Hofmann S, Pette D
Fakultät für Biologie, Universität Konstanz, Germany.
Eur J Biochem. 1994 Jan 15;219(1-2):307-15. doi: 10.1111/j.1432-1033.1994.tb19942.x.
This study followed changes in the capacities of uptake and phosphorylation of glucose in response to contractile activity in low-frequency stimulated (10Hz, 24 h/d) rat fast-twitch muscle. We investigated the intracellular distribution of GLUT-4, the major glucose transporter isoform in muscle, changes in the amounts of its specific mRNA and total cellular protein, as well as changes in its relative synthesis rate. These analyses were complemented by measurements of total hexokinase activity and hexokinase II (HKII) expression at the levels of mRNA content and protein synthesis. Changes in protein synthesis were determined by in vivo labeling with [35S]methionine. Translocation of GLUT-4 into the sarcolemma was an immediate response to contractile activity, whereas changes in its total amount were observed only with ongoing stimulation (5 d and longer). A twofold increase in GLUT-4 content after 5 d and longer stimulation periods was preceded by elevations of its mRNA and by enhanced [35S]methionine incorporation. Conversely, increases in HKII expression with a rise in total hexokinase activity occurred soon after the onset of stimulation (30-fold elevations of HKII mRNA after 12 h and 20-fold increases in [35S]methionine incorporation after 24 h). With ongoing stimulation, HKII mRNA and synthesis returned to lower levels (fivefold elevations). Nevertheless, hexokinase activity continued to rise, stabilizing at fivefold-elevated levels after 3 d. These observation suggested that posttranscriptional mechanisms contributed to the upregulation of HKII, e.g. stabilization by elevated intracellular glucose and mitochondrial binding of the enzyme. This suggestion was supported by experiments with cessation after 24 h where hexokinase activity continued to increase, although the mRNA content and, especially, the [35S]methionine incorporation decayed steeply. The increase in HKII prior to GLUT-4 suggests that phosphorylation may be rate limiting in glucose utilization of glycolytic fibers under conditions of sustained contractile activity. Taken together, the changes in distribution and content of GLUT-4, as well as in HKII represent early metabolic adaptations. In addition, they are related to the overall process of stimulation-induced fiber type transformation.
本研究追踪了低频刺激（10Hz，每天24小时）大鼠快肌中葡萄糖摄取和磷酸化能力随收缩活动的变化。我们研究了肌肉中主要的葡萄糖转运异构体GLUT-4的细胞内分布、其特异性mRNA和总细胞蛋白量的变化，以及其相对合成速率的变化。通过测量总己糖激酶活性以及己糖激酶II（HKII）在mRNA含量和蛋白质合成水平的表达，对这些分析进行补充。蛋白质合成的变化通过用[35S]甲硫氨酸进行体内标记来确定。GLUT-4向肌膜的转位是对收缩活动的即时反应，而其总量的变化仅在持续刺激（5天及更长时间）时才观察到。在5天及更长时间的刺激期后，GLUT-4含量增加两倍之前，其mRNA水平升高且[35S]甲硫氨酸掺入增加。相反，刺激开始后不久，HKII表达增加且总己糖激酶活性升高（12小时后HKII mRNA升高30倍，24小时后[35S]甲硫氨酸掺入增加20倍）。随着持续刺激，HKII mRNA和合成恢复到较低水平（升高5倍）。然而，己糖激酶活性继续上升，3天后稳定在升高5倍的水平。这些观察结果表明，转录后机制有助于HKII的上调，例如细胞内葡萄糖升高导致的稳定以及该酶的线粒体结合。24小时后停止刺激的实验支持了这一观点，尽管mRNA含量尤其是[35S]甲硫氨酸掺入急剧下降，但己糖激酶活性仍继续增加。HKII在GLUT-4之前增加表明，在持续收缩活动的条件下，磷酸化可能是糖酵解纤维葡萄糖利用中的限速步骤。总之，GLUT-4的分布和含量以及HKII的变化代表了早期代谢适应。此外，它们与刺激诱导的纤维类型转变的整体过程有关。
