894. Targeting Adenovirus Mediated Gene Transfer to Chondrocytes In Vitro

Abstract

Metabolically responsive insulin transgenes can be transduced into the livers of diabetic rodents and lead to normalization of randomly sampled blood glucose. Hepatocytes can only produce insulin through a constitutive pathway. Therefore, rapidly changing serum levels of insulin are not possible with this form of gene therapy for diabetes. However, blood glucose remains under better control than can be accounted for by the systemic levels of insulin produced by hepatocytes transduced by an adenoviral vector to express a metabolically sensitive transgene, Ad/(GlRE)3BP-1 2xFur. We hypothesize that intrahepatic carbohydrate metabolism is changed during hepatic insulin gene therapy to contribute to the mechanism of glucose regulation in rats treated with this form of gene therapy. We previously measured in vitro carbohydrate metabolism in primary hepatocytes transduced with Ad/(GlRE)3BP-1 2xFur showing decreased glycogen, decreased gluconeogenesis, and decreased glucose uptake. We now expand the observations to in vivo carbohydrate metabolism. Sprague-Dawley rats were made diabetic with streptozotocin then received Ad/(GlRE)3BP-1 2xFur (2 x 1010 PFU/kg), and compared to non-diabetic rats, diabetic controls, and diabetic rats treated with peripherally administered insulin. Blood glucose and carbohydrate-regulatory hormones were monitored. Glycogen was measured in a group of rats sacrificed during unrestrained feeding. Another group of rats underwent 24 hour fasts with administration of deuterium oxide near the end of the fast to measure pathways of hepatic glucose production by NMR spectroscopy to demonstrate different pathways of gluconeogenesis and glycogenolysis that are active depending on the sites of incorporation of deuterium onto carbon molecules of glucose produced in the liver. Blood glucose was similar in rats producing hepatic insulin and normal rats, with higher levels of glucagon and growth hormone in rats treated with gene therapy. Random levels of hepatic glycogen were highest in normal rats (34.2 + 6.4 mg/g of liver tissue), with less in rats producing insulin within the liver (26.0 + 9.0 mg/g), and lowest in diabetic rats (15.1 + 4.4 mg/g). Normal and gene therapy treated rats had similar blood glucose during the fast, with higher glucose in the diabetic controls. Plasma glucose was extracted and converted to mono-acetylated glucose for evaluation by NMR spectroscopy. The NMR spectra show different relative activity in pathways of hepatic glucose metabolism. Hepatic insulin gene therapy is associated with normalization of random and fasting blood glucose levels, but the mechanism of glucose regulation is different than normal animals due to changes in intrahepatic carbohydrate metabolism and counterregulatory hormones. Potential mechanisms are: (1) autocrine or paracrine activity of hepatic insulin, (2) unique counterregulatory hormone responses due to the anatomical site of hepatic insulin production, (3) changes in hepatic carbohydrate metabolism induced by intracellular insulin, and (4) changes in metabolic enzymes due to transcription factors used by the metabolically responsive transgene.