Porter Marty H, Paveglio Sara A, Zhang Jin-an, Olson Darin E, Campbell Adam G, Thulé Peter M
Department of Internal Medicine and Metabolism Section, Veterans Administration Medical Center, Decatur, GA, USA.
J Investig Med. 2005 May;53(4):201-12. doi: 10.2310/6650.2005.00404.
Hepatic insulin gene therapy (HIGT) restores weight gain and near-normal glycemia in rodent models of insulin-deficient diabetes mellitus. However, the effect of transgenic insulin on endogenous genes and recipient cell function is relatively unexplored. To investigate hepatocellular effects of transgenic insulin expression, we evaluated intermediary glucose metabolism in primary cultured hepatocytes treated with HIGT.
Rat hepatocytes were transduced with adenovirus expressing a glucose-responsive human insulin transgene and cultured in high-glucose and high-insulin conditions. We determined glycogen content in cell cultures and intact liver directly. Glycogenolysis was compared using glucose production of cultured cells. Glucose uptake, oxidative, and glycolytic processing were determined by radiotracer analysis or direct end-product assessment. Quantitative real-time reverse transcriptase polymerase chain reaction was used to determine expression of glucose transporter 2 (GLUT2) and glucokinase genes. GLUT2 protein abundance was determined by Western blot analysis.
HIGT-treated hepatocytes contained significantly less glycogen than either untreated hepatocytes or those treated with an empty virus. Glucose release owing to glycogenolysis remained normal. However, HIGT treatment significantly impaired glucose uptake and processing. Metabolic synthetic processes were not generally inhibited, as indicated by enhanced beta-hydroxybutyrate secretion. While preserving cell viability, HIGT treatment diminished expression of both glucokinase and GLUT2. In HIGT-treated streptozocin-treated diabetic rats, total liver glycogen was intermediate between diabetic animals and normal controls.
These results suggest gene-specific effects in recipient hepatocytes following HIGT treatment and underscore the need for expanded studies examining host cell responses to the transfer of metabolically active transgenes.
肝胰岛素基因疗法（HIGT）可使胰岛素缺乏型糖尿病啮齿动物模型恢复体重增加并实现血糖接近正常。然而，转基因胰岛素对内源基因和受体细胞功能的影响相对未被充分研究。为了探究转基因胰岛素表达对肝细胞的影响，我们评估了经HIGT处理的原代培养肝细胞中的中间葡萄糖代谢。
用表达葡萄糖反应性人胰岛素转基因的腺病毒转导大鼠肝细胞，并在高糖和高胰岛素条件下培养。我们直接测定细胞培养物和完整肝脏中的糖原含量。使用培养细胞的葡萄糖生成比较糖原分解。通过放射性示踪分析或直接终产物评估来测定葡萄糖摄取、氧化和糖酵解过程。采用定量实时逆转录聚合酶链反应来测定葡萄糖转运蛋白2（GLUT2）和葡萄糖激酶基因的表达。通过蛋白质印迹分析测定GLUT2蛋白丰度。
经HIGT处理的肝细胞所含糖原明显少于未处理的肝细胞或用空病毒处理的肝细胞。糖原分解引起的葡萄糖释放保持正常。然而，HIGT处理显著损害了葡萄糖摄取和代谢过程。如β-羟基丁酸分泌增加所示，代谢合成过程一般未受抑制。在保持细胞活力的同时，HIGT处理降低了葡萄糖激酶和GLUT2的表达。在经HIGT处理的链脲佐菌素诱导的糖尿病大鼠中，肝脏总糖原含量介于糖尿病动物和正常对照之间。
这些结果表明HIGT处理后受体肝细胞中存在基因特异性效应，并强调需要开展更多研究来检查宿主细胞对代谢活性转基因转移的反应。
