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Pathogenesis of skeletal muscle insulin resistance in type 2 diabetes mellitus
Pathogenesis of Skeletal Muscle Insulin Resistance in Type 2 Diabetes Mellitus Kitt F. Petersen,
MD,
and Gerald I. Shulman,
MD, PhD
Insulin resistance is a principal feature of type 2 diabetes and precedes the clinical development of the disease by 10 to 20 years. Insulin resistance is caused by the decreased ability of peripheral target tissues (especially muscle) to respond properly to normal circulating concentrations of insulin. Defects in muscle glycogen synthesis play a significant role in insulin resistance, and 3 potentially rate-controlling steps in muscle glucose metabolism have been implicated in its pathogenesis: glycogen synthase, hexokinase, and GLUT4 (the major insulin-stimulated glucose transporter). Results from recent studies using nuclear magnetic resonance (NMR) spectroscopy implicate intracellular defects in glucose transport as the rate-controlling step for insulin-mediated glucose uptake in muscle. These alterations in glucose transport activity are likely the result of dysregulation of intramyocellular fatty acid metabolism, whereby fatty
acids cause insulin resistance by activation of a serine kinase cascade, leading to decreased insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and decreased IRS-1–associated phosphatidylinositol 3-kinase activity, a required step in insulin-stimulated glucose transport into muscle. The thiazolidinedione class of antidiabetic agents directly targets insulin resistance in skeletal muscle by improving glucose transport activity and insulin-stimulated muscle glycogen synthesis. Although the precise mechanism of action is not known, recent NMR studies support the hypothesis that these agents improve insulin action in skeletal muscle and liver by promoting a redistribution of fat out of these tissues and into peripheral adipocytes. 䊚2002 by Excerpta Medica, Inc. Am J Cardiol 2002;90(suppl):11G–18G
he pathophysiology of type 2 diabetes involves defects in tissue sensitivity to insulin and deT creased insulin secretion. It is generally accepted that
events that promote intracellular glucose transport and metabolism.1,2 Insulin resistance is the inability of peripheral target tissues to respond properly to normal circulating concentrations of insulin. To maintain euglycemia, the pancreas compensates by secreting increased amounts of insulin. In patients with type 2 diabetes, insulin resistance precedes the onset of the disease by 1 to 2 decades.3 After a period of compensated insulin resistance, impaired glucose tolerance eventually develops despite elevated insulin concentrations as insulin resistance increases. Finally, pancreatic -cell failure results in decreased insulin secretion. Overt clinical type 2 diabetes results when these 2 defects, insulin resistance and impaired -cell function, occur simultaneously.1,4
resistance to insulin in target tissues (especially muscle and liver) develops initially, followed by decreased insulin secretion as a result of progressive pancreatic -cell dysfunction. This leads to the onset of overt diabetes with fasting hyperglycemia.1 Although several pharmacologic approaches to the treatment of type 2 diabetes are available, investigators are increasingly focusing attention on the intracellular defects that may be causing insulin resistance. Once the underlying cellular defects are identified, novel treatments for type 2 diabetes can be developed and targeted toward the specific defects. This article will discuss the intracellular defects in muscle tissue that contribute to insulin resistance and subsequent development of type 2 diabetes. The role of fatty acids in inducing skeletal muscle insulin resistance will also be examined.
INSULIN RESISTANCE Normally, insulin binds to insulin receptors on target organ cells, resulting in a series of cellular From the Howard Hughes Medical Institute, Department of Internal Medicine, Department of Cellular and Molecular Physiology, General Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut, USA. This work was supported by Grant Nos. R01 DK-49230, P30 DK-45735, M01 RR-00125, a K-23 award (KFP) from the United States Public Health Service and a grant from GlaxoSmithKline (KFP). Gerald I. Shulman was a consultant for GlaxoSmithKline. Address for reprints: Gerald I. Shulman, MD, PhD, Yale University School of Medicine, 295 Congress Avenue, BCMM 254C, New Haven, Connecticut 06510. E-mail: [email protected]. ©2002 by Excerpta Medica, Inc. All rights reserved.
MUSCLE GLYCOGEN SYNTHESIS There are 3 events that take place in a coordinated manner to maintain normal glucose homeostasis: secretion of insulin by the pancreatic  cells, suppression of hepatic glucose production, and stimulation of glucose uptake by the liver and muscle.4 A study was conducted in subjects with type 2 diabetes and in healthy subjects to determine the fate of glucose after it is taken up by muscle cells.5 It is possible to obtain carbon-13 nuclear magnetic resonance (NMR) spectra of human muscle glycogen in vivo. Using this noninvasive technique, glycogen concentrations can be measured accurately with a time resolution of several minutes. In 1 analysis, combined hyperglycemic-hyperinsulinemic clamp studies were performed by infusing carbon-13– enriched glucose and insulin intravenously and measuring the rate of muscle glycogen synthesis.5 Used in combination with indirect calorimetry, the rate of muscle glycogen synthesis could be 0002-9149/02/$ – see front matter PII S0002-9149(02)02554-7
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FIGURE 1. Impaired glucose transport and insulin-stimulated muscle glycogen synthesis in type 2 diabetes: potential rate-controlling steps in muscle glucose metabolism. G6P ⴝ glucose-6-phosphate; Glucoseex ⴝ extracellular glucose; Glucosein ⴝ intracellular glucose; Vglycolysis ⴝ net velocity of the glycolytic flux of glucose-6-phosphate; VGT ⴝ velocity of glucose transport into the muscle cell; VⴚGT ⴝ velocity of glucose transport out of the muscle cell; VHK ⴝ velocity of glucose phosphorylation by hexokinase. (Reprinted with permission from N Engl J Med.6 Copyright 姝1999 Massachusetts Medical Society. All rights reserved.)
related to whole-body glucose uptake and nonoxidative disposal of glucose. In this study, the investigators found that glycogen synthesis represented the primary pathway for nonoxidative glucose disposal in normal subjects and that muscle glycogen synthesis was the major pathway of overall glucose metabolism. In addition, the rate of glycogen formation in subjects with diabetes was 60% lower than the rate in normal subjects, providing evidence that glycogen synthesis was profoundly impaired in persons with type 2 diabetes. Based on data from this study, it was clear that impaired glycogen synthesis was the major intracellular metabolic defect responsible for insulin resistance in subjects with type 2 diabetes.
POTENTIAL RATE-CONTROLLING STEPS IN MUSCLE GLUCOSE METABOLISM Once it was known that defects in muscle glycogen synthesis played a significant part in the insulin resistance that occurs in individuals with type 2 diabetes, subsequent studies attempted to identify the specific biochemical defect responsible for this abnormality. To do this, investigators assessed the potentially ratecontrolling steps for insulin-stimulated muscle glucose metabolism. The following 3 candidate steps were examined: glycogen synthase, hexokinase, and GLUT4, a glucose transporter (Figure 1). Each of these steps has been reported to be defective in patients with type 2 diabetes.6 Glycogen synthase: It has been suggested that glycogen synthase may be the major defect in glycogen 12G THE AMERICAN JOURNAL OF CARDIOLOGY姞
synthesis. If so, then the concentration of glucose-6phosphate (G6P) should be higher in patients with type 2 diabetes than in normal subjects under insulinstimulated conditions (Figure 1). However, if glucose transport and/or hexokinase were rate controlling for insulin-stimulated muscle glycogen synthesis, then G6P concentration would be expected to be no different or lower in the muscle of the subjects with diabetes. To test this hypothesis, we measured the concentration of G6P in muscle using phosphorous-31 NMR spectroscopy during a hyperglycemic-hyperinsulinemic clamp.7 A total of 6 subjects with type 2 diabetes and 6 matched controls were studied at similar steadystate plasma concentrations of insulin and glucose. In addition to measuring G6P, whole-body oxidative and nonoxidative glucose metabolism, which measures muscle glycogen synthesis, was determined. A reduced rate of nonoxidative glucose metabolism was seen in patients with type 2 diabetes compared with normal subjects (13 mol/kg body weight minimum vs 31 mol/kg body weight minimum, respectively; p ⬍0.05). Most importantly, the concentration of G6P was lower in patients with type 2 diabetes than in normal subjects (0.17 mmol/kg muscle vs 0.24 mmol/kg muscle, respectively; p ⬍0.01). These findings indicated that a reduction in the activity of either muscle glucose transport and/or hexokinase activity was most likely responsible for the development of insulin resistance. Studies have shown that insulin resistance can be found in patients more than a decade before they
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develop type 2 diabetes, and insulin resistance is the best predictor for later development of the disease.3,8,9 However, impaired muscle glycogen synthesis and glucose transport/hexokinase activity may be an acquired defect in type 2 diabetes rather than a primary defect. To examine this question, we examined nondiabetic offspring of patients with type 2 diabetes, who are known to be at risk for the development of type 2 diabetes. The rate of muscle glycogen synthesis and the concentration of muscle G6P were measured using carbon-13 and phosphorous-31 NMR spectroscopy in 6 lean, normoglycemic offspring of patients with type 2 diabetes and 7 matched controls.10 The offspring of parents with type 2 diabetes had a 50% reduction in total glucose metabolism, the rate of muscle glycogen synthesis was reduced by 70% (p ⬍0.005), and muscle G6P concentration was reduced by 40% (p ⬍0.003). These findings were similar to those observed in subjects with type 2 diabetes7 and suggested that impaired muscle glucose transport and/or hexokinase activity was the defective biochemical process. The fact that this defect was present in this group of subjects demonstrated that defects in glucose transport/phosphorylation are an early factor in the pathogenesis of type 2 diabetes rather than a consequence of the disease. Distinguishing between potential defects in hexokinase and glucose transport: Intracellular glucose is an
intermediary metabolite between glucose transport and hexokinase activity (Figure 1), and its concentration depends on the relative activities of these 2 processes. For example, if hexokinase activity were reduced in patients with type 2 diabetes, a substantial increase in intracellular glucose would be expected. Therefore, to determine the relative importance of hexokinase and glucose transport in muscle glucose metabolism, Cline et al6 used a novel NMR approach with carbon-13 and phosphorous-31 to measure intracellular glucose, G6P, and glycogen concentrations. Hyperglycemic-hyperinsulinemic clamp studies and NMR measurements were performed in 6 patients with type 2 diabetes and in 7 normal subjects. In the patients with diabetes, rates of whole-body glucose metabolism, muscle glycogen synthesis, and G6P concentrations in muscle were approximately 80% lower than in normal subjects. The mean intracellular glucose concentration was calculated to be 0.11 mmol/L in normal subjects. In patients with diabetes, the concentration was 0.24 mmol/L. This value was 1/25 of what would be expected if hexokinase were the ratecontrolling step in glucose metabolism. Cline et al6 concluded that glucose transport was the rate-controlling step in insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes. As such, investigators now have a target on which to focus. If this defect in glucose transport could be ameliorated, significant improvement in insulin-stimulated glycogen synthesis should be seen in patients with type 2 diabetes. The focus of investigation is now to identify the mechanisms responsible for the defect in the insulin-stimulated GLUT4 transporter activity.
ROLE OF FATTY ACIDS IN GLUCOSE METABOLISM Insulin resistance is the best predictor for the development of type 2 diabetes, and it is related to many other factors, such as age, diet, and exercise. In an attempt to identify the defect in glucose transport, Perseghin et al11 examined a population of young, healthy, lean, nonexercising, normoglucose-tolerant subjects who had 1 parent with type 2 diabetes (offspring). A total of 49 offspring were compared with 29 matched healthy control subjects by means of an intravenous glucose bolus, euglycemic-hyperinsulinemic clamp, and lipid and amino acid profiles. The offspring had higher plasma fatty acid concentrations than did the control subjects (582 vs 470 mol/L, respectively; p ⫽ 0.007); triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein cholesterol levels were comparable between groups. A simple regression analysis between insulin sensitivity and fatty acid concentration was significant in offspring but not in control subjects (Figure 2). The inverse correlation of high fatty acid concentrations with decreased insulin sensitivity suggested that altered fatty acid metabolism may have a primary role in causing insulin resistance in type 2 diabetes. Krssak et al12 have applied noninvasive proton NMR spectroscopy techniques to determine intramyocellular lipid content. In 23 normal-weight, nondiabetic adults, insulin sensitivity was assessed by a euglycemic-hyperinsulinemic clamp test, and intramyocellular lipid concentrations were determined by proton NMR. Insulin-stimulated whole-body glucose was inversely correlated with intramyocellular lipid content (r ⫽ ⫺0.692; p ⫽ 0.0017). A similar inverse relationship between intramyocellular lipid content and insulin stimulant whole-body glucose uptake was found in first-degree relatives of subjects with type 2 diabetes.13 These findings provided additional data implicating lipids as a cause of insulin resistance in the skeletal muscle. Mechanism of fat-induced insulin resistance: Almost 40 years ago, Randle et al14 proposed a mechanism for fat-induced insulin resistance that implicated fatty acid oxidation as causing the inactivation of mitochondrial pyruvate dehydrogenase and ultimately leading to decreased glucose uptake. However, their work was done in isolated rat heart muscle, which may be metabolically very different from resting human skeletal muscle. We examined the mechanism by which lipids cause insulin resistance in human skeletal muscle using carbon-13 and phosphorous-31 NMR spectroscopy techniques.15 In 9 healthy subjects, skeletal muscle glycogen and G6P concentrations were measured simultaneously using NMR spectroscopy under euglycemic-hyperinsulinemic clamp conditions for 6 hours. To examine the effects of fatty acids, the plasma concentration of fatty acids was increased by an intravenous infusion of a triglyceride emulsion combined with heparin to activate lipoprotein lipase. After 3 hours of the lipid infusion, rates of muscle glycogen synthesis decreased by approximately 50% (Figure 3).
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FIGURE 2. High plasma fatty acid (FA) concentration predicts insulin resistance. Simple regression analysis between insulin sensitivity and fasting plasma fatty acids in offspring of parents with type 2 diabetes (solid line: adjusted R2 ⴝ 0.21, p ⴝ 0.0005) and control subjects (dashed line: adjusted R2 ⴝ 0.03, p ⴝ 0.368). E ⴝ control subjects; ⽧ ⴝ offspring. (Reprinted with permission from Diabetes.11)
This effect was preceded by a significant reduction in muscle G6P concentrations. By 6 hours, the rate of whole-body glucose uptake was approximately 46% of control values. Raising fatty acids by infusing lipids made these healthy individuals as insulin resistant as patients with type 2 diabetes. Therefore, in contrast to the mechanism proposed by Randle et al14 in which fatty acids inhibit pyruvate dehydrogenase activity and thereby induce insulin resistance, we found that increases in plasma fatty acid concentrations inhibited glucose transport and/or phosphorylation activity.15 These defects in glucose transport and hexokinase were similar to previous findings in patients with type 2 diabetes7 and in the lean, normoglycemic offspring of diabetic patients.10 This suggested that defects in fatty acid metabolism may have an important role in the pathogenesis of insulin resistance in patients with type 2 diabetes by interfering with insulin activation of glucose transport and/or hexokinase activity. To distinguish between fatty acid–induced inhibition of insulin-stimulated glucose transport and hexokinase activity, Dresner et al16 performed a follow-up study and measured intracellular glucose concentration using the carbon-13 NMR spectroscopy technique previously described. Once again, the assumption was that if increased fatty acid concentrations reduced hexokinase activity, glucose would accumulate inside the muscle cell. Intracellular glucose concentration was measured in 7 healthy subjects during a euglycemic-hyperinsulinemic clamp following a 5-hour infusion of either glycerol or lipid plus heparin. Intracellular glucose was found to be significantly lower after the lipid infusion than after the glycerol infusion (0.04 vs 0.25 mmol/L; p ⫽ 0.04). These data suggested that elevations in plasma fatty acid concen14G THE AMERICAN JOURNAL OF CARDIOLOGY姞
tration caused insulin resistance in skeletal muscle by reducing insulin-stimulated glucose transport activity. The mechanisms by which fatty acids induce changes in glucose transport activity are unknown, but these changes could be a result of the effects of fatty acids on the GLUT4 transporter directly or could result from alterations in the insulin-signaling cascade (Figure 4). Dresner et al16 attempted to answer this question by measuring insulin receptor substrate (IRS)-1–associated phosphatidylinositol 3-kinase (PI3-kinase) activity in muscle biopsy samples using a lipid infusion protocol similar to the aforementioned study. During the control (glycerol) infusion, PI3kinase activity increased 3- to 4-fold after insulin stimulation. However, this effect was abolished during the lipid infusion. This suggested that fatty acid– induced insulin resistance was a consequence of altered insulin signaling through PI3-kinase. Effect of fatty acids on the insulin-signaling cascade:
To further characterize this fat-induced defect in insulin activation of IRS-1–associated PI3-kinase activity, additional studies were conducted in an in vivo animal model of fat-induced insulin resistance. One study examined rats after in vivo insulin stimulation with and without a 5-hour lipid infusion to increase fatty acid concentrations.18 Increased fatty acid concentrations resulted in insulin resistance, which was associated with reduced IRS-1–associated PI3-kinase activity, a blunting in IRS-1 tyrosine phosphorylation, and a 4-fold increase in protein kinase C (PKC)– activity. These data suggested that alterations in the insulin-signaling cascade result in the insulin resistance observed when plasma fatty acids are elevated, and such alterations might be a consequence of PKC– activation. Taken together, these data support the current working model of how elevated plasma fatty
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FIGURE 3. Effect of increasing plasma fatty acid in 9 healthy subjects. Glucose infusion rate, increase in calf muscle glycogen, and increase in calf muscle glucose-6-phosphate (G6P) at low (closed circles) and elevated (open circles) plasma fatty acid concentrations. *p <0.05; †p <0.01; ‡p <0.001. (Reprinted with permission from J Clin Invest.15)
acid concentrations cause insulin resistance (Figure 5). It is theorized that after fatty acids get into the muscle cell, a product of the fatty acids, such as fatty acyl CoA or diacylglycerol, activates PKC-. This leads to a serine/threonine phosphorylation cascade and increased serine phosphorylation of IRS-1 (and possibly IRS-2), which in turn leads to decreased tyrosine phosphorylation of IRS-1, decreased activity of PI3-kinase, and ultimately decreased activation of GLUT4.17,19 Lessons learned from transgenic mice: A mouse model of severe lipodystrophy with virtually no fat tissue (ie, “fatless”) has been developed by Moitra et al.20 These mice develop type 2 diabetes shortly after birth. Kim et al21 have found that these mice are extremely insulin resistant and have defects in insulin action in both the muscle and the liver, which were associated with abnormalities in insulin activation of IRS-1– and IRS-2–associated PI3-kinase activity in muscle and liver, respectively. In addition, the mice
had increased amounts of triglyceride in muscle and liver. After transplanting fat into these fatless mice, triglyceride content in the muscle and liver returned to normal, as did insulin signaling and action in these tissues. This suggested that insulin resistance in lipodystrophic patients may be caused by a similar mechanism in patients with type 2 diabetes associated with obesity in which accumulation of fatty acid– derived metabolites in the liver and muscle leads to defects in insulin signaling and action in these tissues. Recent studies have suggested that circulating fatderived hormones, such as Acrp30, leptin, resistin, and tumor necrosis factor–␣, may have important effects on insulin action in liver and muscle.22–25 However, it is also possible that accumulation of locally derived fat metabolites in skeletal muscle and liver contribute to insulin resistance. To further explore the relative roles of circulating fat-derived hormones versus fatty acid– derived metabolites in mediating insulin resistance in muscles and liver, Kim et al26 studied
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FIGURE 4. Potential mechanisms by which plasma fatty acid inhibits glucose transport activity: changes could result from the effect of fatty acids on GLUT4 or alterations in the insulinsignaling cascade. G6P ⴝ glucose-6-phosphate; HK ⴝ hexokinase; NADⴙ ⴝ nicotinamideadenine dinucleotide; NADH ⴝ reduced nicotinamide-adenine dinucleotide; PDH ⴝ pyruvate dehydrogenase; PFK ⴝ phosphofructokinase. (Reprinted with permission from J Clin Invest.17)
FIGURE 5. Proposed alternative mechanism for fatty acid–induced insulin resistance in human skeletal muscle. IRS ⴝ insulin receptor substrate; PI 3 Kinase ⴝ phosphatidylinositol 3-kinase; PKC ⴝ protein kinase. (Reprinted with permission from J Clin Invest.17)
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mice with tissue-specific (muscle or liver) overexpression of lipoprotein lipase. The investigators hypothesized that overexpression of lipoprotein lipase, the rate-controlling enzyme in triglyceride hydrolysis, in specific insulin-sensitive tissues would increase fatty acid delivery to those tissues. Then by studying the mice during a 2-hour euglycemic-hyperinsulinemic clamp, the effect on insulin action and signaling could be measured in these tissues. Kim et al26 found that mice with lipoprotein lipase overexpressed in muscle had a 3-fold increase in muscle triglyceride content and had decreases in insulinstimulated glucose uptake in skeletal muscle and insulin activation of IRS-1–associated PI3-kinase. Conversely, when lipoprotein lipase was overexpressed in liver, there was a 2-fold increase in liver triglyceride content, and insulin resistance could be attributed to the impaired ability of insulin to suppress endogenous glucose production with defects in IRS-2–associated PI3-kinase. These findings demonstrate that locally derived fatty acid metabolites are the likely culprits responsible for fatty acid–induced insulin resistance in muscle and liver and argue against an important role for circulating fat-derived hormones in this process.
THIAZOLIDINEDIONES TARGET INSULIN RESISTANCE Because insulin resistance in skeletal muscle plays an important role in the development and progression of type 2 diabetes, treatments that increase insulin sensitivity are highly desirable. The thiazolidinediones (TZDs) are a class of antidiabetic agents that directly targets insulin resistance in skeletal muscle.27–30 The peroxisome proliferator-activated receptors (PPARs) are members of the steroid/thyroid hormonereceptor superfamily of transcription factors.31 The TZDs are selective and potent agonists of PPAR-␥, which is most highly expressed in adipose tissue. Although the precise mechanism of action of the TZDs is under investigation, PPAR-␥–responsive genes are involved in the regulation of fatty acid metabolism. One possible action of the TZDs may be to facilitate redistribution of fat from the liver and skeletal muscle and into adipocytes. This hypothesis would explain how TZDs are able to improve insulin sensitivity in muscle and liver tissues where there is a relative paucity of PPAR-␥.17 A study in patients with type 2 diabetes examined the metabolic pathways by which a TZD, troglitazone, improved insulin responsiveness.32 Using phosphorous-31 and carbon-13 NMR spectroscopy, the investigators found that treatment with a TZD facilitated glucose transport activity, which led to increased rates of muscle glycogen synthesis. Another study evaluating the metabolic effects of troglitazone in 93 patients with type 2 diabetes also showed that troglitazone decreased fasting and postprandial glucose levels by augmenting insulin-mediated glucose disposal primarily in skeletal muscle.29 There also was a modest effect of troglitazone in decreasing rates of fasting
hepatic glucose production at the highest dose (600 mg/day). To gain further insights into the mechanism of TZD action in humans, Mayerson et al33 examined the effects of rosiglitazone on whole-body insulin sensitivity assessed by hyperinsulinemic-euglycemic clamp in combination with 1H-NMR measurements of liver and muscle triglyceride content. A total of 9 patients with type 2 diabetes were treated with rosiglitazone 4 mg twice daily for 3 months, and insulin sensitivity was measured using a 2-step hyperinsulinemic-euglycemic clamp. During the low-dosage clamp, treatment with rosiglitazone led to a 68% (p ⬍0.002) improvement in insulin-stimulated glucose metabolism (from 56.2 ⫾ 4.7 to 94.2 ⫾ 5.6 mg/m2 per minute, before and after rosiglitazone), which was associated with ⬃4.0% reductions in plasma fatty acid concentration (p ⬍0.05) and hepatic triglyceride content. Additionally, rosiglitazone increased extramyocellular lipid content by 39% (p ⬍0.05) and was associated with a 52% (p ⫽ 0.04) increase in suppression of glycerol release from adipocytes by insulin, reflecting increased sensitivity of peripheral adipocytes to insulininduced suppression of lipolysis. Taken together, these studies support the hypothesis that TZDs promote increased sensitivity in muscle and liver by activating PPAR-␥ in adipocytes and promoting a redistribution of fat from liver and muscle into adipocytes. Inzucchi et al30 evaluated the metabolic effects of metformin (1,000 mg twice daily) compared with troglitazone (400 mg/day) in 29 patients with type 2 diabetes. Patients were randomly assigned to the 2 treatment groups, and endogenous glucose production and peripheral glucose disposal were measured. In terms of mean endogenous glucose production, a 19% decrease was observed during metformin therapy (p ⫽ 0.001), whereas no change was noted in the troglitazone treatment group. However, the mean glucose disposal rate increased by 54% (p ⫽ 0.006) in the patients treated with troglitazone, compared with only a 13% increase in the metformin group. This study showed that the biguanides acted primarily by decreasing endogenous glucose production, whereas the TZDs act primarily by increasing the rate of peripheral glucose disposal. Recently, Hundal et al34 demonstrated that the mechanism by which metformin reduces endogenous glucose production in patients with type 2 diabetes was through a reduction in gluconeogenesis.
CONCLUSIONS In summary, recent studies using NMR spectroscopy to examine intracellular glucose metabolism have demonstrated that defects in insulin-stimulated muscle glycogen synthesis are responsible for most of the insulin resistance observed in skeletal muscle of patients with type 2 diabetes and that this abnormality can be attributed to defects in insulin-stimulated muscle glucose transport activity. Furthermore, these abnormalities are strongly associated with increased intramyocellular lipid accumulation. Recent NMR stud-
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ies in humans have provided new data that challenge the classic mechanisms of fat-induced insulin resistance in muscle first suggested by Randle et al,14 which implicated fatty acid inhibition of pyruvate dehydrogenase activity. These new data in human skeletal muscle suggest that an increase in intracellular fatty acid metabolites leads to activation of a serine kinase cascade that results in decreased insulin activation of IRS-1 tyrosine phosphorylation, resulting in decreased IRS-1–associated PI3-kinase activity and decreased glucose transport activity. This hypothesis, which is supported by both human and transgenic mouse data, would also explain the mechanism of insulin resistance in patients with severe lipodystrophy in whom the lack of adipocytes leads to fatty acid metabolite accumulation in liver and muscle, resulting in insulin resistance in these tissue beds through the same mechanism.35 Finally, this hypothesis would also explain the insulin-sensitizing effects of the novel class of insulin-sensitizing agents, the TZDs. It is proposed that these PPAR-␥ agonists improve insulin action in muscle and liver through an indirect mechanism whereby they directly increase insulin sensitivity in the adipocyte, which has the highest density of PPAR-␥, resulting in a redistribution of fat away from liver and muscle and into peripheral adipocytes. Overall, these data provide new potential targets for treatment of type 2 diabetes. 1. Powers AC. Diabetes mellitus. In: Braunwald E, Fauci AS, Kasper DL, Hauser
SL, Longo DL, Jameson JL, eds. Harrison’s Principles of Internal Medicine, 15th ed. New York: McGraw-Hill, 2001:2109 –2137. 2. Morris F, White C, Kahn R. Molecular aspects of insulin action. In: Kahn CR, Weir GC, eds. Joslin’s Diabetes Mellitus, 13th ed. Philadelphia: Lippincott Williams & Wilkins, 1994:139 –162. 3. Warram JH, Martin BC, Krolewski AS, Soeldner JS, Kahn CR. Slow glucose removal rate and hyperinsulinemia precede the development of type II diabetes in the offspring of diabetic parents. Ann Intern Med 1990;113:909 –915. 4. DeFronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM: a balanced overview. Diabetes Care 1992;15:318 –368. 5. Shulman GI, Rothman DL, Jue T, Stein P, DeFronzo RA, Shulman RG. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non–insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy. N Engl J Med 1990;322:223–228. 6. Cline GW, Falk Petersen K, Krssak M, Shen J, Hundal RS, Trajanoski Z, Inzucchi S, Dresner A, Rothman DL, Shulman GI. Impaired glucose transport as a cause of decreased insulin-stimulated muscle glycogen synthesis in type 2 diabetes. N Engl J Med 1999;341:240 –246. 7. Rothman DL, Shulman RG, Shulman GI. 31P nuclear magnetic resonance measurements of muscle glucose-6-phosphate: evidence for reduced insulindependent muscle glucose transport or phosphorylation activity in non–insulindependent diabetes mellitus. J Clin Invest 1992;89:1069 –1075. 8. Eriksson J, Franssila-Kallunki A, Ekstrand A, Saloranta C, Widen E, Schalin C, Groop L. Early metabolic defects in persons at increased risk for non–insulindependent diabetes mellitus. N Engl J Med 1989;321:337–343. 9. Lillioja S, Mott DM, Howard BV, Bennett PH, Yki-Jarvinen H, Freymond D, Nyomba BL, Zurlo F, Swinburn B, Bogardus C. Impaired glucose tolerance as a disorder of insulin action: longitudinal and cross-sectional studies in Pima Indians. N Engl J Med 1988;318:1217–1225. 10. Rothman DL, Magnusson I, Cline G, Gerard D, Kahn CR, Shulman RG, Shulman GI. Decreased muscle glucose transport/phosphorylation is an early defect in the pathogenesis of non–insulin-dependent diabetes mellitus. Proc Natl Acad Sci USA 1995;92:983–987. 11. Perseghin G, Ghosh S, Gerow K, Shulman GI. Metabolic defects in lean nondiabetic offspring of NIDDM parents: a cross-sectional study. Diabetes 1997;46:1001–1009.
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12. Krssak M, Falk Petersen K, Dresner A, DiPietro L, Vogel SM, Rothman DL,
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Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI. Effects of free fatty acids on glucose transport and IRS-1–associated phosphatidylinositol 3-kinase activity. J Clin Invest 1999;103:253–259. 17. Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest 2000; 106:171–176. 18. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI. Free fatty acid–induced insulin resistance is associated with activation of protein kinase C and alterations in the insulin signaling cascade. Diabetes 1999;48:1270 –1274. 19. Yu C, Chen Y, Zong H, Wang Y, Bergerson R, Kim JK, Cline GW, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, Shulman GI. Mechanism by which fatty acids inhibit insulin activation of IRS-1 associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 2002 (in press). 20. Moitra J, Mason MM, Olive M, Krylov D, Gavrilova O, Marcus-Samuels B, Feigenbaum L, Lee E, Aoyama T, Eckhaus M, Reitman ML, Vinson C. Life without white fat: a transgenic mouse. Genes Dev 1998;12:3168 –3181. 21. Kim JK, Gavrilova O, Chen Y, Reitman ML, Shulman GI. Mechanism of insulin resistance in A-ZIP/F-1 fatless mice. J Biol Chem 2000;275:8456 –8460. 22. Hotamisligil GS. The role of TNF alpha and TNF receptors in obesity and insulin resistance. J Intern Med 1999;245:621–625. 23. Mohamed-Ali V, Pinkney JH, Coppack SW. Adipose tissue as an endocrine and paracrine organ. Int J Obes Relat Metab Disord 1998;22:1145–1158. 24. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature 1999;401:73–76. 25. Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA. The hormone resistin links obesity to diabetes. Nature 2001;409:307–312. 26. Kim JK, Fillmore JJ, Chen Y, Yu C, Moore IK, Pypaert M, Lutz EP, Kako Y, Velez-Carrasco W, Goldberg IJ, Breslow JL, Shulman GI. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance. Proc Natl Acad Sci USA 2001;98:7522–7527. 27. Saltiel AR, Olefsky JM. Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 1996;45:1661–1669. 28. Yu JG, Kruszynska YT, Mulford MI, Olefsky JM. A comparison of troglitazone and metformin on insulin requirements in euglycemic intensively insulintreated type 2 diabetic patients. Diabetes 1999;48:2414 –2421. 29. Maggs DG, Buchanan TA, Burant CF, Cline G, Gumbiner B, Hsueh WA, Inzucchi S, Kelly D, Nolan J, Olefsky JM, Polonsky KS, Silver D, Valiquett TR, Shulman GI. Metabolic effects of troglitazone monotherapy in type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 1998;128:176 –185. 30. Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, Shulman GI. Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med 1998;338:867–872. 31. Saltiel AR, Olefsky JM. Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 1996;45:1661–1669. 32. Petersen KF, Krssak M, Inzucchi S, Cline GW, Dufour S, Shulman GI. Mechanism of troglitazone action in type 2 diabetes. Diabetes 2000;49:827–831. 33. Mayerson AB, Hundal R, Dufour S, Lebon V, Befroy D, Cline GW, Enocksson S, Inzucchi SE, Shulman GI, Petersen KF. The effects of rosiglitazone on insulin sensitivity, lipolysis, and hepatic and skeletal muscle triglyceride content in patients with type 2 diabetes. Diabetes 2002;51:797–802. 34. Hundal RS, Krssak M, Dufour S, Laurent D, Lebon V, Chandramouli V, Inzucchi SE, Schumann WC, Petersen KF, Landau BR, Shulman GI. Mechanism by which metformin reduces glucose production in type 2 diabetes. Diabetes 2000;49:2063–2069. 35. Peterson KF, Oral EA, Dufour S, Befroy D, Ariyan C, Yu C, Cline GW, DePaoli AM, Taylor SI, Gordon P, Shulman GJ. Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest 2002;109:1345–1350.
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MD,
and Gerald I. Shulman,
MD, PhD
Insulin resistance is a principal feature of type 2 diabetes and precedes the clinical development of the disease by 10 to 20 years. Insulin resistance is caused by the decreased ability of peripheral target tissues (especially muscle) to respond properly to normal circulating concentrations of insulin. Defects in muscle glycogen synthesis play a significant role in insulin resistance, and 3 potentially rate-controlling steps in muscle glucose metabolism have been implicated in its pathogenesis: glycogen synthase, hexokinase, and GLUT4 (the major insulin-stimulated glucose transporter). Results from recent studies using nuclear magnetic resonance (NMR) spectroscopy implicate intracellular defects in glucose transport as the rate-controlling step for insulin-mediated glucose uptake in muscle. These alterations in glucose transport activity are likely the result of dysregulation of intramyocellular fatty acid metabolism, whereby fatty
acids cause insulin resistance by activation of a serine kinase cascade, leading to decreased insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation and decreased IRS-1–associated phosphatidylinositol 3-kinase activity, a required step in insulin-stimulated glucose transport into muscle. The thiazolidinedione class of antidiabetic agents directly targets insulin resistance in skeletal muscle by improving glucose transport activity and insulin-stimulated muscle glycogen synthesis. Although the precise mechanism of action is not known, recent NMR studies support the hypothesis that these agents improve insulin action in skeletal muscle and liver by promoting a redistribution of fat out of these tissues and into peripheral adipocytes. 䊚2002 by Excerpta Medica, Inc. Am J Cardiol 2002;90(suppl):11G–18G
he pathophysiology of type 2 diabetes involves defects in tissue sensitivity to insulin and deT creased insulin secretion. It is generally accepted that
events that promote intracellular glucose transport and metabolism.1,2 Insulin resistance is the inability of peripheral target tissues to respond properly to normal circulating concentrations of insulin. To maintain euglycemia, the pancreas compensates by secreting increased amounts of insulin. In patients with type 2 diabetes, insulin resistance precedes the onset of the disease by 1 to 2 decades.3 After a period of compensated insulin resistance, impaired glucose tolerance eventually develops despite elevated insulin concentrations as insulin resistance increases. Finally, pancreatic -cell failure results in decreased insulin secretion. Overt clinical type 2 diabetes results when these 2 defects, insulin resistance and impaired -cell function, occur simultaneously.1,4
resistance to insulin in target tissues (especially muscle and liver) develops initially, followed by decreased insulin secretion as a result of progressive pancreatic -cell dysfunction. This leads to the onset of overt diabetes with fasting hyperglycemia.1 Although several pharmacologic approaches to the treatment of type 2 diabetes are available, investigators are increasingly focusing attention on the intracellular defects that may be causing insulin resistance. Once the underlying cellular defects are identified, novel treatments for type 2 diabetes can be developed and targeted toward the specific defects. This article will discuss the intracellular defects in muscle tissue that contribute to insulin resistance and subsequent development of type 2 diabetes. The role of fatty acids in inducing skeletal muscle insulin resistance will also be examined.
INSULIN RESISTANCE Normally, insulin binds to insulin receptors on target organ cells, resulting in a series of cellular From the Howard Hughes Medical Institute, Department of Internal Medicine, Department of Cellular and Molecular Physiology, General Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut, USA. This work was supported by Grant Nos. R01 DK-49230, P30 DK-45735, M01 RR-00125, a K-23 award (KFP) from the United States Public Health Service and a grant from GlaxoSmithKline (KFP). Gerald I. Shulman was a consultant for GlaxoSmithKline. Address for reprints: Gerald I. Shulman, MD, PhD, Yale University School of Medicine, 295 Congress Avenue, BCMM 254C, New Haven, Connecticut 06510. E-mail: [email protected]. ©2002 by Excerpta Medica, Inc. All rights reserved.
MUSCLE GLYCOGEN SYNTHESIS There are 3 events that take place in a coordinated manner to maintain normal glucose homeostasis: secretion of insulin by the pancreatic  cells, suppression of hepatic glucose production, and stimulation of glucose uptake by the liver and muscle.4 A study was conducted in subjects with type 2 diabetes and in healthy subjects to determine the fate of glucose after it is taken up by muscle cells.5 It is possible to obtain carbon-13 nuclear magnetic resonance (NMR) spectra of human muscle glycogen in vivo. Using this noninvasive technique, glycogen concentrations can be measured accurately with a time resolution of several minutes. In 1 analysis, combined hyperglycemic-hyperinsulinemic clamp studies were performed by infusing carbon-13– enriched glucose and insulin intravenously and measuring the rate of muscle glycogen synthesis.5 Used in combination with indirect calorimetry, the rate of muscle glycogen synthesis could be 0002-9149/02/$ – see front matter PII S0002-9149(02)02554-7
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FIGURE 1. Impaired glucose transport and insulin-stimulated muscle glycogen synthesis in type 2 diabetes: potential rate-controlling steps in muscle glucose metabolism. G6P ⴝ glucose-6-phosphate; Glucoseex ⴝ extracellular glucose; Glucosein ⴝ intracellular glucose; Vglycolysis ⴝ net velocity of the glycolytic flux of glucose-6-phosphate; VGT ⴝ velocity of glucose transport into the muscle cell; VⴚGT ⴝ velocity of glucose transport out of the muscle cell; VHK ⴝ velocity of glucose phosphorylation by hexokinase. (Reprinted with permission from N Engl J Med.6 Copyright 姝1999 Massachusetts Medical Society. All rights reserved.)
related to whole-body glucose uptake and nonoxidative disposal of glucose. In this study, the investigators found that glycogen synthesis represented the primary pathway for nonoxidative glucose disposal in normal subjects and that muscle glycogen synthesis was the major pathway of overall glucose metabolism. In addition, the rate of glycogen formation in subjects with diabetes was 60% lower than the rate in normal subjects, providing evidence that glycogen synthesis was profoundly impaired in persons with type 2 diabetes. Based on data from this study, it was clear that impaired glycogen synthesis was the major intracellular metabolic defect responsible for insulin resistance in subjects with type 2 diabetes.
POTENTIAL RATE-CONTROLLING STEPS IN MUSCLE GLUCOSE METABOLISM Once it was known that defects in muscle glycogen synthesis played a significant part in the insulin resistance that occurs in individuals with type 2 diabetes, subsequent studies attempted to identify the specific biochemical defect responsible for this abnormality. To do this, investigators assessed the potentially ratecontrolling steps for insulin-stimulated muscle glucose metabolism. The following 3 candidate steps were examined: glycogen synthase, hexokinase, and GLUT4, a glucose transporter (Figure 1). Each of these steps has been reported to be defective in patients with type 2 diabetes.6 Glycogen synthase: It has been suggested that glycogen synthase may be the major defect in glycogen 12G THE AMERICAN JOURNAL OF CARDIOLOGY姞
synthesis. If so, then the concentration of glucose-6phosphate (G6P) should be higher in patients with type 2 diabetes than in normal subjects under insulinstimulated conditions (Figure 1). However, if glucose transport and/or hexokinase were rate controlling for insulin-stimulated muscle glycogen synthesis, then G6P concentration would be expected to be no different or lower in the muscle of the subjects with diabetes. To test this hypothesis, we measured the concentration of G6P in muscle using phosphorous-31 NMR spectroscopy during a hyperglycemic-hyperinsulinemic clamp.7 A total of 6 subjects with type 2 diabetes and 6 matched controls were studied at similar steadystate plasma concentrations of insulin and glucose. In addition to measuring G6P, whole-body oxidative and nonoxidative glucose metabolism, which measures muscle glycogen synthesis, was determined. A reduced rate of nonoxidative glucose metabolism was seen in patients with type 2 diabetes compared with normal subjects (13 mol/kg body weight minimum vs 31 mol/kg body weight minimum, respectively; p ⬍0.05). Most importantly, the concentration of G6P was lower in patients with type 2 diabetes than in normal subjects (0.17 mmol/kg muscle vs 0.24 mmol/kg muscle, respectively; p ⬍0.01). These findings indicated that a reduction in the activity of either muscle glucose transport and/or hexokinase activity was most likely responsible for the development of insulin resistance. Studies have shown that insulin resistance can be found in patients more than a decade before they
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develop type 2 diabetes, and insulin resistance is the best predictor for later development of the disease.3,8,9 However, impaired muscle glycogen synthesis and glucose transport/hexokinase activity may be an acquired defect in type 2 diabetes rather than a primary defect. To examine this question, we examined nondiabetic offspring of patients with type 2 diabetes, who are known to be at risk for the development of type 2 diabetes. The rate of muscle glycogen synthesis and the concentration of muscle G6P were measured using carbon-13 and phosphorous-31 NMR spectroscopy in 6 lean, normoglycemic offspring of patients with type 2 diabetes and 7 matched controls.10 The offspring of parents with type 2 diabetes had a 50% reduction in total glucose metabolism, the rate of muscle glycogen synthesis was reduced by 70% (p ⬍0.005), and muscle G6P concentration was reduced by 40% (p ⬍0.003). These findings were similar to those observed in subjects with type 2 diabetes7 and suggested that impaired muscle glucose transport and/or hexokinase activity was the defective biochemical process. The fact that this defect was present in this group of subjects demonstrated that defects in glucose transport/phosphorylation are an early factor in the pathogenesis of type 2 diabetes rather than a consequence of the disease. Distinguishing between potential defects in hexokinase and glucose transport: Intracellular glucose is an
intermediary metabolite between glucose transport and hexokinase activity (Figure 1), and its concentration depends on the relative activities of these 2 processes. For example, if hexokinase activity were reduced in patients with type 2 diabetes, a substantial increase in intracellular glucose would be expected. Therefore, to determine the relative importance of hexokinase and glucose transport in muscle glucose metabolism, Cline et al6 used a novel NMR approach with carbon-13 and phosphorous-31 to measure intracellular glucose, G6P, and glycogen concentrations. Hyperglycemic-hyperinsulinemic clamp studies and NMR measurements were performed in 6 patients with type 2 diabetes and in 7 normal subjects. In the patients with diabetes, rates of whole-body glucose metabolism, muscle glycogen synthesis, and G6P concentrations in muscle were approximately 80% lower than in normal subjects. The mean intracellular glucose concentration was calculated to be 0.11 mmol/L in normal subjects. In patients with diabetes, the concentration was 0.24 mmol/L. This value was 1/25 of what would be expected if hexokinase were the ratecontrolling step in glucose metabolism. Cline et al6 concluded that glucose transport was the rate-controlling step in insulin-stimulated muscle glycogen synthesis in patients with type 2 diabetes. As such, investigators now have a target on which to focus. If this defect in glucose transport could be ameliorated, significant improvement in insulin-stimulated glycogen synthesis should be seen in patients with type 2 diabetes. The focus of investigation is now to identify the mechanisms responsible for the defect in the insulin-stimulated GLUT4 transporter activity.
ROLE OF FATTY ACIDS IN GLUCOSE METABOLISM Insulin resistance is the best predictor for the development of type 2 diabetes, and it is related to many other factors, such as age, diet, and exercise. In an attempt to identify the defect in glucose transport, Perseghin et al11 examined a population of young, healthy, lean, nonexercising, normoglucose-tolerant subjects who had 1 parent with type 2 diabetes (offspring). A total of 49 offspring were compared with 29 matched healthy control subjects by means of an intravenous glucose bolus, euglycemic-hyperinsulinemic clamp, and lipid and amino acid profiles. The offspring had higher plasma fatty acid concentrations than did the control subjects (582 vs 470 mol/L, respectively; p ⫽ 0.007); triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein cholesterol levels were comparable between groups. A simple regression analysis between insulin sensitivity and fatty acid concentration was significant in offspring but not in control subjects (Figure 2). The inverse correlation of high fatty acid concentrations with decreased insulin sensitivity suggested that altered fatty acid metabolism may have a primary role in causing insulin resistance in type 2 diabetes. Krssak et al12 have applied noninvasive proton NMR spectroscopy techniques to determine intramyocellular lipid content. In 23 normal-weight, nondiabetic adults, insulin sensitivity was assessed by a euglycemic-hyperinsulinemic clamp test, and intramyocellular lipid concentrations were determined by proton NMR. Insulin-stimulated whole-body glucose was inversely correlated with intramyocellular lipid content (r ⫽ ⫺0.692; p ⫽ 0.0017). A similar inverse relationship between intramyocellular lipid content and insulin stimulant whole-body glucose uptake was found in first-degree relatives of subjects with type 2 diabetes.13 These findings provided additional data implicating lipids as a cause of insulin resistance in the skeletal muscle. Mechanism of fat-induced insulin resistance: Almost 40 years ago, Randle et al14 proposed a mechanism for fat-induced insulin resistance that implicated fatty acid oxidation as causing the inactivation of mitochondrial pyruvate dehydrogenase and ultimately leading to decreased glucose uptake. However, their work was done in isolated rat heart muscle, which may be metabolically very different from resting human skeletal muscle. We examined the mechanism by which lipids cause insulin resistance in human skeletal muscle using carbon-13 and phosphorous-31 NMR spectroscopy techniques.15 In 9 healthy subjects, skeletal muscle glycogen and G6P concentrations were measured simultaneously using NMR spectroscopy under euglycemic-hyperinsulinemic clamp conditions for 6 hours. To examine the effects of fatty acids, the plasma concentration of fatty acids was increased by an intravenous infusion of a triglyceride emulsion combined with heparin to activate lipoprotein lipase. After 3 hours of the lipid infusion, rates of muscle glycogen synthesis decreased by approximately 50% (Figure 3).
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FIGURE 2. High plasma fatty acid (FA) concentration predicts insulin resistance. Simple regression analysis between insulin sensitivity and fasting plasma fatty acids in offspring of parents with type 2 diabetes (solid line: adjusted R2 ⴝ 0.21, p ⴝ 0.0005) and control subjects (dashed line: adjusted R2 ⴝ 0.03, p ⴝ 0.368). E ⴝ control subjects; ⽧ ⴝ offspring. (Reprinted with permission from Diabetes.11)
This effect was preceded by a significant reduction in muscle G6P concentrations. By 6 hours, the rate of whole-body glucose uptake was approximately 46% of control values. Raising fatty acids by infusing lipids made these healthy individuals as insulin resistant as patients with type 2 diabetes. Therefore, in contrast to the mechanism proposed by Randle et al14 in which fatty acids inhibit pyruvate dehydrogenase activity and thereby induce insulin resistance, we found that increases in plasma fatty acid concentrations inhibited glucose transport and/or phosphorylation activity.15 These defects in glucose transport and hexokinase were similar to previous findings in patients with type 2 diabetes7 and in the lean, normoglycemic offspring of diabetic patients.10 This suggested that defects in fatty acid metabolism may have an important role in the pathogenesis of insulin resistance in patients with type 2 diabetes by interfering with insulin activation of glucose transport and/or hexokinase activity. To distinguish between fatty acid–induced inhibition of insulin-stimulated glucose transport and hexokinase activity, Dresner et al16 performed a follow-up study and measured intracellular glucose concentration using the carbon-13 NMR spectroscopy technique previously described. Once again, the assumption was that if increased fatty acid concentrations reduced hexokinase activity, glucose would accumulate inside the muscle cell. Intracellular glucose concentration was measured in 7 healthy subjects during a euglycemic-hyperinsulinemic clamp following a 5-hour infusion of either glycerol or lipid plus heparin. Intracellular glucose was found to be significantly lower after the lipid infusion than after the glycerol infusion (0.04 vs 0.25 mmol/L; p ⫽ 0.04). These data suggested that elevations in plasma fatty acid concen14G THE AMERICAN JOURNAL OF CARDIOLOGY姞
tration caused insulin resistance in skeletal muscle by reducing insulin-stimulated glucose transport activity. The mechanisms by which fatty acids induce changes in glucose transport activity are unknown, but these changes could be a result of the effects of fatty acids on the GLUT4 transporter directly or could result from alterations in the insulin-signaling cascade (Figure 4). Dresner et al16 attempted to answer this question by measuring insulin receptor substrate (IRS)-1–associated phosphatidylinositol 3-kinase (PI3-kinase) activity in muscle biopsy samples using a lipid infusion protocol similar to the aforementioned study. During the control (glycerol) infusion, PI3kinase activity increased 3- to 4-fold after insulin stimulation. However, this effect was abolished during the lipid infusion. This suggested that fatty acid– induced insulin resistance was a consequence of altered insulin signaling through PI3-kinase. Effect of fatty acids on the insulin-signaling cascade:
To further characterize this fat-induced defect in insulin activation of IRS-1–associated PI3-kinase activity, additional studies were conducted in an in vivo animal model of fat-induced insulin resistance. One study examined rats after in vivo insulin stimulation with and without a 5-hour lipid infusion to increase fatty acid concentrations.18 Increased fatty acid concentrations resulted in insulin resistance, which was associated with reduced IRS-1–associated PI3-kinase activity, a blunting in IRS-1 tyrosine phosphorylation, and a 4-fold increase in protein kinase C (PKC)– activity. These data suggested that alterations in the insulin-signaling cascade result in the insulin resistance observed when plasma fatty acids are elevated, and such alterations might be a consequence of PKC– activation. Taken together, these data support the current working model of how elevated plasma fatty
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FIGURE 3. Effect of increasing plasma fatty acid in 9 healthy subjects. Glucose infusion rate, increase in calf muscle glycogen, and increase in calf muscle glucose-6-phosphate (G6P) at low (closed circles) and elevated (open circles) plasma fatty acid concentrations. *p <0.05; †p <0.01; ‡p <0.001. (Reprinted with permission from J Clin Invest.15)
acid concentrations cause insulin resistance (Figure 5). It is theorized that after fatty acids get into the muscle cell, a product of the fatty acids, such as fatty acyl CoA or diacylglycerol, activates PKC-. This leads to a serine/threonine phosphorylation cascade and increased serine phosphorylation of IRS-1 (and possibly IRS-2), which in turn leads to decreased tyrosine phosphorylation of IRS-1, decreased activity of PI3-kinase, and ultimately decreased activation of GLUT4.17,19 Lessons learned from transgenic mice: A mouse model of severe lipodystrophy with virtually no fat tissue (ie, “fatless”) has been developed by Moitra et al.20 These mice develop type 2 diabetes shortly after birth. Kim et al21 have found that these mice are extremely insulin resistant and have defects in insulin action in both the muscle and the liver, which were associated with abnormalities in insulin activation of IRS-1– and IRS-2–associated PI3-kinase activity in muscle and liver, respectively. In addition, the mice
had increased amounts of triglyceride in muscle and liver. After transplanting fat into these fatless mice, triglyceride content in the muscle and liver returned to normal, as did insulin signaling and action in these tissues. This suggested that insulin resistance in lipodystrophic patients may be caused by a similar mechanism in patients with type 2 diabetes associated with obesity in which accumulation of fatty acid– derived metabolites in the liver and muscle leads to defects in insulin signaling and action in these tissues. Recent studies have suggested that circulating fatderived hormones, such as Acrp30, leptin, resistin, and tumor necrosis factor–␣, may have important effects on insulin action in liver and muscle.22–25 However, it is also possible that accumulation of locally derived fat metabolites in skeletal muscle and liver contribute to insulin resistance. To further explore the relative roles of circulating fat-derived hormones versus fatty acid– derived metabolites in mediating insulin resistance in muscles and liver, Kim et al26 studied
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FIGURE 4. Potential mechanisms by which plasma fatty acid inhibits glucose transport activity: changes could result from the effect of fatty acids on GLUT4 or alterations in the insulinsignaling cascade. G6P ⴝ glucose-6-phosphate; HK ⴝ hexokinase; NADⴙ ⴝ nicotinamideadenine dinucleotide; NADH ⴝ reduced nicotinamide-adenine dinucleotide; PDH ⴝ pyruvate dehydrogenase; PFK ⴝ phosphofructokinase. (Reprinted with permission from J Clin Invest.17)
FIGURE 5. Proposed alternative mechanism for fatty acid–induced insulin resistance in human skeletal muscle. IRS ⴝ insulin receptor substrate; PI 3 Kinase ⴝ phosphatidylinositol 3-kinase; PKC ⴝ protein kinase. (Reprinted with permission from J Clin Invest.17)
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mice with tissue-specific (muscle or liver) overexpression of lipoprotein lipase. The investigators hypothesized that overexpression of lipoprotein lipase, the rate-controlling enzyme in triglyceride hydrolysis, in specific insulin-sensitive tissues would increase fatty acid delivery to those tissues. Then by studying the mice during a 2-hour euglycemic-hyperinsulinemic clamp, the effect on insulin action and signaling could be measured in these tissues. Kim et al26 found that mice with lipoprotein lipase overexpressed in muscle had a 3-fold increase in muscle triglyceride content and had decreases in insulinstimulated glucose uptake in skeletal muscle and insulin activation of IRS-1–associated PI3-kinase. Conversely, when lipoprotein lipase was overexpressed in liver, there was a 2-fold increase in liver triglyceride content, and insulin resistance could be attributed to the impaired ability of insulin to suppress endogenous glucose production with defects in IRS-2–associated PI3-kinase. These findings demonstrate that locally derived fatty acid metabolites are the likely culprits responsible for fatty acid–induced insulin resistance in muscle and liver and argue against an important role for circulating fat-derived hormones in this process.
THIAZOLIDINEDIONES TARGET INSULIN RESISTANCE Because insulin resistance in skeletal muscle plays an important role in the development and progression of type 2 diabetes, treatments that increase insulin sensitivity are highly desirable. The thiazolidinediones (TZDs) are a class of antidiabetic agents that directly targets insulin resistance in skeletal muscle.27–30 The peroxisome proliferator-activated receptors (PPARs) are members of the steroid/thyroid hormonereceptor superfamily of transcription factors.31 The TZDs are selective and potent agonists of PPAR-␥, which is most highly expressed in adipose tissue. Although the precise mechanism of action of the TZDs is under investigation, PPAR-␥–responsive genes are involved in the regulation of fatty acid metabolism. One possible action of the TZDs may be to facilitate redistribution of fat from the liver and skeletal muscle and into adipocytes. This hypothesis would explain how TZDs are able to improve insulin sensitivity in muscle and liver tissues where there is a relative paucity of PPAR-␥.17 A study in patients with type 2 diabetes examined the metabolic pathways by which a TZD, troglitazone, improved insulin responsiveness.32 Using phosphorous-31 and carbon-13 NMR spectroscopy, the investigators found that treatment with a TZD facilitated glucose transport activity, which led to increased rates of muscle glycogen synthesis. Another study evaluating the metabolic effects of troglitazone in 93 patients with type 2 diabetes also showed that troglitazone decreased fasting and postprandial glucose levels by augmenting insulin-mediated glucose disposal primarily in skeletal muscle.29 There also was a modest effect of troglitazone in decreasing rates of fasting
hepatic glucose production at the highest dose (600 mg/day). To gain further insights into the mechanism of TZD action in humans, Mayerson et al33 examined the effects of rosiglitazone on whole-body insulin sensitivity assessed by hyperinsulinemic-euglycemic clamp in combination with 1H-NMR measurements of liver and muscle triglyceride content. A total of 9 patients with type 2 diabetes were treated with rosiglitazone 4 mg twice daily for 3 months, and insulin sensitivity was measured using a 2-step hyperinsulinemic-euglycemic clamp. During the low-dosage clamp, treatment with rosiglitazone led to a 68% (p ⬍0.002) improvement in insulin-stimulated glucose metabolism (from 56.2 ⫾ 4.7 to 94.2 ⫾ 5.6 mg/m2 per minute, before and after rosiglitazone), which was associated with ⬃4.0% reductions in plasma fatty acid concentration (p ⬍0.05) and hepatic triglyceride content. Additionally, rosiglitazone increased extramyocellular lipid content by 39% (p ⬍0.05) and was associated with a 52% (p ⫽ 0.04) increase in suppression of glycerol release from adipocytes by insulin, reflecting increased sensitivity of peripheral adipocytes to insulininduced suppression of lipolysis. Taken together, these studies support the hypothesis that TZDs promote increased sensitivity in muscle and liver by activating PPAR-␥ in adipocytes and promoting a redistribution of fat from liver and muscle into adipocytes. Inzucchi et al30 evaluated the metabolic effects of metformin (1,000 mg twice daily) compared with troglitazone (400 mg/day) in 29 patients with type 2 diabetes. Patients were randomly assigned to the 2 treatment groups, and endogenous glucose production and peripheral glucose disposal were measured. In terms of mean endogenous glucose production, a 19% decrease was observed during metformin therapy (p ⫽ 0.001), whereas no change was noted in the troglitazone treatment group. However, the mean glucose disposal rate increased by 54% (p ⫽ 0.006) in the patients treated with troglitazone, compared with only a 13% increase in the metformin group. This study showed that the biguanides acted primarily by decreasing endogenous glucose production, whereas the TZDs act primarily by increasing the rate of peripheral glucose disposal. Recently, Hundal et al34 demonstrated that the mechanism by which metformin reduces endogenous glucose production in patients with type 2 diabetes was through a reduction in gluconeogenesis.
CONCLUSIONS In summary, recent studies using NMR spectroscopy to examine intracellular glucose metabolism have demonstrated that defects in insulin-stimulated muscle glycogen synthesis are responsible for most of the insulin resistance observed in skeletal muscle of patients with type 2 diabetes and that this abnormality can be attributed to defects in insulin-stimulated muscle glucose transport activity. Furthermore, these abnormalities are strongly associated with increased intramyocellular lipid accumulation. Recent NMR stud-
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ies in humans have provided new data that challenge the classic mechanisms of fat-induced insulin resistance in muscle first suggested by Randle et al,14 which implicated fatty acid inhibition of pyruvate dehydrogenase activity. These new data in human skeletal muscle suggest that an increase in intracellular fatty acid metabolites leads to activation of a serine kinase cascade that results in decreased insulin activation of IRS-1 tyrosine phosphorylation, resulting in decreased IRS-1–associated PI3-kinase activity and decreased glucose transport activity. This hypothesis, which is supported by both human and transgenic mouse data, would also explain the mechanism of insulin resistance in patients with severe lipodystrophy in whom the lack of adipocytes leads to fatty acid metabolite accumulation in liver and muscle, resulting in insulin resistance in these tissue beds through the same mechanism.35 Finally, this hypothesis would also explain the insulin-sensitizing effects of the novel class of insulin-sensitizing agents, the TZDs. It is proposed that these PPAR-␥ agonists improve insulin action in muscle and liver through an indirect mechanism whereby they directly increase insulin sensitivity in the adipocyte, which has the highest density of PPAR-␥, resulting in a redistribution of fat away from liver and muscle and into peripheral adipocytes. Overall, these data provide new potential targets for treatment of type 2 diabetes. 1. Powers AC. Diabetes mellitus. In: Braunwald E, Fauci AS, Kasper DL, Hauser
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Dresner A, Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL, Petersen KF, Shulman GI. Effects of free fatty acids on glucose transport and IRS-1–associated phosphatidylinositol 3-kinase activity. J Clin Invest 1999;103:253–259. 17. Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest 2000; 106:171–176. 18. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI. Free fatty acid–induced insulin resistance is associated with activation of protein kinase C and alterations in the insulin signaling cascade. Diabetes 1999;48:1270 –1274. 19. Yu C, Chen Y, Zong H, Wang Y, Bergerson R, Kim JK, Cline GW, Cushman SW, Cooney GJ, Atcheson B, White MF, Kraegen EW, Shulman GI. Mechanism by which fatty acids inhibit insulin activation of IRS-1 associated phosphatidylinositol 3-kinase activity in muscle. J Biol Chem 2002 (in press). 20. 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Kim JK, Fillmore JJ, Chen Y, Yu C, Moore IK, Pypaert M, Lutz EP, Kako Y, Velez-Carrasco W, Goldberg IJ, Breslow JL, Shulman GI. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance. Proc Natl Acad Sci USA 2001;98:7522–7527. 27. Saltiel AR, Olefsky JM. Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 1996;45:1661–1669. 28. Yu JG, Kruszynska YT, Mulford MI, Olefsky JM. A comparison of troglitazone and metformin on insulin requirements in euglycemic intensively insulintreated type 2 diabetic patients. Diabetes 1999;48:2414 –2421. 29. Maggs DG, Buchanan TA, Burant CF, Cline G, Gumbiner B, Hsueh WA, Inzucchi S, Kelly D, Nolan J, Olefsky JM, Polonsky KS, Silver D, Valiquett TR, Shulman GI. Metabolic effects of troglitazone monotherapy in type 2 diabetes mellitus: a randomized, double-blind, placebo-controlled trial. Ann Intern Med 1998;128:176 –185. 30. 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