Acquired Defects of Glycogen Synthase Activity in Cultured Human Skeletal Muscle Cells: Influence of High Glucose and Insulin Levels
Acquired Defects of Glycogen Synthase Activity in Cultured Human Skeletal Muscle Cells: Influence of High Glucose and Insulin Levels
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DOI：
10.2337/diab.45.4.400
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发表时间：
1996-04
期刊：
影响因子：
7.7
通讯作者：
R. Henry;T. Ciaraldi;S. Mudaliar;L. Abrams;S. Nikoulina
中科院分区：
文献类型：
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作者：
R. Henry;T. Ciaraldi;S. Mudaliar;L. Abrams;S. Nikoulina
为了确定肌糖原合成酶（GS）活性的缺陷是否可以通过暴露于升高的葡萄糖或胰岛素水平而获得，从正常对照对象中通过针活检获得的人类骨骼肌细胞在培养中生长4 - 6周，然后在含有正常（5.5 mmol/l葡萄糖和22 pmol/l胰岛素）或增加浓度的葡萄糖（20 mmol/l）、胰岛素（30微mol/l）或两者的培养基中融合和分化4天。在正常培养基中融合后，33 nmol/l胰岛素刺激1 h，使GS分数速度（FV）增加约2倍（从9.01±1.26增加到16.31±2.40,P < 0.05）。在融合过程中，将培养基中葡萄糖浓度单独增加到20 mmol/l对基础FV没有影响，但会对胰岛素刺激的GS反应造成轻微损害（从8.51±1.33到12.99±1.90,P = 0.08）。在5.5 mmol/l葡萄糖的融合过程中，将培养基胰岛素浓度增加到30微mol/l也没有改变基础GS FV(10.61±1.69%)，但完全消除了正常胰岛素刺激的GS活性增加（11.63±1.55%，NS）。融合过程中高胰岛素（30微mol/l）和高葡萄糖（20 mmol/l）联合使用对基础（11.66±2.16%，NS）或胰岛素刺激（9.20±1.80%，NS） GS活性的FV影响均不大于单独使用高胰岛素。高胰岛素血症培养基中的融合改变了GS的动力学参数，尿苷二磷酸-葡萄糖的基础Km0.1和Vmax0.1几乎增加了一倍。高胰岛素血症也完全阻止了正常胰岛素刺激下Vmax0.1的三倍增长和葡萄糖-6-磷酸A0.5的65%下降。通过RNase保护实验和免疫印迹检测，GS mRNA和蛋白的表达不受培养基条件变化的影响。我们得出结论，暴露于高胰岛素环境的人类骨骼肌细胞通过多种获得性翻译后缺陷诱导严重的胰岛素抵抗，从而影响GS酶的动力学特性和绝对活性。
To determine whether defects of muscle glycogen synthase (GS) activity can be acquired by exposure to elevated glucose or insulin levels, human skeletal muscle cells obtained by needle biopsy from normal control subjects were grown in culture for 4–6 weeks followed by 4 days of fusion and differentiation in media containing either normal (5.5 mmol/l glucose and 22 pmol/l insulin) or increased concentrations of glucose (20 mmol/l), insulin (30 micromol/l), or both. After fusion in normal media, acute stimulation by 33 nmol/l insulin for 1 h increased GS fractional velocity (FV) ∼ twofold (from 9.01 ± 1.26 to 16.31 ± 2.40, P < 0.05). Increasing the media glucose concentration alone to 20 mmol/l during fusion had no effect on basal FV but caused a marginal impairment of the insulin-stimulated GS response (from 8.51 ± 1.33 to 12.99 ± 1.90, P = 0.08). Increasing the media insulin concentration to 30 micromol/l during fusion at 5.5 mmol/l glucose also did not alter basal GS FV (10.61 ± 1.69%) but completely abolished the normal insulin-stimulated increase in GS activity (to 11.63 ± 1.55%, NS). The combination of high insulin (30 micromol/l) and high glucose (20 mmol/l) during fusion had no greater effect on the FV of either basal (11.66 ± 2.16%, NS) or insulin-stimulated (9.20 ± 1.80%, NS) GS activity than high insulin alone. Fusion in hyperinsulinemic media altered the kinetic parameters of GS with a near doubling of the basal Km0.1 and Vmax0.1 for uridinediphospho-glucose. Hyperinsulinemia also totally prevented the normal insulin-stimulated threefold increase in the Vmax0.1 and the 65% decrease in the A0.5 for glucose-6-phosphate. GS mRNA and protein expression, determined by RNase protection assay and immunoblotting, respectively, were unaffected by changes in media conditions. We conclude that exposure of human skeletal muscle cells primarily to high insulin induces severe insulin resistance through multiple acquired posttranslational defects, which affect both the kinetic characteristics and absolute activity of the GS enzyme.