Gómez-Foix A M, Coats W S, Baqué S, Alam T, Gerard R D, Newgard C B
Gifford Laboratories for Diabetes Research, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1992 Dec 15;267(35):25129-34.
The muscle isozyme of glycogen phosphorylase is potently activated by the allosteric ligand AMP, whereas the liver isozyme is not. In this study we have investigated the metabolic impact of expression of muscle phosphorylase in liver cells. To this end, we constructed a replication-defective, recombinant adenovirus containing the muscle glycogen phosphorylase cDNA (termed AdCMV-MGP) and used this system to infect hepatocytes in culture. AMP-activatable glycogen phosphorylase activity was increased 46-fold 6 days after infection of primary liver cells with AdCMV-MGP. Despite large increases in phosphorylase activity, glycogen levels were only slightly reduced in AdCMV-MGP-infected liver cells compared to uninfected cells or cells infected with wild-type adenovirus. The lack of correlation of phosphorylase activity and glycogen content suggests that the liver cell environment can inhibit the muscle phosphorylase isozyme. This inhibition can be overcome, however, by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which increases AMP levels by 30-fold and causes a much larger decrease in glycogen levels in AdCMV-MGP-infected cells than in uninfected or wild-type adenovirus-infected controls. CCCP treatment also caused a preferential decrease in glycogen content relative to glucagon treatment in AdCMV-MGP-infected hepatocytes (74% versus 11%, respectively), even though the two drugs caused equal increases in phosphorylase a activity. Introduction of muscle phosphorylase into hepatocytes therefore confers a capacity for glycogenolytic response to effectors that is not provided by the endogenous liver phosphorylase isozyme. The remarkable efficiency of adenovirus-mediated gene transfer into primary hepatocytes and the demonstration of altered regulation of glycogen metabolism as a consequence of expression of a non-cognate phosphorylase isozyme may have implications for gene therapy of glycogen storage diseases.
糖原磷酸化酶的肌肉同工酶可被变构配体AMP有效激活，而肝脏同工酶则不然。在本研究中，我们调查了肌肉磷酸化酶在肝细胞中表达的代谢影响。为此，我们构建了一种复制缺陷型重组腺病毒，其包含肌肉糖原磷酸化酶cDNA（称为AdCMV - MGP），并使用该系统感染培养中的肝细胞。用AdCMV - MGP感染原代肝细胞6天后，AMP可激活的糖原磷酸化酶活性增加了46倍。尽管磷酸化酶活性大幅增加，但与未感染细胞或感染野生型腺病毒的细胞相比，AdCMV - MGP感染的肝细胞中的糖原水平仅略有降低。磷酸化酶活性与糖原含量缺乏相关性表明，肝细胞环境可抑制肌肉磷酸化酶同工酶。然而，通过添加羰基氰化物间氯苯腙（CCCP）可以克服这种抑制作用，CCCP可使AMP水平增加30倍，并导致AdCMV - MGP感染细胞中的糖原水平比未感染或野生型腺病毒感染的对照细胞有更大幅度的降低。在AdCMV - MGP感染的肝细胞中，CCCP处理相对于胰高血糖素处理也导致糖原含量优先降低（分别为74%和11%），尽管这两种药物导致磷酸化酶a活性同等增加。因此，将肌肉磷酸化酶引入肝细胞赋予了对效应物的糖原分解反应能力，而内源性肝脏磷酸化酶同工酶则不具备这种能力。腺病毒介导的基因转移到原代肝细胞中的显著效率以及非同源磷酸化酶同工酶表达导致糖原代谢调节改变的证明可能对糖原贮积病的基因治疗有影响。
