Triglyceride-rich lipoproteins and insulin resistance

A major pathophysiological feature of type 2-Diabetes Mellitus (type 2-DM) is insulin resistance which is defined as the diminished ability of insulin sensitive tissues to take up and metabolize glucose. In addition to its critical role in the development of type 2-DM, insulin resistance confounds insulin treatment in type 2-DM patients and probably plays a critical role in the development of the devastating complications in type 2-DM. The molecular basis underlying insulin resistance has not been identified, but it probably involves a defect in the complex signaling cascade of insulin. Not only the mechanisms of insulin resistance, but also the major factors that induce and/or aggravate insulin resistance have not been characterized yet. Yet, evidence is accumulating that lipids, in particular NEFAs and triglycerides may be an important factor in inducing and/or worsening insulin resistance. Aim of this study is to investigate whether triglycerides as they occur in plasma, i.e. in the form of TGRL, may affect insulin action in cell culture. This will be achieved by studying the effects of TGRL fractions, i.e. chylomicrons and VLDL, isolated from human subjects on insulin signaling steps in cultured skeletal muscle cells. Skeletal muscle cells have been selected, since it has been shown that skeletal muscle is the major tissue responsible for impaired glucose metabolism in type 2-DM. Pure lipoprotein fractions will be isolated by the method of zonal ultracentrifugation and their effect on the insulin signaling cascade will be studied in intact cells. Specifically, we will investigate whether different lipoprotein fractions may induce changes in expression, in protein-protein interaction, in phosphorylation pattern and activity of insulin signaling components. We will study both, proximal (insulin receptors, IRS proteins, Shc, Grb2-Sos) as well as more distal signaling events (PI 3-kinase, MAPK pathway). The specific experiments planned may enable us to gain considerable insight both into the pathogenesis of insulin resistance and type 2-DM and into the underlying molecular mechanisms.

The basic mechanisms in the development of insulin resistance, a principal defect in type 2 diabetes, are unclear. Since in Western societies a major part of the day is spent in the phase of postprandial lipemia with elevated plasma levels of triglyceride-rich lipoproteins (TGRL), we tested the novel hypothesis that these lipoproteins might be an important factor in inducing an acute state of insulin resistance. And indeed, our study revealed that postprandial TGRL are capable of bringing about insulin resistance both in vitro and in vivo. First, we performed an in vitro study using cultured skeletal muscle cells since skeletal muscle is a major site of insulin resistance. In this cell culture system, TGRL induced insulin resistance as evidenced by an impairment of insulin signalling and glucose metabolism. Of the steps in glucose metabolism studied, we found TGRL to diminish insulin-stimulated glycogen synthesis, glycogen synthase activity and glucose uptake. For the calculation of glycogen synthase activity we established a novel assay system employing a gel filtration method to improve accuracy and precision of the assay. In addition to glucose metabolism, incubation of our cells with TGRL also impaired several important steps in the insulin signalling pathway. The effects observed were independent of non- esterified fatty acid levels in the incubation media and, therefore, exclusively attributable to intact lipoprotein particles. Based on these intriguing results in a cell culture system we initiated a clinical study involving 10 healthy men to test our hypothesis in vivo. Employing a crossover design we studied whether two nearly isocaloric fat-rich meals may affect insulin sensitivity in the postprandial state. Two meals were designed in a way that both meals give a postprandial rise in TGRL but only one meal gives a postprandial rise in non-esterified fatty acids. This clinical trial revealed that either of the two meals administered resulted in an impairment of postprandial insulin sensitivity suggesting that, in accordance with our in vitro study, elevated levels of postprandial TGRL induce insulin resistance independently of non-esterified fatty acid levels. With the results of these studies, a mechanism underlying the development of insulin resistance has been established that has not been appreciated to date.