Gary W. Cline

Mechanism of free fatty acid-induced insulin resistance in humans.

To examine the mechanism by which lipids cause insulin resistance in humans, skeletal muscle glycogen and glucose-6-phosphate concentrations were measured every 15 min by simultaneous 13C and 31P nuclear magnetic resonance spectroscopy in nine healthy subjects in the presence of low (0.18 +/- 0.02 mM [mean +/- SEM]; control) or high (1.93 +/- 0.04 mM; lipid infusion) plasma free fatty acid levels under euglycemic (approximately 5.2 mM) hyperinsulinemic (approximately 400 pM) clamp conditions for 6 h. During the initial 3.5 h of the clamp the rate of whole-body glucose uptake was not affected by lipid infusion, but it then decreased continuously to be approximately 46% of control values after 6 h (P < 0.00001). Augmented lipid oxidation was accompanied by a approximately 40% reduction of oxidative glucose metabolism starting during the third hour of lipid infusion (P < 0.05). Rates of muscle glycogen synthesis were similar during the first 3 h of lipid and control infusion, but thereafter decreased to approximately 50% of control values (4.0 +/- 1.0 vs. 9.3 +/- 1.6 mumol/[kg.min], P < 0.05). Reduction of muscle glycogen synthesis by elevated plasma free fatty acids was preceded by a fall of muscle glucose-6-phosphate concentrations starting at approximately 1.5 h (195 +/- 25 vs. control: 237 +/- 26 mM; P < 0.01). Therefore in contrast to the originally postulated mechanism in which free fatty acids were thought to inhibit insulin-stimulated glucose uptake in muscle through initial inhibition of pyruvate dehydrogenase these results demonstrate that free fatty acids induce insulin resistance in humans by initial inhibition of glucose transport/phosphorylation which is then followed by an approximately 50% reduction in both the rate of muscle glycogen synthesis and glucose oxidation.

Effects of free fatty acids on glucose transport and IRS-1–associated phosphatidylinositol 3-kinase activity

To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was also measured in muscle biopsy samples obtained from seven additional subjects before and after an identical protocol. Rates of insulin stimulated whole-body glucose uptake. Glucose oxidation and muscle glycogen synthesis were 50%-60% lower following the lipid infusion compared with the glycerol infusion and were associated with a approximately 90% decrease in the increment in intramuscular glucose-6-phosphate concentration, implying diminished glucose transport or phosphorylation activity. To distinguish between these two possibilities, intracellular glucose concentration was measured and found to be significantly lower in the lipid infusion studies, implying that glucose transport is the rate-controlling step. Insulin stimulation, during the glycerol infusion, resulted in a fourfold increase in PI 3-kinase activity over basal that was abolished during the lipid infusion. Taken together, these data suggest that increased concentrations of plasma FFA induce insulin resistance in humans through inhibition of glucose transport activity; this may be a consequence of decreased IRS-1-associated PI 3-kinase activity.

Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy

Lipodystrophy is a rare disorder that is characterized by selective loss of subcutaneous and visceral fat and is associated with hypertriglyceridemia, hepatomegaly, and disordered glucose metabolism. It has recently been shown that chronic leptin treatment ameliorates these abnormalities. Here we show that chronic leptin treatment improves insulin-stimulated hepatic and peripheral glucose metabolism in severely insulin-resistant lipodystrophic patients. This improvement in insulin action was associated with a marked reduction in hepatic and muscle triglyceride content. These data suggest that leptin may represent an important new therapy to reverse the severe hepatic and muscle insulin resistance and associated hepatic steatosis in patients with lipodystrophy.

Increased Glucose Transport–Phosphorylation and Muscle Glycogen Synthesis after Exercise Training in Insulin-Resistant Subjects

BACKGROUND Insulin resistance in the offspring of parents with non-insulin-dependent diabetes mellitus (NIDDM) is the best predictor of development of the disease and probably plays an important part in its pathogenesis. We studied the mechanism and degree to which exercise training improves insulin sensitivity in these subjects. METHODS Ten adult children of parents with NIDDM and eight normal subjects were studied before starting an aerobic exercise-training program, after one session of exercise, and after six weeks of exercise. Insulin sensitivity was measured by the hyperglycemic-hyperinsulinemic clamp technique combined with indirect calorimetry, and the rate of glycogen synthesis in muscle and the intramuscular glucose-6-phosphate concentration were measured by carbon-13 and phosphorus-31 nuclear magnetic resonance spectroscopy, respectively. RESULTS During the base-line study, the mean (+/-SE) rate of muscle glycogen synthesis was 63 +/- 9 percent lower in the offspring of diabetic parents than in the normal subjects (P < 0.001). The mean value increased 69 +/- 10 percent (P = 0.04) and 62 +/- 11 percent (P = 0.04) after the first exercise session and 102 +/- 11 percent (P = 0.02) and 97 +/- 9 percent (P = 0.008) after six weeks of exercise training in the offspring and the normal subjects, respectively. The increment in glucose-6-phosphate during hyperglycemic-hyperinsulinemic clamping was lower in the offspring than in the normal subjects (0.039 +/- 0.013 vs. 0.089 +/- 0.009 mmol per liter, P = 0.005), reflecting reduced glucose transport-phosphorylation, but this increment was normal in the offspring after the first exercise session and after exercise training. Basal and stimulated insulin secretion was higher in the offspring than the normal subjects and was not altered by the exercise training program. CONCLUSIONS Exercise increases insulin sensitivity in both normal subjects and the insulin-resistant offspring of diabetic parents because of a twofold increase in insulin-stimulated glycogen synthesis in muscle, due to an increase in insulin-stimulated glucose transport-phosphorylation.

IMPAIRED GLUCOSE TRANSPORT AS A CAUSE OF DECREASED INSULIN- STIMULATED MUSCLE GLYCOGEN SYNTHESIS IN TYPE 2 DIABETES

BACKGROUND Insulin resistance, a major factor in the pathogenesis of type 2 diabetes mellitus, is due mostly to decreased stimulation of glycogen synthesis in muscle by insulin. The primary rate-controlling step responsible for the decrease in muscle glycogen synthesis is not known, although hexokinase activity and glucose transport have been implicated. METHODS We used a novel nuclear magnetic resonance approach with carbon-13 and phosphorus-31 to measure intramuscular glucose, glucose-6-phosphate, and glycogen concentrations under hyperglycemic conditions (plasma glucose concentration, approximately 180 mg per deciliter [10 mmol per liter]) and hyperinsulinemic conditions in six patients with type 2 diabetes and seven normal subjects. In vivo microdialysis of muscle tissue was used to determine the gradient between plasma and interstitial-fluid glucose concentrations, and open-flow microperfusion was used to determine the concentrations of insulin in interstitial fluid. RESULTS The time course and concentration of insulin in interstitial fluid were similar in the patients with diabetes and the normal subjects. The rates of whole-body glucose metabolism and muscle glycogen synthesis and the glucose-6-phosphate concentrations in muscle were approximately 80 percent lower in the patients with diabetes than in the normal subjects under conditions of matched plasma insulin concentrations. The mean (+/-SD) intracellular glucose concentration was 2.0+/-8.2 mg per deciliter (0.11+/-0.46 mmol per liter) in the normal subjects. In the patients with diabetes, the intracellular glucose concentration was 4.3+/-4.9 mg per deciliter (0.24+/-0.27 mmol per liter), a value that was 1/25 of what it would be if hexokinase were the rate-controlling enzyme in glucose metabolism. CONCLUSIONS Impaired insulin-stimulated glucose transport is responsible for the reduced rate of insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes mellitus.

Functional polarization of tumour-associated macrophages by tumour-derived lactic acid

Macrophages have an important role in the maintenance of tissue homeostasis. To perform this function, macrophages must have the capacity to monitor the functional states of their ‘client cells’: namely, the parenchymal cells in the various tissues in which macrophages reside. Tumours exhibit many features of abnormally developed organs, including tissue architecture and cellular composition. Similarly to macrophages in normal tissues and organs, macrophages in tumours (tumour-associated macrophages) perform some key homeostatic functions that allow tumour maintenance and growth. However, the signals involved in communication between tumours and macrophages are poorly defined. Here we show that lactic acid produced by tumour cells, as a by-product of aerobic or anaerobic glycolysis, has a critical function in signalling, through inducing the expression of vascular endothelial growth factor and the M2-like polarization of tumour-associated macrophages. Furthermore, we demonstrate that this effect of lactic acid is mediated by hypoxia-inducible factor 1α (HIF1α). Finally, we show that the lactate-induced expression of arginase 1 by macrophages has an important role in tumour growth. Collectively, these findings identify a mechanism of communication between macrophages and their client cells, including tumour cells. This communication most probably evolved to promote homeostasis in normal tissues but can also be engaged in tumours to promote their growth.

The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome

We examined the hypothesis that insulin resistance in skeletal muscle promotes the development of atherogenic dyslipidemia, associated with the metabolic syndrome, by altering the distribution pattern of postprandial energy storage. Following ingestion of two high carbohydrate mixed meals, net muscle glycogen synthesis was reduced by ≈60% in young, lean, insulin-resistant subjects compared with a similar cohort of age–weight–body mass index–activity-matched, insulin-sensitive, control subjects. In contrast, hepatic de novo lipogenesis and hepatic triglyceride synthesis were both increased by >2-fold in the insulin-resistant subjects. These changes were associated with a 60% increase in plasma triglyceride concentrations and an ≈20% reduction in plasma high-density lipoprotein concentrations but no differences in plasma concentrations of TNF-α, IL-6, adiponectin, resistin, retinol binding protein-4, or intraabdominal fat volume. These data demonstrate that insulin resistance in skeletal muscle, due to decreased muscle glycogen synthesis, can promote atherogenic dyslipidemia by changing the pattern of ingested carbohydrate away from skeletal muscle glycogen synthesis into hepatic de novo lipogenesis, resulting in an increase in plasma triglyceride concentrations and a reduction in plasma high-density lipoprotein concentrations. Furthermore, insulin resistance in these subjects was independent of changes in the plasma concentrations of TNF-α, IL-6, high-molecular-weight adiponectin, resistin, retinol binding protein-4, or intraabdominal obesity, suggesting that these factors do not play a primary role in causing insulin resistance in the early stages of the metabolic syndrome.

Metformin suppresses gluconeogenesis by inhibiting mitochondrial glycerophosphate dehydrogenase

Metformin is considered to be one of the most effective therapeutics for treating type 2 diabetes because it specifically reduces hepatic gluconeogenesis without increasing insulin secretion, inducing weight gain or posing a risk of hypoglycaemia. For over half a century, this agent has been prescribed to patients with type 2 diabetes worldwide, yet the underlying mechanism by which metformin inhibits hepatic gluconeogenesis remains unknown. Here we show that metformin non-competitively inhibits the redox shuttle enzyme mitochondrial glycerophosphate dehydrogenase, resulting in an altered hepatocellular redox state, reduced conversion of lactate and glycerol to glucose, and decreased hepatic gluconeogenesis. Acute and chronic low-dose metformin treatment effectively reduced endogenous glucose production, while increasing cytosolic redox and decreasing mitochondrial redox states. Antisense oligonucleotide knockdown of hepatic mitochondrial glycerophosphate dehydrogenase in rats resulted in a phenotype akin to chronic metformin treatment, and abrogated metformin-mediated increases in cytosolic redox state, decreases in plasma glucose concentrations, and inhibition of endogenous glucose production. These findings were replicated in whole-body mitochondrial glycerophosphate dehydrogenase knockout mice. These results have significant implications for understanding the mechanism of metformin’s blood glucose lowering effects and provide a new therapeutic target for type 2 diabetes.

Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and whole-animal insulin resistance

Lipid infusion or ingestion of a high-fat diet results in insulin resistance, but the mechanism underlying this phenomenon remains unclear. Here we show that, in rats fed a high-fat diet, whole-animal, muscle and liver insulin resistance is ameliorated following hepatic overexpression of malonyl–coenzyme A (CoA) decarboxylase (MCD), an enzyme that affects lipid partitioning. MCD overexpression decreased circulating free fatty acid (FFA) and liver triglyceride content. In skeletal muscle, levels of triglyceride and long-chain acyl-CoA (LC-CoA)—two candidate mediators of insulin resistance—were either increased or unchanged. Metabolic profiling of 36 acylcarnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, β-OH-butyrate, in muscle of MCD-overexpressing animals. The best explanation for our findings is that hepatic expression of MCD lowered circulating FFA levels, which led to lowering of muscle β-OH-butyrate levels and improvement of insulin sensitivity.

Reversal of diet-induced hepatic steatosis and hepatic insulin resistance by antisense oligonucleotide inhibitors of acetyl-CoA carboxylases 1 and 2

Hepatic steatosis is a core feature of the metabolic syndrome and type 2 diabetes and leads to hepatic insulin resistance. Malonyl-CoA, generated by acetyl-CoA carboxylases 1 and 2 (Acc1 and Acc2), is a key regulator of both mitochondrial fatty acid oxidation and fat synthesis. We used a diet-induced rat model of nonalcoholic fatty liver disease (NAFLD) and hepatic insulin resistance to explore the impact of suppressing Acc1, Acc2, or both Acc1 and Acc2 on hepatic lipid levels and insulin sensitivity. While suppression of Acc1 or Acc2 expression with antisense oligonucleotides (ASOs) increased fat oxidation in rat hepatocytes, suppression of both enzymes with a single ASO was significantly more effective in promoting fat oxidation. Suppression of Acc1 also inhibited lipogenesis whereas Acc2 reduction had no effect on lipogenesis. In rats with NAFLD, suppression of both enzymes with a single ASO was required to significantly reduce hepatic malonyl-CoA levels in vivo, lower hepatic lipids (long-chain acyl-CoAs, diacylglycerol, and triglycerides), and improve hepatic insulin sensitivity. Plasma ketones were significantly elevated compared with controls in the fed state but not in the fasting state, indicating that lowering Acc1 and -2 expression increases hepatic fat oxidation specifically in the fed state. These studies suggest that pharmacological inhibition of Acc1 and -2 may be a novel approach in the treatment of NAFLD and hepatic insulin resistance.