Interleukin-18 enhances glucose uptake in 3T3-L1 adipocytes

In order to characterize the potential causative effects of interleukin-18 (IL-18) on insulin resistance, we measured glucose uptake in 3T3-L1 adipocytes treated with mouse recombinant IL-18. IL-18 surprisingly enhanced, rather than reduced insulin-mediated glucose uptake in adipocytes. Moreover IL-18 could counteract the glucose uptake suppression caused by tumor necrosis factor α in 3T3-L1 adipocytes. The mechanism dissection showed that the IL-18 upregulated phosphorylated Akt and downregulated phosphorylated P38 MAPK. These findings indicated that the elevated serum IL-18 levels in obesity and diabetes might be a compensatory response to insulin resistance.     

Effects of crude drugs on glucose uptake in 3T3-L1 adipocytes

In this study, various water-extracted crude drugs from Radix Asparagi, Radix Ginseng, Radix Scutellariae, Cortex Lycii Radicis, Cortex Phellodendri and Radix Ophiopogonis were investigated in their effects on [3H]-2-deoxyglucose uptake in 3T3-L1 adipocytes. Following treatment of cells with various crude drugs for 60 mim, the basal [3H]-2-deoxyglucose uptake in cultured 3T3-L1 cells was changed by Radix Asparagi from 140 pmole/min/mg protein of control to 513 (0.1 mg/ml), 201 (1 mg/ml) and 97 (10 mg/ml). Glucose uptake was changed to 324 (0.1 mg/ml), 146 (1 mg/ml) and 46 (10 mg/ml) with Radix Ginseng. In the presence of Radix Scutellariae, glucose uptake was changed to 215 (0.1 mg/ml), 213 (1 mg/ml) and 34 (10 mg/ml). In the presence of Cortex Lycii Radicis, glucose uptake was 230 (0.1 mg/ml), 188 (1 mg/ml) and 38 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, uptake was changed to 142 (0.1 mg/ml), 132 (1 mg/ml), 24 (10 mg/ml) and 489 (0.1 mg/ml), 374 (1 mg/ml), 344 (10 mg/ml), respectively. In insulin-stimulated cells, the [3H]-2-deoxyglucose uptake was changed by Radix Asparagi from 570 pmole/min/mg protein of the control to 816 (0.1 mg/ml), 674 (1 mg/ml) and 532 (10 mg/ml). After incubation with Radix Ginseng, the glucose uptake was changed to 254 (0.1 mg/mi), 123 (1 mg/mi) to 76 (10 mg/mi). In the presence of Radix Scutellariae, the glucose uptake was changed to 315 (0.1 mg/ml), 265 (1 mg/ml) and 33 (10 mg/ml). After incubation of Cortex Lycii Radicis, the uptake activity was changed to 281 (0.1 mg/ml), 248 (1 mg/ml) and 37 (10 mg/ml). In the case of Cortex Phellodendri and Radix Ophiopogonis, the activity of glucose uptake was measured as 747 (0.1 mg/ml), 523 (1 mg/ml), 33 (10 mg/ml) and 753 (0.1 mg/ml), 740 (1 mg/ml), and 421 (10 mg/ml), respectively. These results indicate that the water-extracted materials of Radix Asparagi and Radix Ophiopogonis increase the glucose uptake in basal and insulin-stimulated 3T3-L1 adipocytes.     

Enhanced long-chain fatty acid uptake contributes to overaccumulation of triglyceride in hyperinsulinemic insulin-resistant 3T3-L1 adipocytes

The precise pathogenesis of obesity remains controversial. In obesity, diminished adipose glucose utilization suggests that some other substrates may be responsible for the adipose triglyceride (TG) overaccumulation. Here we attempted to evaluate if long-chain fatty acid (LCFA) flux was modulated by a physiologically relevant condition of hyperinsulinemia in 3T3-L1 adipocytes and if the altered LCFA influx might eventually contribute to the TG overaccumulation in obesity. The effects of prolonged insulin exposure to adipocytes on basal, insulin-stimulated LCFA uptake as well as intracellular LCFA metabolism were measured. Prolonged insulin exposure was found to induce insulin resistance (IR) yet enhance basal and insulin-stimulated LCFA uptake in normoglycemic condition, and the addition of high glucose exacerbated these abnormalities of both glucose and LCFA influx. Along with the enhanced LCFA uptake was an increase in the rates of intracellular LCFA deposition and incorporation into TG; but a decrease was found in basal and insulin-suppressive LCFA oxidation, as well as in isoproterenol-induced fatty acid efflux. Inhibition of either phosphatidylinositol 3-kinase or mitogen-activated protein kinase (MAPK) pathway did not prevent the induction of IR, whereas the enhanced basal and insulin-stimulated LCFA uptake was abrogated by inhibition of MAPK pathway. In hyperinsulinemic insulin-resistant 3T3-L1 adipocytes, basal and insulin-stimulated LCFA uptake tends to increase via a MAPK-dependent mechanism. The increment of LCFA influx predominantly accounts for TG overaccumulation, but not for mitochondrial oxidation, and is prone to retain within adipocytes. These findings may interpret the plausible mechanism of pathogenesis for obesity in hyperinsulinemia-associated IR.     

Enhancement of glucose uptake in 3T3-L1 adipocytes by Toona sinensis leaf extract

The effects of substances extracted from Toona sinensis leaves, using 50% alcohol/water, on cellular [3H]-2-deoxyglucose uptake in differentiated cultured 3T3-L1 adipocytes were investigated. Following treatment of cells with 0.001, 0.01, or 0.1 mg/mL extracts for 60 minutes, [3H]-2-deoxyglucose uptake increased from a basal value of 0.23 nmol/min/mg protein to 0.30, 0.33, and 0.38 nmol/min/mg protein, respectively. In insulin-stimulated cells, cellular [3H]-2-deoxyglucose uptake was enhanced by Toona sinensis leaf extract from a basal value of 0.35 nmol/min/mg protein to 0.41, 0.46, and 0.52 nmol/min/mg protein, respectively. Cellular glucose uptake was also enhanced by Toona sinensis leaf extract after incubation of cells with 20 mM glucose for 48 hours. Cellular glucose uptake with a combination of Toona sinensis leaf extract and insulin was significantly inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide and the protein kinase C inhibitor calphostin C in normal-, medium- and high-glucose media. However, the glucose uptake-enhancing effect of Toona sinensis leaf extract was not diminished by cycloheximide and calphostin C in the absence of insulin. These results indicate that enhancement of cellular glucose uptake by Toona sinensis leaf extract in basal and insulin-stimulated 3T3-L1 adipocytes may be mediated by distinct mechanisms.     

The p38 mitogen-activated protein kinase inhibitor SB203580 reduces glucose turnover by the glucose transporter-4 of 3T3-L1 adipocytes in the insulin-stimulated state

Insulin induces a profound increase in glucose uptake in 3T3-L1 adipocytes through the activity of the glucose transporter-4 (GLUT4). Apart from GLUT4 translocation toward the plasma membrane, there is also an insulin-induced p38 MAPK-dependent step involved in the regulation of glucose uptake. Consequently, treatment with the p38 MAPK inhibitor SB203580 reduces insulin-induced glucose uptake by approximately 30%. Pretreatment with SB203580 does not alter the apparent K(m) of GLUT4-mediated glucose uptake but reduces the maximum velocity by approximately 30%. Insulin-induced GLUT4 translocation and exposure of the transporter to the extracellular environment was not altered by pretreatment with SB203580, as evidenced by a lack of effect of the inhibitor on the amount of GLUT4 present in the plasma membrane, as assessed by subcellular fractionation, the amount of GLUT4 that is able to undergo biotinylation on intact adipocytes and the level of extracellular exposure of an ectopically expressed GLUT-green fluorescence protein construct with a hemagglutinin tag in its first extracellular loop. In contrast, labeling of GLUT4 after insulin stimulation by a membrane-impermeable, mannose moiety-containing, photoaffinity-labeling agent [2-N-4(1-azido-2,2,2-trifluoroethyl)benzoyl-1,3-bis(d-mannose-4-yloxy)-2-propylamine] that binds to the extracellular glucose acceptor domain was markedly reduced by SB203580, although photolabeling with this compound in the absence of insulin was unaffected by SB203580. These data suggest that SB203580 affects glucose turnover by the insulin-responsive GLUT4 transporter in 3T3-L1 adipocytes.     

Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb(1) has been shown to regulate peroxisome proliferator-activated receptor gamma activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb(1) on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb(1) significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 microM and a time of 3 h. In adipocytes, Rb(1) promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb(1) increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb(1)-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb(1) stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.     

Chemerin过表达致3T3-L1脂肪细胞葡萄糖消耗减少

目的 应用重组慢病毒构建3T3-L1脂肪细胞chemerin过表达模型并进一步探讨其对糖代谢的影响及可能机制.方法 构建鼠chemerin过表达重组慢病毒,并设对照慢病毒,感染3T3-L1细胞,实时定量聚合酶链反应（RT-PCR）法检测转染后chemerin表达水平;应用胰岛素、3-异丁基1-甲基黄嘌呤、地塞米松诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色鉴定;诱导分化第8天加入慢病毒重组体,继续培养5d,葡萄糖氧化酶法检测各组葡萄糖消耗;RT-PCR法检测各组胰岛素受体底物1（IRS1）、胰岛素受体底物2（IRS2）、蛋白激酶B1 （Akt1）、叉头状转录因子O1 （FoxO1）基因表达水平;Western-blotting检测chemerin、丝氨酸/苏氨酸蛋白激酶（Akt）、磷酸化丝氨酸/苏氨酸蛋白激酶（pAkt）、FoxO1、磷酸化叉头状转录因子O1 （pFoxO1）蛋白水平.两组数据比较应用t检验.结果 Chemerin过表达慢病毒感染3T3-L1细胞72 h后细胞中可见红色荧光,RT-PCR结果显示：过表达组与空载对照组相比chemerin基因表达明显增加（分别为3.04±0.19比1.01±0.11,t=15.65,P＜0.05）;chemerin过表达组葡萄糖消耗减少[分别为（3.30± 1.44）比（6.07±1.15） mmol/L,t=-0.35,P＜0.05];RT-PCR结果显示：IRS1、IRS2基因水平无明显变化（均P＞0.05）,Akt1基因表达下降（分别为0.76±0.08比1.07±0.15,t=-3.11,P＜0.05）,FoxO1基因表达上调（分别为1.53±0.30与1.03±0.21,t=2.34,P＜0.05）.Western-blotting结果显示：Chemerin过表达后chemerin蛋白水平增加（相对表达量分别为1.08±0.06比0.72±0.03,t=-10.12;P＜0.05）;Akt、pAkt蛋白水平均降低（分别为0.74±0.21比1.23±0.20,0.58±0.17比0.92±0.07;t=2.81、3.17,均P＜0.05）,FoxO1蛋白水平升高（分别为1.04±0.09比0.76±0.14,t=-2.91,P＜0.05）、pFoxO1蛋白水平降低（分别为0.61±0.13比0.89±0.10,t=2.93,P＜0.05）.结论 Chemerin可能通过下调Akt1 mRNA使3T3-L1脂肪细胞葡萄糖消耗减少.     

Leptin-mediated inhibition of the insulin-stimulated increase in fatty acid uptake in differentiated 3T3-L1 adipocytes

The effects of insulin and leptin on fatty acid uptake in differentiated (adipocytes) and undifferentiated 3T3-L1 cells were investigated. It was demonstrated that in undifferentiated 3T3-L1 cells, insulin and leptin have no effect on fatty acid uptake. In differentiated 3T3-L1 adipocytes, insulin had a concentration-dependent stimulatory effect on fatty acid uptake, whereas leptin on its own had no effect. Leptin, when coincubated with 10 nmol/L insulin, resulted in a concentration-dependent inhibition of the insulin-stimulated fatty acid uptake in differentiated 3T3-L1 cells. These results indicate that leptin has a direct inhibitory effect on the stimulation of fatty acid uptake by insulin in differentiated murine adipocytes.     

小檗碱对3T3-L1胰岛素抵抗细胞模型PI-3K p85蛋白表达的影响

目的:研究小檗碱对3T3-L1胰岛素抵抗细胞模型PI-3K p85蛋白表达的影响,探讨小檗碱改善胰岛素抵抗的分子机制.方法:分别以0.5 mmol/L软脂酸与25 mmol/L葡萄糖加0.6 nmmol/L胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,予以小檗碱进行干预,同时以阿司匹林作为阳性对照,以2-脱氧-[3H]-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测PI-3K p85蛋白的表达.结果:0.5 mmol/L软脂酸作用24 h或25 mmol/L葡萄糖加0.6 nmmol/L胰岛素作用18 h分别使3T3-L1脂肪细胞胰岛素刺激的葡萄糖转运抑制67%和60%,Westem blot显示PI-3K p85蛋白表达减少,与正常对照组比较有统计学意义(P<0.01);同时加入小檗碱则可逆转上述效应使P1-3K p85蛋白表达增加,与模型组比较有明显差异(P<0.01),并且PI-3K p85蛋白的表达与小檗碱的剂量和作用时间呈依赖关系.结论:小檗碱可以明显改善游离脂肪酸和高糖诱导的胰岛素抵抗,其分子机制可能与小檗碱提高PI-3K p85蛋白的表达有关.     

JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢的影响

目的 探讨JAZF1基因抑制对3T3-L1脂肪细胞糖、脂代谢相关基因的影响.方法 构建JAZF1小发夹RNA (shRNA)表达载体并转染3T3-L1细胞,实时荧光定量PCR(RT-QPCR)和蛋白印迹法检测JAZF1 mRNA和蛋白水平的表达;氢三放射示踪法检测3T3-L1细胞糖摄取率;蛋白印记法检测糖、脂代谢相关基因蛋白水平;油红O染色检测脂肪细胞甘油三酯(TG)含量变化.结果 成功构建JAZF1-shRNA;转染脂肪细胞48 h后,JAZF1 mRNA和蛋白水平明显低于对照组(P＜0.05);氢3放射性示踪法显示转染组葡萄糖摄取率明显降低(P＜0.05);PPAR-γ蛋白表达升高(P＜0.05),激素敏感脂肪酶(HSL)、内脏脂肪素(Visfatin)、胰岛素诱导基囚-2 (Insig-2)蛋白表达降低(均P＜0.05);油红O染色显示JAZF1转染组细胞内脂质积聚明显,比对照组升高约25％(P＜0.05).结论 JAZF1基因抑制可减少基础糖转运,增加脂质与胆固醇合成,减少脂质分解并减少相关脂肪细胞因子的表达.     

Regulation of insulin-stimulated glucose transport by chronic glucose exposure in 3T3-L1 adipocytes.

Chronic hyperglycemia causes insulin resistance, termed glucose toxicity. Herein we studied chronic glucose-dependent regulation of the glucose transport system in adipocytes. 3T3-L1 adipocytes were incubated for up to 24 h with low (1 mM) or high (25 mM) glucose, and glucose transport was subsequently analyzed. 100 nM insulin was present throughout the experiments. 24 h incubation with 1 mM glucose caused a 2.3+/-0.4 fold increase in glucose transport activity, compared to the values obtained with 25 mM glucose. This difference was not observed when 24 h incubation was carried out without insulin. Glucose transport activity was not increased at 3 or 6 h incubation with 1 mM glucose, but was increased at 12 h, which closely paralleled increased expression of GLUT1. In addition to increased GLUT1 expression, more efficient translocation of GLUT1 to the plasma membrane was observed when incubated with 1 mM glucose compared to 25 mM glucose. The addition of azaserin or deprivation of glutamine at 25 mM glucose did not increase the glucose transport activity to the level obtained with 1 mM glucose. PD98059 did not affect glucose transport activity when incubated with 1 mM or 25 mM glucose. In conclusion, the present study is the first to show that, in 3T3-L1 adipocytes, chronic exposure to low (1 mM) and high (25 mM) glucose leads to different insulin-stimulated glucose transport activities. These differences result from the difference in the expression and plasma membrane distribution of GLUT1, but not of GLUT4, and the hexosamine biosynthesis pathway or extracellular signal-regulated protein kinase is not involved.     

Orexin A stimulates glucose uptake, lipid accumulation and adiponectin secretion from 3T3-L1 adipocytes and isolated primary rat adipocytes

Aims/hypothesis

Orexin A (OXA) modulates body weight, food intake and energy expenditure. In vitro, OXA increases PPAR?? (also known as PPARG) expression and inhibits lipolysis, suggesting direct regulation of lipid metabolism. Here, we characterise the metabolic effects and mechanisms of OXA action in adipocytes.

Methods

Isolated rat adipocytes and differentiated murine 3T3-L1 adipocytes were exposed to OXA in the presence or absence of phosphoinositide 3-kinase (PI3K) inhibitors. Ppar?? expression was silenced using small interfering RNA. Glucose uptake, GLUT4 translocation, phosphatidylinositol (3,4,5)-trisphosphate production, lipogenesis, lipolysis, and adiponectin secretion were measured. Adiponectin plasma levels were determined in rats treated with OXA for 4?weeks.

Results

OXA PI3K-dependently stimulated active glucose uptake by translocating the glucose transporter GLUT4 from cytoplasm into the plasma membrane. OXA increased cellular triacylglycerol content via PI3K. Cellular triacylglycerol accumulation resulted from increased lipogenesis as well as from a decrease of lipolysis. Adiponectin levels in chow- and high-fat diet-fed rats treated chronically with OXA were increased. OXA stimulated adiponectin expression and secretion in adipocytes. Both pharmacological blockade of peroxisome proliferator-activated receptor ?? (PPAR??) activity or silencing Ppar?? expression prevented OXA from stimulating triacylglycerol accumulation and adiponectin production.

Conclusions/interpretation

Our study demonstrates that OXA stimulates glucose uptake in adipocytes and that the evolved energy is stored as lipids. OXA increases lipogenesis, inhibits lipolysis and stimulates the secretion of adiponectin. These effects are conferred via PI3K and PPAR??2. Overall, OXA??s effects on lipids and adiponectin secretion resemble that of insulin sensitisers, suggesting a potential relevance of this peptide in metabolic disorders.     

Insulin-mediated cellular insulin resistance decreases osmotic shock-induced glucose transport in 3T3-L1 adipocytes

Similar to insulin, osmotic shock treatment of 3T3-L1 adipocytes causes translocation of GLUT4 protein to the plasma membrane and an increase in glucose transport activity. In our study, we evaluated the effect of chronic insulin treatment on the osmotic shock signaling pathway leading to GLUT4 translocation and glucose uptake. We found that chronic administration of insulin to the adipocytes induced cellular resistance to osmotic shock-stimulated GLUT4 translocation and glucose transport. We found that chronic insulin treatment attenuated shock-induced Gab-1 tyrosine phosphorylation. Furthermore, chronic insulin exposure led to a marked impairment in the ability of Gab-1 to associate with p85 subunit of PI 3-kinase in response to acute shock and insulin stimulation. Cells that were chronically treated with insulin showed a 70% and a 61% decrease in Gab-1 associated PI 3-kinase activity in shock- vs. insulin-treated cells, respectively. In addition, we found that chronic insulin treatment inhibited both insulin- and osmotic shock-induced membrane ruffling, indicating that two PI 3-kinase dependent effects, GLUT4 translocation and membrane ruffling are decreased in chronically insulin-treated cells. The results described above clearly demonstrate that chronic insulin treatment induces a state of cellular resistance to osmotic shock signal transduction.     

Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport

Aims/hypothesis. Insulin stimulates glucose transport in adipose and muscle tissue by the translocation of a specialised pool of intracellular GLUT4-containing vesicles to the cell surface. It is well established that defective insulin-stimulated GLUT4 translocation is associated with insulin resistance. Long-term insulin treatment (500 nmol/l for 24 h) of 3T3-L1 adipocytes has previously been shown to decrease cellular GLUT4 content and reduce insulin-stimulated GLUT4 translocation. Here, we test the hypothesis that the insulin resistance observed after long-term insulin treatment arises by the selective loss of GLUT4 from a specific intracellular compartment.¶Methods. Using iodixanol gradient centrifugation we have separated intracellular GLUT4 containing membranes into two distinct populations corresponding to recycling endosomes and a distinct intracellular compartment which probably represents GLUT4 storage vesicles (GSVs).¶Results. A short-term insulin stimulation reduced the content of GLUT4 in the GSV fraction (51 ± 3.5 %) with only a modest decrease from the endosomal fraction (23 ± 2.6 %). Long-term insulin treatment decreased cellular GLUT4 content by about 40 % and diminished the ability of a short-term insulin challenge to promote GLUT4 translocation. We further show that this depletion of cellular GLUT4 is selectively from the GSV fraction (68 ± 7 % decrease compared to untreated cells).¶Conclusions/interpretation. Such data argue that long-term insulin treatment results in the mis-targeting of GLUT4 such that it no longer accesses the GSV compartment. These data imply that defective targeting of GLUT4 away from the GSV compartment plays an important role in the aetiology of insulin resistance. [Diabetologia (2000) 43: 1273–1281]     

Suppressed intrinsic catalytic activity of GLUT1 glucose transporters in insulin-sensitive 3T3-L1 adipocytes.

Previous studies indicated that the erythroidtype (GLUT1) glucose transporter isoform contributes to basal but not insulin-stimulated hexose transport in mouse 3T3-L1 adipocytes. In the present studies it was found that basal hexose uptake in 3T3-L1 adipocytes was about 50% lower than that in 3T3-L1 or CHO-K1 fibroblasts. Intrinsic catalytic activities of GLUT1 transporters in CHO-K1 and 3T3-L1 cells were compared by normalizing these hexose transport rates to GLUT1 content on the cell surface, as measured by two independent methods. Cell surface GLUT1 levels in 3T3-L1 fibroblasts and adipocytes were about 10- and 25-fold higher, respectively, than in CHO-K1 fibroblasts, as assessed with an anti-GLUT1 exofacial domain antiserum, delta. The large excess of cell surface GLUT1 transporters in 3T3-L1 adipocytes relative to CHO-K1 fibroblasts was confirmed by GLUT1 protein immunoblot analysis and by photoaffinity labelling (with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n) of glucose transporters in isolated plasma membranes. Thus, GLUT1 intrinsic activity is markedly reduced in 3T3-L1 fibroblasts compared with the CHO-K1 fibroblasts, and further reduction occurs upon differentiation to adipocytes. Intrinsic catalytic activities specifically associated with heterologously expressed human GLUT1 protein in transfected CHO-K1 versus 3T3-L1 cells were determined by subtracting appropriate control cell values for hexose transport and delta-antibody binding from those determined in the transfected cells expressing high levels of human GLUT1. The results confirmed a greater than 90% inhibition of the intrinsic catalytic activity of human GLUT1 transporters on the surface of mouse 3T3-L1 adipocytes relative to CHO-K1 fibroblasts. We conclude that a mechanism that markedly suppresses basal hexose transport catalyzed by GLUT1 is a major contributor to the dramatic insulin sensitivity of glucose uptake in 3T3-L1 adipocytes.     

胰岛素、葡萄糖对3T3-L1脂肪细胞脂肪水孔蛋白表达的调节

利用半定量RT PCR技术及Western印迹法研究胰岛素、葡萄糖对成熟脂肪细胞脂肪水孔蛋白 (AQPap)基因表达的影响。结果表明 ,胰岛素对AQPap的表达具有抑制作用 ;而高浓度葡萄糖则对AQPap的表达具有促进作用     

Nitric oxide stimulates glucose transport through insulin-independent GLUT4 translocation in 3T3-L1 adipocytes

OBJECTIVE: It is well known that nitric oxide synthase (NOS) is expressed and that it modulates glucose transport in skeletal muscles. Recent studies have shown that adipose tIssues also express inducible and endothelial nitric oxide synthase (eNOS). In the present study, we investigated whether nitric oxide (NO) induces glucose uptake in adipocytes, and the signaling pathway involved in the NO-stimulated glucose uptake in 3T3-L1 adipocytes. METHODS: First, we determined the expression of eNOS in 3T3-L1 adipocytes, and then these cells were treated with the NO donor sodium nitroprusside (SNP) and/or insulin, and glucose uptake and phosphorylation of insulin receptor substrate (IRS)-1 and Akt were evaluated. Moreover, we examined the effects of a NO scavenger, a guanylate cyclase inhibitor or dexamethasone on SNP-stimulated glucose uptake and GLUT4 translocation. RESULTS: SNP at a concentration of 50 mmol/l increased 2-deoxyglucose uptake (1.8-fold) without phosphorylation of IRS-1 and Akt. Treatment with the NO scavenger or guanylate cyclase inhibitor decreased SNP-stimulated glucose uptake to the basal level. Dexamethasone reduced both insulin- and SNP-stimulated glucose uptake with impairment of GLUT4 translocation. CONCLUSION: NO is capable of stimulating glucose transport through GLUT4 translocation in 3T3-L1 adipocytes, via a mechanism different from the insulin signaling pathway.     

Rottlerin inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes by uncoupling mitochondrial oxidative phosphorylation

There is increasing evidence that protein kinase C (PKC) isoforms modulate insulin-signaling pathways in both positive and negative ways. Recent reports have indicated that the novel PKCdelta mediates some of insulin's actions in muscle and liver cells. Many studies use the specific inhibitor rottlerin to demonstrate the involvement of PKCdelta. In this study, we investigated whether PKCdelta might play a role in 3T3-L1 adipocytes. We found that PKCdelta is highly expressed in mouse adipose tissue and increased on 3T3-L1 adipocyte differentiation, and insulin-stimulated glucose transport is blocked by rottlerin. The phosphorylation state and activity of PKCdelta are not altered by insulin, but the protein translocates to membranes following insulin treatment. In contrast to the results with rottlerin, inhibition of PKCdelta activity or expression has no effect on glucose transport in adipocytes, unlike muscle cells. Lastly, we found that rottlerin lowers adenosine triphosphate levels in 3T3-L1 cells by acting as a mitochondrial uncoupler, and this is responsible for the observed inhibition of glucose transport.     

未知功能基因JAZF1过表达对3T3-L1脂肪细胞糖脂代谢的影响

目的 观察JAZF1基因(Juxtaposed with another zincfinger gene 1)过表达对3T3-L1脂肪细胞糖脂代谢相关基因的影响.方法 采用实时荧光定量PCR(RT-QPCR)法检测JAZF1 mRNA在健康C57BL/6J小鼠多种组织表达分布情况;构建JAZF1真核表达载体并瞬时转染3T3-L1细胞,RT-QPCR法检测JAZF1、糖脂代谢相关基因mRNA的表达;用Western印迹法测定各组细胞JAZF1蛋白水平;油红O染色比色法检测细胞内脂质积聚的变化.结果 转染48 h后,JAZF1转染组脂肪细胞中,JAZF1 mRNA及蛋白表达明显高于阴性对照和空载组,激素敏感脂肪酶(HSL)mRNA表达水平明显增加(P<0.05),脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)、类固醇调节元件结合蛋白1(SREBP1)mRNA相对表达量明显降低(均P<0.01),脂肪细胞甘油三酯酶(ATGL)、葡萄糖转运子1(GLUT1)、GLUT4 mRNA表达并无明显改变;油红O染色显示JAZF1转染组细胞内脂质积聚明显降低(P<0.05).结论 JAZFl在C57BL/6J小鼠多种组织均有表达,提示其可能在维持正常生理功能中起着一定作用.3T3-L1细胞过表达JAZF1可减少脂质合成、增加脂解,并可明显改善脂质积聚,可能是肥胖和糖尿病治疗的一个潜在靶点.     

Cooperative activation of lipolysis by protein kinase A and protein kinase C pathways in 3T3-L1 adipocytes

In the present study, we investigate the coherence of signaling pathways leading to lipolysis in 3T3-L1 adipocytes. We observe two linear signaling pathways: one well known, acting via cAMP and protein kinase A (PKA) activation, and a second one induced by phorbol 12-myristate 13-acetate treatment involving protein kinase C (PKC) and MAPK. We demonstrate that both the PKA regulatory subunits RIalpha and RIIbeta are expressed in 3T3-L1 adipocytes and are responsible for the lipolytic effect mediated via the cAMP/PKA pathway. Inhibition of the PKA pathway by the selective PKA inhibitor Rp-8-CPT-cAMPS does not impair lipolysis induced by PKC activation, and neither PD98059 nor U0126, as known MAPK kinase inhibitors, changes the level of glycerol release caused by PKA activation, indicating no cross-talk between these two pathways when only one is activated. However, when both are activated, they act synergistically on glycerol release. Additional experiments focusing on this synergy show no involvement of MAPK phosphorylation and cAMP formation. Phosphorylation of hormone-sensitive lipase is similar upon stimulation of either pathway, but we demonstrate a difference in the ability of both PKA and the PKC pathway activation to phosphorylate perilipin, which in turn may be an explanation for the different maximal lipolytic effect of both pathways.