2型糖尿病是一种由遗传和环境因素相互作用，以靶组织对胰岛素敏感性降低(胰岛素抵抗)、临床上以高血糖为主要特征的慢性全身性代谢性疾病。传统的抗2型糖尿病药物存在价格昂贵，副作用大等缺点，而微量元素三价铬是价格低廉，副作用小的抗糖尿病活性元素。但是到目前为止三价铬治疗糖尿病的作用机制尚不明确，限制了三价铬在临床的应用。 本研究采用三价铬化合物中生物活性最强及机体吸收率最高的吡啶羧酸铬(CrPic)作为研究药物，在胰岛素主要靶细胞之一的3T3-L1脂肪细胞中，模拟2型糖尿病患者体内高糖高胰岛素的胰岛素抵抗病理生理状态，采用高糖(25 mM)高胰岛素(100 nM)作用24 h诱导胰岛素抵抗的3T3-L1脂肪细胞模型，运用放射性³H葡萄糖及¹⁴C棕榈酸摄取实验，ELISA，Western Blot, real-time PCR,免疫荧光等方法观察CrPic对正常及胰岛素抵抗3T3-L1脂肪细胞的糖代谢、葡萄糖转运体4 (GLUT4)、相关脂肪因子分泌、脂肪酸代谢及脂肪酸转位酶FAT/CD36的影响及其机制，从而探讨CrPic可能的抗糖尿病作用机制。 第一部分 吡啶羧酸铬对正常及胰岛素抵抗3T3-L1脂肪细胞糖代谢的影响及其机制研究 本部分研究主要目的是观察CrPic对正常及胰岛素抵抗3T3-L1脂肪细胞糖代谢的作用及机制。研究采用高糖(25 mM)和高胰岛素(100 nM)作用24 h造成胰岛素抵抗的3T3-L1脂肪细胞模型。葡萄糖消耗实验显示，加入CrPic可显著降低正常及胰岛素抵抗3T3-L1脂肪细胞培液中的葡萄糖含量。³H-葡萄糖摄取实验结果表明，CrPic可增加基础及胰岛素刺激状态下的正常及胰岛素抵抗3T3-L1脂肪细胞对葡萄糖的摄取。免疫荧光结果显示，CrPic可促进基础及胰岛素刺激状态下的正常及胰岛素抵抗3T3-L1脂肪细胞的葡萄糖转运体GLUT4 从胞浆转位到胞膜。随后，我们研究了 CrPic产生这些作用的分子机制。Western Blot结果显示，CrPic对基础及胰岛素刺激状态下的正常及胰岛素抵抗3T3-L1脂肪细胞的胰岛素受体β (IR-β)、蛋白激酶B (Akt)、c-Cbl、细胞外调节蛋白激酶(ERK)及c-Jun的蛋白磷酸化都没有影响；real-time PCR研究发现，CrPic同样不会增加c-Cbl结合蛋白(CAP)的基因表达。有趣的是，CrPic被发现可显著增加基础及胰岛素刺激状态下的正常及胰岛素抵抗3T3-L1脂肪细胞的p38丝裂原素激活的蛋白激酶(p38 MAPK)磷酸化蛋白的表达，即激活p38 MAPK。加入p38 MAPK通路的特异性抑制剂SB203580之后，结果显示SB203580可抑制CrPic对p38 MAPK磷酸化的增强作用。同时CrPic对葡萄糖摄取的促进作用也能被SB203580抑制，但CrPic对GLUT4转位的促进作用却不会随着抑制剂的加入而消除。以上研究表明，CrPic通过激活P38 MAPK促进葡萄糖摄取，且这种作用不依赖于GLUT4的转位。 第二部分 吡啶羧酸铬对正常及胰岛素抵抗3T3-L1脂肪细胞中脂联素及抵抗素分泌的影响及其机制研究 脂肪细胞作为机体重要的内分泌器官，它分泌的细胞因子如脂联素、抵抗素等在胰岛素抵抗和2型糖尿病的发生发展中起着重要作用。抵抗素的分泌与胰岛素抵抗成正相关，而脂联素则可改善胰岛素抵抗。CrPic作为可改善脂质代谢异常的抗糖尿病药物，是否影响这些脂肪因子的分泌呢？本部分研究将通过ELISA及real-time PCR方法观察CrPic对正常及胰岛素抵抗3T3-L1脂肪细胞脂联素、抵抗素分泌及其基因表达的影响，并通过Western Blot研究CrPic对与脂联素和抵抗素分泌有关的信号通路AMP激活的蛋白激酶(AMPK)的作用。研究结果显示，CrPic不能影响脂联素和抵抗素的mRNA表达，但CrPic可抑制正常及胰岛素抵抗3T3-L1脂肪细胞的抵抗素分泌，且差异具有统计学意义。CrPic对脂联素的分泌及总蛋白表达没有影响。进一步的机制研究表明， CrPic可显著提高正常及胰岛素抵抗3T3-L1脂肪细胞中AMPK及乙酰辅酶A羧化酶(ACC)的磷酸化蛋白表达水平。但CrPic并不影响AMPK α1及AMPK α2的mRNA水平。加入AMPK通路的特异性抑制剂compound C之后，随着CrPic对磷酸化AMPK及磷酸化ACC蛋白表达的增强作用被抑制，CrPic对正常及胰岛素抵抗3T3-L1脂肪细胞抵抗素分泌的抑制作用也消失。研究显示， CrPic通过AMPK通路抑制正常及胰岛素抵抗3T3-L1脂肪细胞的抵抗素分泌。 第三部分 吡啶羧酸铬对正常3T3-L1脂肪细胞脂肪酸代谢及脂肪酸转位酶CD36的影响及其机制研究 长链脂肪酸(LCFA)是维持和产生能量的重要底物。研究显示脂肪酸转位酶CD36在转运长链脂肪酸中起着重要作用。近年来，CD36在2型糖尿病及胰岛素抵抗中的作用受到越来越多的关注。本部分研究通过Western Blot及免疫荧光方法研究CrPic及胰岛素对3T3-L1脂肪细胞中脂肪酸转位酶CD36转位的影响及机制，并通过¹⁴C-棕榈酸摄取实验观察CrPic及胰岛素对3T3-L1脂肪细胞脂肪酸摄取的影响。免疫荧光及Western Blot结果表明，CD36蛋白可表达在分化成熟的3T3-L1脂肪细胞，而在未分化的前脂肪细胞并没有表达。免疫荧光结果显示，CrPic和胰岛素可促进3T3-L1脂肪细胞中的CD36转位到胞膜；胞膜蛋白的Western Blot结果也表明，加入CrPic和胰岛素可使胞膜的CD36表达量较未加入CrPic前显著提高，但CrPic比胰岛素的作用弱。CrPic对3T3-L1脂肪细胞的脂肪酸摄取没有影响，而胰岛素可以增加3T3-L1脂肪细胞的脂肪酸摄取。加入PI3K通路抑制剂wortmannin之后，胰岛素对CD36的转位及脂肪酸摄取的促进作用都被抑制。而加入AMPK通路抑制剂compound C之后，CrPic对3T3-L1脂肪细胞CD36的转位作用被抑制。以上研究表明，胰岛素通过PI3K通路促进3T3-L1脂肪细胞的CD36转位，并促进相应的脂肪酸摄取；而CrPic通过激活AMPK促进CD36的转位，但是CrPic对CD36转位的促进作用却不足以增加脂肪酸的摄取。 结论： 1.CrPic通过激活p38 MAPK促进正常及胰岛素抵抗3T3-L1脂肪细胞的葡萄糖摄取，且这种作用不依赖于GLUT4的转位。 2.CrPic通过AMPK通路的介导抑制正常及胰岛素抵抗3T3-L1脂肪细胞抵抗素的分泌。 3.CrPic促进3T3-L1脂肪细胞中的脂肪酸转位酶CD36转位至胞膜，这种作用是通过AMPK通路介导的。但是CrPic对CD36的转位作用却不足以使脂肪酸的转运增加。胰岛素通过PI3K通路的介导促进3T3-L1脂肪细胞中CD36的转位及脂肪酸的摄取。 关键词：2型糖尿病:3T3-L1脂肪细胞；AMPK； FAT/CD36； GLUT4； p38 MAPK；吡啶羧酸铬；抵抗素；葡萄糖转运；胰岛素抵抗；脂肪酸转运 中图分类号:R96

 Type 2 diabetes is a progressive chronic disease resulting from a dynamic interaction between defects in insulin secretion and action (insulin resistance). Traditional anti-diabetic drugs are expensive and have many side effects. As a supplemental or alternative medication for type 2 diabetes, trivalent chromium (Cr) is an essential nutrient with low price and toxicity. But till now, its anti-diabetic mechanisms are not well clarified. Understanding of these mechanisms is necessary for more effective use of trivalent Cr in the treatment of type 2 diabetes. In the present study, we selected chromium picolinate (CrPic) which is the most bioavailable and clinical effective in both human and animal studies in all forms of Cr as the representative. Insulin resistance plays a key role in the development of type 2 diabetes mellitus characterized by high glucose and insulin levels in diabetic patients. To induce insulin resistance model in 3T3-L1 adipocytes, the cells were incubated for a period of 24 h in high glucose(25 mM) and insulin (100 nM) as insulin-resistant cells. By using ³H-glucose and ¹⁴C-palmitate uptake experiments, ELISA, Western Blot, real-time PCR, immunofluorescence microscopy and so on, we explored the effects and mechanisms of CrPic on glucose metabolism and uptake, glucose transporter 4 (GLUT4) translocation, adipokines secretion, long-chain fatty acid (LCFA) uptake and fatty acid translocase (FAT)/CD36 translocation in control and insulin-resistant 3T3-L1 adipocytes. Part 1 The effects and mechanisms of CrPic on glucose metabolism and uptake in control and insulin-resistant 3T3-L1 adipocytes The present study was to investigate the effects and mechanisms of CrPic on glucose metabolism and uptake in control and insulin-resistant 3T3-L1 adipocytes. The insulin-resistant model was induced by treatment with high glucose (25 mM) and high insulin (100 nM) for 24 h. Glucose consumption experiment showed that CrPic reduced the glucose concentration in the medium significantly in control and insulin-resistant 3T3-L1 adipocytes. 2-DOG uptake showed that incubation of 3T3-L1 adipocytes with CrPic increased the basal and insulin-stimulated glucose transport activity in both control and insulin-resistant 3T3-L1 adipocytes. From immunofluorescence microscopy, it was revealed that CrPic increased the basal and insulin-stimulated translocation of GLUT4 from intracellular pools to the plasma membrane (PM) in both cells. Then we investigated the signaling cascades that contribute to CrPic-induced glucose metabolism and uptake. Western Blot and real-time PCR revealed that CrPic did not have any effects on insulin receptor β (IR-β), protein kinase B (Akt), c-Cbl, extracellular signal-regulated kinase (ERK), c-Jun phosphorylation, and c-Cbl-associated protein (CAP) mRNA levels. Interestingly, CrPic was able to increase the basal and insulin-stimulated levels of p38 MAPK activation in the control and insulin-resistant cells. Pre-treatment with the specific p38 MAPK inhibitor SB203580 inhibited the CrPic-induced p38 MAPK activation and glucose transport, but CrPic-activated translocation of GLUT4 was not inhibited by SB203580. This study provides experimental evidence of the effects of chromium picolinate on glucose uptake through the activation of p38 MAPK and it is independent of the effect on GLUT4 translocation. Part 2 The effects and mechanisms of CrPic on adiponectin and resistin secretion in control and insulin-resistant 3T3-L1 adipocytes As an important endocrine tissue, adipose tissue secretes adipokines such as adiponectin and resistin that have been linked to the development of insulin resistance and type 2 diabetes. Resistin levels were found to be markedly elevated in patients with obesity and type 2 diabetes, but adiponectin is an anti-diabetic hormone. CrPic has been shown to have therapeutic effects for treating type 2 diabetes and improving abnormal lipid metabolism; however, the effects of CrPic on the secretion of adipokines are unknown. Using ELISA and real-time PCR, the present study was to investigate the effects of CrPic on gene transcription and secretion of adiponectin and resistin in control and insulin-resistant 3T3-L1 adipocytes. Then we investigated whether CrPic could activate AMP activated protein kinase (AMPK), which contributes to the regulation of adiponectin and resistin expression and secretion. We found that CrPic did not modulate the expression of adiponectin and resistin; however, it significantly inhibited the secretion of resistin, but not adiponectin, in control and insulin-resistant 3T3-L1 adipocytes in vitro. Furthermore, CrPic markedly elevated the levels of phosphorylated AMPK and acetyl-CoA carboxylase (ACC), but did not change the levels of AMPK α-1 and α-2 mRNA transcripts in 3T3-L1 adipocytes. Importantly, inhibition of AMPK by compound C completely abolished CrPic-induced suppression of resistin secretion. Our data suggest that CrPic inhibits resistin secretion via the activation of AMPK in control and insulin-resistant 3T3-L1 adipocytes. Part 3 The effects and mechanisms of CrPic on LCFA uptake and fatty acid translocase (FAT)/CD36 in 3T3-L1 adipocytes Long-chain fatty acid (LCFA) is one of the major substrates generating energy to sustain life process. CD36 is one of the LCFA transporter proteins extensively studied, and identified to be a key determinant in cellular LCFA uptake. Through Western Blot and immunofluorescence microscopy, the present study was to investigate the effects and mechanisms of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the plasma membrane (PM) in 3T3-L1 adipocytes. And the effects of CrPic and insulin on LCFA uptake were determined by ¹⁴C-palmitate uptake experiment. Immunofluorescence microscopy and Western Blot revealed that CD36 was expressed in 3T3-L1 adipocytes, but not in 3T3-L1 preadipocytes. Immunofluorescence microscopy revealed that CD36 was primarily located intracellularly, and upon insulin or CrPic stimulation PM expression of CD36 increased; Western Blot of PM protein showed that the CrPic-stimulated PM expression was however weaker than that of insulin. But CrPic did not have any effects on LCFA ([1-¹⁴C] palmitate) uptake. The increase in PM localization for CD36 by insulin paralleled LCFA ([1-¹⁴C]palmitate) uptake in 3T3-L1 adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of CD36 and prevented the increased uptake of LCFA in these cells. And AMPK inhibitor compound C could abolish the effects of CrPic on CD36 transcolation. Taken together, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes, that their translocation-inducing effects are not additive, and that the signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36. CrPic increased the translocation of CD36 via the activation of AMPK in 3T3-L1 adipocytes, but this effect on CD36 was insufficient to increase the uptake of LCFA. Summary: 1.CrPic enhanced glucose uptake in control and insulin-resistant 3T3-L1 adipocytes through the activation of p38 MAPK and it is independent of the effect on GLUT4 translocation. 2.CrPic inhibited resistin secretion via the activation of AMPK in both control and insulin-resistant 3T3-L1 adipocytes. 3.CrPic increased the translocation of CD36 via the activation of AMPK in 3T3-L1 adipocytes, but this effect on CD36 was insufficient to increase the uptake of LCFA. Insulin enhanced the translocation of CD36 and LCFA uptake through PI3K pathway. Key words: 3T3-L1 adipocytes; AMPK; Chromium picolinate; FAT/CD36; Glucose transporter 4; Glucose uptake; Insulin resistance; p38 MAPK; Palmitate uptake; Resistin; Type 2 diabetes 中图分类号:R96