灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响

目的 研究灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响,探讨灵芝多糖改善胰岛素抵抗的分子机制.方法 3T3-L1前脂肪细胞经1-甲基-3-异丁基-黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,以葡萄糖氧化酶法测定培养液中残余的葡萄糖含量.比较二甲双胍组,检测培养液中葡萄糖含量及PI-3K p85和GLUT4蛋白表达变化.结果 地塞米松联合胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,细胞对葡萄糖的摄取量减少.灵芝多糖可改善3T3-L1脂肪细胞胰岛素抵抗.胰岛素抵抗细胞的PI-3K p85和GLUT4蛋白表达明显减少;应用灵芝多糖后,相关蛋白表达增加.结论 灵芝多糖通过提高PI-3K p85和GLUT4蛋白的表达,参与胰岛素抵抗状态下3T3-L1细胞的葡萄糖代谢.     

Pioglitazone upregulates adiponectin receptor 2 in 3T3-L1 adipocytes

AimsPioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes.Main methodsGlucose uptake was evaluated by 2-[³H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT–PCR.Key findingsWe first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ.SignificanceOur results suggest that pioglitazone increases insulin sensitivity, at least partly, by PPARγ-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. In conclusion, the upregulation of AdipoR2 expression may be one of the mechanisms by which pioglitazone improves insulin resistance in 3T3-L1 adipocytes.     

Effects of adrenal hormones on the expression of adiponectin and adiponectin receptors in adipose tissue, muscle and liver

Background

Adiponectin, an insulin-sensitive hormone that is primarily synthesized in adipose tissue, exerts its effects by binding to two receptors, adipoR1 and adipoR2. Little is known regarding the effects of glucocorticoids on the expression of adiponectin receptors.

Methods

Male Wistar rats were bilaterally adrenalectomized and treated with dexamethasone (0.2 mg/100 g) twice daily for 3 days. To analyze the potential effects of glucocorticoids, rats received two daily injections of the glucocorticoid receptor antagonist (RU-486, 5.0 mg) over the course of 3 days. Additionally, 3T3-L1 adipocytes and C2C12 myotubes were treated with dexamethasone, adrenaline or RU-486. The gene expression of adiponectin, adipoR1 and adipoR2 was determined by real-time PCR, and protein secretion was examined by Western blotting using lysates from retroperitoneal, epididymal and subcutaneous adipose tissue depots, liver and muscle.

Results

In rats, excess glucocorticoids increased the levels of insulin in serum and decreased serum adiponectin concentrations, whereas adrenalectomy decreased the mRNA expression of adiponectin (3-fold) and adipoR2 (7-fold) in epididymal adipose tissue and increased adipoR2 gene expression in muscle (3-fold) compared to control group sham-operated. Dexamethasone treatment did not reverse the effects of adrenalectomy, and glucocorticoid receptor blockade did not reproduce the effects of adrenalectomy. In 3T3-L1 adipocytes, dexamethasone and adrenaline both increased adipoR2 mRNA levels, but RU-486 reduced adipoR2 gene expression in vitro.

Conclusion

Dexamethasone treatment induces a state of insulin resistance but does not affect adiponectin receptor expression in adipose tissue. However, the effects of catecholamines on insulin resistance may be due to their effects on adipoR2.     

Co-treatment with dexamethasone and octanoate induces adipogenesis in 3T3-L1 cells

We report here that octanoate, a medium chain fatty acid, induces adipocyte differentiation in 3T3-L1 cells by co-treatment with dexamethasone, although octanoate has been known not to stimulate 3T3-L1 adipogenesis. A low concentration of exogenous glucose prevented 3T3-L1 adipogenesis induced by 1-methyl 3-isobutylxanthine, dexamethasone, and insulin (MDI) treatment (a common protocol for adipocyte differentiation). In contrast, co-treatment with dexamethasone and octanoate (D-OCT) induced adipogenesis under the same conditions. These findings imply that octanoate, rather than glucose, is the source of accumulated lipids in D-OCT-induced adipogenesis. D-OCT increased expression of the differentiation markers peroxisome proliferator-activated receptor (PPAR)gamma2 and caveolin-1. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase inhibited D-OCT-induced adipogenesis. These results suggest that the p38 MAP kinase pathway followed by up-regulation of PPARgamma2 may be involved in 3T3-L1 adipocyte differentiation induced by D-OCT, as well as by MDI.     

Apelin stimulates glucose uptake through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.     

Possible involvement of pertussis toxin-sensitive GTP-binding protein(s) in c-fos expression during differentiation of 3T3-L1 fibroblasts to adipocytes.

Following the differentiation of 3T3-L1 fibroblasts by insulin/dexamethasone/methylisobutylxanthine, marked increases in cAMP levels by isoproterenol but not forskolin and in 2-deoxyglucose uptake by insulin occurred. Pertussis toxin-pretreatment prior to addition of insulin/dexamethasone/methylisobutylxanthine and exposure of cells to pertussis toxin during differentiation attenuated glycerophosphate dehydrogenase activity as a differentiation marker enzyme and the responses to isoproterenol and insulin by approximately 50% of those in pertussis toxin-untreated cells. On the other hand, insulin/dexamethasone/methylisobutylxanthine caused induction of c-fos proto-oncogene in confluent 3T3-L1 fibroblasts. This induction was also reduced in pertussis toxin-pretreated cells. These results suggested that pertussis toxin-sensitive GTP-binding protein(s) is involved in expression of c-fos mRNA accompanied by differentiation. In addition, accumulation of c-fos mRNA by insulin/dexamethasone/methylisobutylxanthine was enhanced in protein kinase C-depleted cells pretreated with phorbol 12-myristate 13-acetate, indicating that protein kinase C may negatively regulate c-fos expression induced by insulin/dexamethasone/methylisobutylxanthine.     

Glucose, dexamethasone, and the unfolded protein response regulate TRB3 mRNA expression in 3T3-L1 adipocytes and L6 myotubes

Tribbles 3 (TRB3) is a recently recognized atypical inactive kinase that negatively regulates Akt activity in hepatocytes, resulting in insulin resistance. Recent reports link TRB3 to nutrient sensing and regulation of cell survival under stressful conditions. We studied the regulation of TRB3 by glucose, insulin, dexamethasone (Dex), and the unfolded protein response (UPR) in 3T3-L1 adipocytes and in L6 myotubes. In 3T3-L1 adipocytes, incubation in high glucose with insulin did not increase TRB3 mRNA expression. Rather, TRB3 mRNA increased fourfold with glucose deprivation and two- to threefold after incubation with tunicamcyin (an inducer of the UPR). Incubation of cells in no glucose or in tunicamcyin stimulated the expression of CCAAT/enhancer-binding protein homologous protein. In L6 myotubes, absent or low glucose induced TRB3 mRNA expression by six- and twofold, respectively. The addition of Dex to 5 mM glucose increased TRB3 mRNA expression twofold in 3T3-L1 adipocytes but decreased it 16% in L6 cells. In conclusion, TRB3 is not the mediator of high glucose or glucocorticoid-induced insulin resistance in 3T3-L1 adipocytes or L6 myotubes. TRB3 is induced by glucose deprivation in both cell types as a part of the UPR, where it may be involved in regulation of cell survival in response to glucose depletion.     

胰岛素对3T3-L1脂肪细胞中极低密度脂蛋白受体基因表达的影响

体外培养3T3-L1细胞分化模型,研究不同浓度胰岛素及慢性胰岛素刺激对3T3-L1脂肪细胞中极低密度脂蛋白受体（VLDLR）基因表达的影响.在不同浓度胰岛素及胰岛素慢性刺激的干预下,用半定量RT-PCR检测细胞VLDLR mRNA水平的变化.微量化GOD-PAP法检测培养基中残存的葡萄糖.在细胞诱导分化过程中,胰岛素浓度的增高促进VLDLR的表达;胰岛素慢性刺激下,VLDLR表达因浓度差异呈现不同变化.研究结果表明,胰岛素的浓度及慢性刺激对3T3-L1脂肪细胞的成熟和VLDLR基因的表达有显著作用,而胰岛素抵抗明显减低成熟脂肪细胞VLDLR的表达.     

The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis.

The receptors for insulin and insulin-like growth factor I (IGF-I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF-I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA-synthesis, and expression of proto-oncogene c-fos in 3T3-L1 adipocytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, KI of IGF-I greater than 500 nM; IGF-I receptor, KD = 1.1 nM, KI of insulin = 191 nM). Induction of proto-oncogene c-fos mRNA by insulin and IGF-I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF-I, 3.9 nM). Similarly, both insulin and IGF-I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF-I, 4.0 nM), glucose transport activity (EC50 of insulin, 1.7 nM; IGF-I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3-L1 cells the alpha-subunits of insulin and IGF-I receptors have an equal potential to stimulate a metabolic and a mitogenic response.