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Cellular Mechanisms of Insulin Resistance

Research Summary

Gerald Shulman's group examines insulin resistance in patients with diabetes and in transgenic mouse models of insulin resistance. Their long-term objectives are to elucidate the cellular mechanisms of insulin resistance and to identify new therapeutic targets to reverse insulin resistance in patients with type 2 diabetes.

Diabetes is a major health care problem in the United States, affecting ~6 percent of the population over age 18 and ~15 percent of the population over 65. The leading cause of blindness and nontraumatic loss of limb, diabetes accounts for 25 percent of all new cases of end-stage renal failure. More than 90 percent of diabetics have type 2 diabetes. Although the primary factors causing this disease are unknown, it is clear that insulin resistance has a primary role in its development.

Our objective is to elucidate the molecular mechanisms behind insulin resistance in patients with type 2 diabetes, in the hope that this will enable the development of therapeutic agents to reverse this pathologic condition, and assist with identification of genes that make individuals prone to this disease. Since liver and muscle are the two key insulin-responsive organs that account for most of the glucose metabolized in humans, we are using magnetic resonance spectroscopy (MRS), in combination with gas chromatography mass spectrometry, to focus on these tissues in normal and diabetic subjects. Our approach has major advantages over existing techniques: it is noninvasive, it involves no ionizing radiation, and repeated measurements of biochemical metabolites in plasma and tissue can be performed that then yield localized metabolic flux rates and information on rate-controlling steps of glucose metabolism.

Insulin Resistance and Muscle Glucose Metabolism
Using 13C MRS to monitor the rate of [1-13C] glucose incorporation into muscle glycogen, we found that defective muscle glycogen synthesis plays a major role in causing insulin resistance in patients with type 2 diabetes. In subsequent studies we performed 13C and 31P MRS to assess intramyocellular concentrations of glucose-6-phosphate and identify the rate-controlling steps responsible for decreased muscle glycogen synthesis and type 2 diabetes. We determined that the rate-controlling step in this process was at the level of glucose transport/phosphorylation. To examine whether this defect is a primary defect or an acquired defect secondary to other factors, we used a similar protocol to study lean, normoglycemic insulin-resistant offspring of parents with type 2 diabetes (IR offspring). These IR offspring, who have a high likelihood of developing diabetes later in life, had a 50 percent reduction in the rate of insulin-stimulated whole-body glucose metabolism, which could be attributed to a decrease in the rate of muscle glycogen synthesis. This reduction in insulin-stimulated muscle glycogen synthesis is associated with a blunted increment of insulin-stimulated intramuscular glucose-6-phosphate concentration. These data suggest that defects in insulin-stimulated muscle glucose transport/phosphorylation activity are an early event in the pathogenesis of type 2 diabetes.

We next examined whether we could reverse this defect in glucose transport/phosphorylation activity with chronic exercise training. A similar cohort of IR offspring was recruited to exercise four times a week at 65 percent VO2 max for 40 minutes on a StairMaster for 6 weeks. With this exercise regimen, the IR offspring normalized their rates of insulin-stimulated muscle glycogen synthesis; this could be attributed to correction of their defect in glucose transport/phosphorylation activity. These data suggest that aerobic exercise might be useful in reversing insulin resistance in these prediabetic individuals and thus prevent the development of type 2 diabetes.

Finally, to determine whether glucose transport or hexokinase II activity is rate controlling for insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes, we developed a novel 13C MRS method to assess intracellular glucose concentrations in muscle noninvasively. Intracellular glucose is an intermediary metabolite between glucose transport and glucose phosphorylation, and its concentration reflects the relative activities of glucose transporters (particularly GLUT4) and of hexokinase II. If hexokinase II activity is reduced relative to glucose transport activity in type 2 diabetes, one would predict a substantial increase in the intracellular glucose concentration, whereas if glucose transport is primarily responsible for rate control of intracellular glucose metabolism, intracellular glucose and glucose-6-phosphate should change proportionately. We found that the intracellular glucose concentration is far lower in the diabetic subjects than the concentration expected if hexokinase II is the primary rate-controlling step for glycogen synthesis. These data suggest a predominant role for glucose transport control of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes.

Fatty Acid–Induced Insulin Resistance in Muscle
Increased plasma free fatty acid concentrations are typically associated with many insulin-resistant states, including obesity and type 2 diabetes mellitus. In a cross-sectional study of young, normal-weight offspring of type 2 diabetic patients, we found an inverse relationship between fasting plasma fatty acid concentrations and insulin sensitivity, consistent with the hypothesis that altered fatty acid metabolism contributes to insulin resistance in patients with type 2 diabetes. Furthermore, recent studies measuring intramyocellular triglyceride content by 1H MRS have shown an even stronger relationship between accumulation of triglyceride and insulin resistance. Approximately 40 years ago, Philip Randle and his colleagues demonstrated that fatty acids compete with glucose for substrate oxidation in isolated rat heart and diaphragm muscle preparations. They speculated that increased fat oxidation was responsible for the insulin resistance associated with obesity. The mechanism they proposed to explain this resistance was that an increase in fatty acids caused an increase in the intramitochondrial acetyl-CoA/CoA and NADH/NAD+ ratios, with subsequent inactivation of pyruvate dehydrogenase. This in turn would cause intracellular citrate concentrations to increase, leading to inhibition of phosphofructokinase, a key rate-controlling enzyme in glycolysis. Subsequent accumulation of glucose-6-phosphate would inhibit hexokinase II activity, resulting in an increase in intracellular glucose concentrations and decreased glucose uptake. A recent series of studies by our group have challenged this hypothesis.

We applied 13C and 31P MRS to measure skeletal muscle glycogen and glucose-6-phosphate concentrations in healthy subjects. The subjects were maintained at euglycemic, hyperinsulinemic conditions, with either low or high levels of plasma fatty acids. The increment of the plasma fatty acid concentration caused a 50 percent reduction in insulin-stimulated rates of muscle glycogen synthesis compared to the control studies. In contrast to Randle's model, which predicted that fat-induced insulin resistance would result in an increase in intramuscular glucose-6-phosphate concentrations, we found that the drop in muscle glycogen synthesis was preceded by a fall in intramuscular glucose-6-phosphate, suggesting that increases in plasma fatty acid concentrations initially induce insulin resistance by inhibiting glucose transport or phosphorylation activity, and that the reduction in muscle glycogen synthesis and glucose oxidation follows. The reduction in insulin-activated glucose transport/phosphorylation activity in normal subjects maintained at high plasma fatty acid levels is similar to that seen in obese individuals, patients with type 2 diabetes, and healthy, lean, normoglycemic insulin-resistant offspring of type 2 diabetic patients. Hence, accumulation of intramuscular fatty acid metabolites appears to play an important role in the pathogenesis of insulin resistance seen in obese individuals and patients with type 2 diabetes.

To distinguish between possible effects of fatty acids on glucose transport activity and on hexokinase II activity, we used 13C MRS to measure intracellular concentrations of glucose in muscle and found that elevated plasma fatty acid concentrations caused a significant reduction in intracellular glucose concentration in the lipid infusion studies compared to the control studies. These data imply that the rate-controlling step for fatty acid–induced insulin resistance in humans is glucose transport, and they offer further evidence against the Randle mechanism, which predicts an increase in both intracellular glucose-6-phosphate and glucose concentrations. This reduced glucose transport activity could be the result of fatty acid effects on the GLUT4 transporter directly—alterations in the trafficking, budding, fusion, or activity of GLUT4—or it could result from fatty acid–induced alterations in upstream insulin-signaling events, resulting in decreased GLUT4 translocation to the plasma membrane. To explore the latter possibility, we examined IRS-1 (insulin receptor substrate-1)-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in muscle biopsy samples, using the identical lipid infusion protocol. We found that elevations in plasma fatty acid concentrations similar to the previous MRS studies abolished insulin-stimulated, IRS-1–associated PI 3-kinase activity compared with a fourfold insulin stimulation observed in the control studies. The reduced insulin-stimulated PI 3-kinase activity may be due to a direct effect of intracellular free fatty acids (or some fatty acid metabolite) on PI 3-kinase, or may be secondary to alterations in upstream insulin-signaling events. Consistent with an indirect effect, we found that a similar lipid infusion protocol in rats resulted in a reduction of insulin-stimulated IRS-1 tyrosine phosphorylation, which was associated with activation of protein kinase C-θ, a known serine kinase that is activated by diacylglycerol.

A Unifying Hypothesis for Common Forms of Insulin Resistance in Humans
An attractive hypothesis for common forms of insulin resistance in humans that we are now pursuing is that any perturbation that results in an increase in the concentration of intracellular fatty acid metabolites (e.g., diacylglycerol, fatty acyl-CoAs) leads to activation of a serine/threonine kinase cascade (possibly initiated by protein kinase C) leading to phosphorylation of serine/threonine sites on insulin receptor substrates, which in turn reduces the ability of the insulin receptor substrates to associate and activate PI 3-kinase, resulting in decreased activation of glucose transport activity and other downstream events. Therefore, defects in the adipocyte, leading to increased fat delivery to liver and muscle or defects in intracellular fatty acid oxidation, might be expected to induce insulin resistance through this mechanism. Supporting evidence for both of these possibilities comes from recent studies by our group.

Studies in lipodystrophic humans and mice have demonstrated severe hepatic and muscle insulin resistance where primary abnormalities in the adipocyte lead to increased fatty acid delivery to both of these organs. More recent 13C/31P MRS studies have demonstrated reduced mitochondrial oxidative-phosphorylation activity in insulin-resistant healthy elderly subjects as well as in young, lean, insulin-resistant offspring of parents with type 2 diabetes associated with increases in intramyocellular lipid content. These data suggest that alterations in nuclear-encoded genes that regulate mitochondrial biogenesis may form the genetic basis for inheritance of type 2 diabetes.

Grants from the National Institutes of Health and the American Diabetes Association provided support for some of this work.

As of May 30, 2012

Scientist Profile

Investigator
Yale University
Medicine and Translational Research, Physiology