A simple and effective model to study insulin resistance in obesity and diabetes and the processes and mechanisms of in vitro aging using lymphocyte culture
Matthews, Kathleen S.
Doctor of Philosophy
Cell culture provides a simple and effective system to study metabolic and cellular processes required for cellular growth in vitro. In this study, we demonstrate that lymphocyte culture can be used as a model system to study the mechanism of insulin resistance (obesity/diabetes) as well as the process of aging of cells from the immune system. Our results indicate that metabolic differences can be observed in lymphocytes from obese subjects and that different degrees of alteration in response can be detected between obese nondiabetic and diabetic subjects. Furthermore different effects of insulin resistance can be reflected faithfully in lymphocyte biochemistry in culture. In isolated lymphocytes from normal weight subjects, G3PDH (an enzyme at the intersection of glycolytic and lipogenic pathways) activity increases in the presence of insulin. Augmented G3PDH activity requires new protein synthesis and involves the inositol triphosphate-diacylglycerol signalling system. However, in obese subjects for whom insulin resistance in vitro can be demonstrated, the extent of insulin stimulation of G3PDH activity is decreased compared to normal weight individuals. These results suggest that G3PDH activity is dependent upon the metabolic state of the subjects from which the cells are obtained. Dietary restriction for obese subjects normalizes insulin and glycerol responses and insulin effects on G3PDH activity. These results demonstrate that the lymphocyte can serve in vitro as a model to reflect organismal metabolism in obesity/diabetes. Alterations of lymphocytes due to aging contribute significantly to changes in immune function. In present study, an artificial aging environment was introduced using whole blood. In this environment, we demonstrate that lymphocytes undergo degeneration and aging processes similar to those observed in vivo. Similar changes in growth capacity, lipid peroxidation, surface antigens on T-cells, and adherent cell suppression were observed in lymphocytes aged in vivo or in vitro. Based on these results, we suggest that blood aged in vitro may provide a simple but effective model to study many aspects of cellular aging in vivo. To further examine this hypothesis and to further understand the aging process, vitamin E was used to examine its effect on in vitro aging. The results from lymphocyte growth capacity assays show that vitamin E provides a protective effect against in vitro aging similar to that observed for in vivo aging. Examination of lymphocytes in culture allows monitoring of biochemical alterations, nutrient requirements, and other parameters in vitro, without effect on the subject. This technique therefore provides an opportunity to examine a wide variety of factors and a system eventually in which mechanisms to prevent or impede diseases can be tested.