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Host Cells Reduce Glucose Uptake and Glycogen Deposition in Response to Hepatic Insulin Gene Therapy
  1. Marty H. Porter,
  2. Sara A. Paveglio,
  3. Jin-an Zhang,
  4. Darin E. Olson,
  5. Adam G. Campbell,
  6. Peter M. Thulé
  1. From the Department of Internal Medicine (M.H.P., S.A.P., D.E.O., A.G.C., P.M.T.), Endocrinology and Metabolism Section, Veterans Administration Medical Center and Emory University, Decatur, GA, and the Department of Endocrinology, The First Hospital of Xi'an Jiaotong (J.Z.), University, Xi'an, China. This work was supported by the following grants: research grant, Juvenile Diabetes Research Foundation International 1-2000-401; Innovative Award, American Diabetes Association; Georgia Tech/Emory Center for Engineering of Living Tissues/National Science Foundation EEC-9731643; Veterans Affairs Merit Review Award to P.M.T.; and National Institutes of Health/National Research Service Award DKO 7298 and a Veterans Award VISN7 Career Development Award to D.E.O.
  1. Address correspondence to: Dr. Peter M. Thulé, Veterans Administration Medical Center, Room 5A170, Clinical Addition, 1670 Clairmont Road, Decatur, GA 30033; tel: 404-321-6111 ext. 2079; fax: 404-728-7780; e-mail: pthule{at}emory.edu.

Abstract

Background 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.

Methods 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.

Results 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 β-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.

Conclusions 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.

Key Words
  • adenovirus
  • liver
  • glycogen

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