling in diet-induced hyperglycaemic mice

Although clinical studies suggested that blockade of the renin-angiotensin system may prevent diabetes, the mechanism is uncertain. As a follow-up to an earlier study, we investigated how des-aspartate-angiotensin-1 (DAA-1) and its metabolite, angiotensin IV (Ang-IV) improved glucose tolerance in diet-induced hyperglycaemic mice. Male C57BL/6J mice were fed a high-fat-high-sucrose (HFD) or normal (ND) diet for 52 weeks. HFD animals were orally administered either DAA-I (600 nmol/kg/day), Ang-IV (400 nmol/kg/day) or distilled water. Body weight, blood glucose and insulin were measured fortnightly. Inflammatory and insulin signalling transducers that are implicated in hyperglycaemia were analyzed in skeletal muscles at 52 weeks. HFD animals developed hyperglycemia, hyperinsulinemia and obesity. DAA-I and Ang-IV improved glucose tolerance but had no effect on hyperinsulinemia and obesity. Skeletal muscles of HFD animals showed increased level of ROS, gp91 of NADPH oxidase, pJNK and AT₁R-JAK-2-IRS-1 complex. Both DAA-I and Ang-IV attenuated these increases. Insulin-induced activation of IR, IRS-1, IRS-1-PI3K coupling, phosphorylation of Akt, and GLUT4 translocation were attenuated in skeletal muscles of HFD animals. The attenuation was significantly ameliorated in DAA-I-treated HFD animals. In corresponding Ang-IV treated animals, insulin induced IRAP and PI3K interaction, activation of pAkt and GLUT4 translocation, but no corresponding activation of IR, IRS-1 and IRS-1-PI3 K coupling were observed. DAA-I and Ang-IV improved glucose tolerance, insulin signalling, and para-inflammatory processes linked to hyperglycaemia. DAA-I acts via the angiotensin AT₁ receptor and activates the insulin pathway. Ang-IV acts via IRAP, which couples PI3K and activates the later part of the insulin pathway.     

新化合物结构蜕皮甾酮衍生物TAPA降糖作用与机制的初步研究

目的:评价新化合物结构蜕皮甾酮衍生物TAPA的体内降糖作用,并对其作用机制从细胞水平上进行初步研究。方法:取大鼠随机分为正常对照组、模型组、TAPA-1(1 mg/kg)组、TAPA-5(5 mg/kg)组、TAPA-25(25 mg/kg)组和罗格列酮(2 mg/kg)组,每组10只,除正常对照组外其余各组大鼠建立2型糖尿病模型,后4组即为给药组,每日灌胃1次,连续给药28 d,给药期间每周测定大鼠摄食量、体质量、饮水量及随机血糖水平,末次给药5 h后灌胃葡萄糖测定2 h内的血糖浓度,计算血糖-时间曲线下面积(AUC)考察糖耐量。建立胰岛素抵抗肝癌细胞Hep G2,采用3H-d-葡萄糖掺入实验评价在1×10-9、1×10-7mol/L胰岛素环境下,1×10-5mol/L的TAPA和罗格列酮分别对Hep G2细胞和胰岛素抵抗Hep G2细胞的葡萄糖掺入率。取胰岛β细胞瘤细胞系3(βTC3)细胞,随机分为空白对照组、TAPA-Ⅰ(1×10-10mol/L)组、TAPA-Ⅱ(1×10-8mol/L)组、TAPA-Ⅲ(1×10-6mol/L)组和格列齐特(1×10-5mol/L)组,给药孵育24 h后测定各组细胞液中胰岛素含量。结果:与正常对照组比较,给药组大鼠的摄食量、体质量、饮水量均无明显变化;与模型组比较,给药组大鼠血糖水平在给药结束时均明显降低、糖耐量均降低(P<0.05),且降糖效果、糖耐量与TAPA剂量和给药时间均呈正相关。TAPA和罗格列酮对Hep G2细胞的葡萄糖掺入率无明显影响,能明显提高对胰岛素抵抗Hep G2细胞的葡萄糖掺入率(P<0.01)。与空白对照组比较,TAPA各剂量组βTC3细胞的胰岛素释放量无明显变化,格列齐特组βTC3细胞的胰岛素释放量明显增加(P<0.01)。结论:TAPA可降低2型糖尿病模型大鼠的血糖水平和糖耐量,体外试验认为其可增加胰岛素敏感性,但不促进胰岛素分泌。     

Insulin-sensitizing effects of a novel alpha-methyl- alpha -phenoxylpropionate derivative in vitro

AIM: To examine the insulin sensitizing effects of a novel alpha-methyl-alpha- phenoxylpropionate derivative YY20 in insulin-sensitive cell lines. METHODS: The peroxisome proliferator-activated receptor gamma (PPAR gamma) agonist bioactivities of YY20 were detected by a preadipocyte differentiation assay. RT-PCR and Western blotting analysis were used to detect the expression of the target gene or protein. The effects of YY20 on insulin-mediated glucose consumption were determined in the HepG2 human hepatocellular carcinoma line. RESULTS: YY20 could enhance the differentiation of preadipocytes to adipocytes and upregulate the gene expression of PPAR gamma 2, as well as the protein expression of insulin receptor substrate- 1 (IRS-1), glucose transporter-4 (GLUT4), and adiponectin (ACRP30). The effects on GLUT4 and ACRP30 could be reversed by the PPAR gamma inhibitor SR-202. Furthermore, YY20 efficiently reduced glucose consumptions in HepG2 cells after 24 h culture, and the effects were related to insulin and YY20 concentrations. CONCLUSION: YY20, a potential insulin-sensitizing agent like rosiglitazone, could enhance glucose consumption in HepG2 cells in a concentration- and insulindependent manner. It may improve the insulin resistance associated with type 2 diabetes.     

过表达SOCS3对西格列汀改善胰岛素抵抗作用的影响

目的:探究过表达SOCS3对西格列汀(sitagliptin,SITA)在改善脂质代谢和氧化应激,以减轻棕榈酸(palrnitic acid,PA)介导的HepG2细胞胰岛素抵抗(insulin resistance,IR)中的作用。方法:MTT法检测PA与SITA对肝癌细胞HepG2增殖活性的影响;将HepG2分为4组:Control组,PA组,PA+SITA+pEX-RB-NC组和PA+SITA+pEX-RB-SOCS3组;油红O染色检测细胞中脂质的积聚水平,RT-PCR检测细胞中SREBP1c和PPARα的mRNA表达,DCFH-DA法检测细胞中ROS的水平,RT-PCR与试剂盒检测细胞中CAT与GPx的mRNA表达与酶活性;Western blot试验检测细胞IR通路中p-IRS-1^Ser307/IRS-1、p-AKT^Ser473/AKT、p-GSK3β^Ser9/GSK3β的比值。结果:与Control组相比,SITA能削弱PA诱导的HepG2细胞活力损伤(P＜0.05),且PA组、PA+SITA+pEX-RB-NC组和PA+SITA+pEX-RB-SOCS3组中富含大量红色脂滴,SREBP1c与PPARα的mRNA水平、ROS、CAT与GPx表达及p-IRS-1^Ser307/IRS-1、p-AKT^Ser473/AKT、p-GSK3β^Ser9/GSK3β比值均明显升高(P＜0.05);但与PA组相比,PA+SITA+pEX-RB-NC组除CAT、GPx及p-IRS-1^Ser307/IRS-1、p-AKT^Ser473/AKT、p-GSK3β^Ser9/GSK3β比值升高外(P＜0.05),其他检测指标均显著降低(P＜0.05),PA+SITA+pEX-RB-SOCS3组中上述指标均无明显改变(P＞0.05)。结论:SITA可抑制脂质代谢和氧化应激,从而以减轻PA介导的肝细胞IR,而该作用能够被过表达SOCS3所抵消。     

葡萄糖对HepG2细胞酰基辅酶A:胆固醇酰基转移酶2表达的影响

目的 探讨葡萄糖对HepG2细胞酰基辅酶A:胆固醇酰基转移酶2(acyl-CoA:cholesterol acyltransferase2,ACAT2)表达的调节作用及可能机制.方法 用不同浓度的葡萄糖(5.5、12.5、25.0 mmol/L)作用HepG2细胞12 h,RTPCR检测ACAT2和人肝细胞核因子-1α(hepatocyte nuclear factor 1α,HNF1α) mRNA的表达水平.将HNF1α的真核表达质粒瞬时转染HepG2,利用RT-PCR和Western blot检测高表达HNF1α对ACAT2表达的影响.利用凝胶迁移率实验(electrophoretic mobility shift assay,EMSA)观察葡萄糖对HNF1α结合于ACAT2上游调控序列活性的影响.结果 25.0 mmol/L葡萄糖可上调HepG2细胞HNF1α和ACAT2的转录水平;HepG2细胞内过表达HNF1α可增强ACAT2的表达;高糖组HNF1α与ACAT2上游调控序列的结合活性明显增高.结论 高糖可上调ACAT2的表达,其机制可能与高糖增强了HNF1α与ACAT2上游调控区域的结合活性有关.     

Mechanism of suppression of insulin signalling with lignocaine

Lignocaine suppresses insulin-stimulated glucose transport into the cells and insulin-stimulated glycogenesis at doses equivalent to that used in the treatment of muscle pain disorder. We evaluated the direct effect of lignocaine on insulin receptor (IR) kinase activity. After lignocaine (40 mM, approximately equivalent to 1%) or an equal volume (100 microl) saline had been injected into the tibialis anterior muscle of rat, insulin (50 mM g-1 body weight) was administered into the portal vein in vivo. Immunoprecipitation and immunoblotting were used to detect insulin-mediated tyrosine phosphorylation of both IR-beta and insulin receptor substrate (IRS)-1, and insulin-stimulated binding of IRS-1 to p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K) in the extracted muscle. In the in vitro study, purified IR from rat liver and/or recombinant IRS-1 protein with adenosine triphosphate were incubated with lignocaine (4 or 40 mM). Lignocaine reduced insulin-stimulated tyrosine phosphorylation of IR-beta to 12.6+/-5.7% (P<0.001), and IRS-1 to 32.1+/-18.8% (P<0.01), and also reduced insulin-stimulated binding of IRS-1 to p85 to 27.4+/-12.7% (P<0.001) relative to control (100%) in muscle in vivo. The in vitro study revealed that lignocaine directly inhibited both basal and insulin-stimulated tyrosine phosphorylation of IR. These results indicate that clinically used doses of lignocaine inhibit insulin signalling in skeletal muscle. The inhibitory effect of lignocaine on tyrosine kinase activity of the IR underlies the suppression of insulin signalling with lignocaine.