Host cells reduce glucose uptake and glycogen deposition in response to hepatic insulin gene therapy.
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 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.


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.
Department of Internal Medicine and Metabolism Section, Veterans Administration Medical Center, Decatur, GA, USA.
Marty H
MH
Porter
Sara A
SA
Paveglio
Jin-an
JA
Zhang
Darin E
DE
Olson
Adam G
AG
Campbell
Peter M
PM
Thulé
D000818:Animals; D015316:Genetic Therapy; D005941:Glucokinase / Q000235:genetics; D005943:Gluconeogenesis; D005947:Glucose / Q000378:metabolism*; D051273:Glucose Transporter Type 2; D006003:Glycogen / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism; D007328:Insulin / Q000235:genetics*; D008099:Liver / Q000378:metabolism*; D008297:Male; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D051273:Glucose Transporter Type 2; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D006003:Glycogen; D005941:Glucokinase; D005947:Glucose