ABSTRACT
Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector-mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 +/- 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 +/- 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 +/- 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 +/- 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 +/- 291 pg/ml; vehicle, 1,861 +/- 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.
Subject(s)
Diabetes Mellitus/genetics , Gene Expression/physiology , Glucokinase/genetics , Adenoviridae/genetics , Animals , Blood Glucose/analysis , Diabetes Mellitus/physiopathology , Eating , Fasting/blood , Gene Transfer Techniques , Genetic Vectors , Glucokinase/metabolism , Glycogen/metabolism , Humans , Insulin/blood , Liver/enzymology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Phenotype , Triglycerides/metabolismABSTRACT
The enzyme glucokinase (GK) plays a central role in glucose homeostasis. Hepatic GK activity is acutely controlled by the action of the GK regulatory protein (GKRP). In vitro evidence suggests that GKRP reversibly binds to GK and inhibits its activity; however, less is known about the in vivo function of GKRP. To further explore the physiological role of GKRP in vivo, we used an E1/E2a/E3-deficient adenoviral vector containing the cDNA encoding human GKRP (Av3hGKRP). High fat diet-induced diabetic mice were administered Av3hGKRP or a control vector lacking a transgene (Av3Null). Surprisingly, the Av3hGKRP-treated mice showed a significant improvement in glucose tolerance and had lower fasting blood glucose levels than Av3Null-treated mice. A coincident decrease in insulin levels indicated that the Av3hGKRP-treated mice had sharply improved insulin sensitivity. These mice also exhibited lower leptin levels, reduced body weight, and decreased liver GK activity. In vitro experiments indicated that GKRP was able to increase both GK protein and enzymatic activity levels, suggesting that another role for GKRP is to stabilize and/or protect GK. These data are the first to indicate the ability of GKRP to treat type 2 diabetes and therefore have significant implications for future therapies of this disease.
Subject(s)
Carrier Proteins , Diabetes Mellitus, Type 2/therapy , Genetic Therapy , Proteins/genetics , Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Avian Sarcoma Viruses/genetics , Blood Glucose/metabolism , Body Weight , Cells, Cultured , Diabetes Mellitus, Type 2/etiology , Dietary Fats/adverse effects , Fasting , Genetic Vectors , Glucokinase/antagonists & inhibitors , Glucose Intolerance/etiology , Glucose Intolerance/therapy , Glucose Tolerance Test , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Liver/physiology , Liver Glycogen/metabolism , Male , Mice , Mice, Inbred C57BL , Organ Size , Rats , Rats, Sprague-Dawley , Simian virus 40/genetics , Transfection , Tumor Cells, CulturedABSTRACT
The coding region of the gene for Taq DNA polymerase has been cloned into the common vector pUC18. Using a single-step procedure, large amounts of active enzyme can be purified from Escherichia coli carrying this construct. This procedure takes advantage of the thermostable properties of the DNA polymerase. This simple procedure gives very high yields of essentially homogeneous, highly active enzyme suitable for use in molecular biological applications. Yields are over two orders of magnitude greater than available with current methods.
