EXERCISE AND INSULIN SENSITIVITY

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1 EXERCISE AND INSULIN SENSITIVITY INCREASED GLUCOSE TRANSPORT PHOSPHORYLATION AND MUSCLE GLYCOGEN SYNTHESIS AFTER EXERCISE TRAINING IN INSULIN-RESISTANT SUBJECTS GIANLUCA PERSEGHIN, M.D., THOMAS B. PRICE, PH.D., KITT FALK PETERSEN, M.D., MICHAEL RODEN, M.D., GARY W. CLINE, PH.D., KARYNN GEROW, R.N., DOUGLAS L. ROTHMAN, PH.D., AND GERALD I. SHULMAN, M.D., PH.D. ABSTRACT 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 percent (P 0.04) and percent (P 0.04) after the first exercise session and 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 ( vs 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. (N Engl J Med 1996; 335: ) 1996, Massachusetts Medical Society. FIRST-DEGREE relatives of patients with noninsulin-dependent diabetes mellitus (NIDDM) have a lifetime risk of diabetes of approximately 40 percent. 1 In these relatives, insulin resistance is the best predictor of the development of diabetes and probably plays an important part in its pathogenesis. 2-4 The most important site of peripheral insulin resistance is the skeletal muscle, and in this tissue there are several steps involved in insulin-mediated glucose uptake in which insulin resistance might occur (Fig. 1). Previous studies using carbon-13 nuclear magnetic resonance ( 13 C NMR) spectroscopy to measure the glycogen content of muscle demonstrated that a defect in insulin-stimulated glycogen synthesis in muscle plays a major part in slowing the rate of glucose disposal in patients with NIDDM. 5 Recent studies using phosphorus-31 ( 31 P) NMR spectroscopy to measure intracellular glucose-6-phosphate indicate that a defect in glucose transport phosphorylation in muscle is responsible for this abnormality in patients with NIDDM 6 as well as in lean offspring who are normoglycemic but insulin-resistant. 7 Exercise is well known to improve insulin sensitivity 8-12 and can prevent or delay the onset of NIDDM. 13 However, the mechanism by which exercise improves insulin sensitivity in humans is unknown. This study was undertaken to examine the degree to which exercise improves insulin sensitivity in both normal subjects and the insulin-resistant adult children of parents with NIDDM and the mechanism by which this improvement occurs. Screening Procedures METHODS We initially recruited for the study 55 young (age range, 19 to 43 years), lean offspring of parents with NIDDM (ascertained by history) and 8 subjects with similar characteristics who had no family history of NIDDM or hypertension traced to their grandparents. All the subjects were in good health as assessed by medical history, physical examination, routine blood counts, biochemical tests, and urinalysis, and all had normal oral glucose tolerance. Habitual physical activity at work, during sports, and at leisure was assessed with a questionnaire. 14 Subjects were excluded from the study if they were not within 8 percent of their ideal body weights (according to the 1959 Metropolitan Life Insurance tables) or if From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Conn. Address reprint requests to Dr. Shulman at the Department of Internal Medicine, Yale University School of Medicine, P.O. Box , Fitkin 104, New Haven, CT Volume 335 Number

2 Insulin tion Committee of Yale University School of Medicine, and all subjects gave written informed consent. Experimental Protocol Insulin receptor Glucose GLUT-4 GLUT-4 GLUT-4 Glucose Hexokinase II Glucose-6-phosphate We studied the effect of a single session of exercise and then six weeks of exercise training on insulin-stimulated rates of wholebody glucose metabolism, oxidative and nonoxidative glucose metabolism, glycogen synthesis in muscle, and intracellular glucose-6-phosphate in both groups, using 13C and 31P NMR spectroscopy during a hyperglycemic hyperinsulinemic clamp procedure at base line, 48 hours after the first session of exercise, and 48 hours after the last session of a six-week exercise-training program. In addition, the effect of exercise training on the first and second phases of insulin secretion was studied in both groups during separate hyperglycemic-clamp studies performed at base line and one week after the last NMR clamp study, during which time the subjects continued to exercise. Maximal aerobic capacity was measured in each subject on a bicycle ergometer at base line and after six weeks of exercise, as previously described.9 The workload was increased by 20 W every minute until the subject was exhausted, while oxygen consumption was monitored continuously with a gas analyzer (Sensor Medics, Yorba Linda, Calif.). Exercise-Training Protocol Glucose-1-phosphate Glycolysis UDP-glucose Glycogen synthase Glycogen Figure 1. Schematic Representation of the Metabolic Pathways for Intramuscular Glucose Metabolism. The key insulin-regulated steps of the metabolic pathway are shown, including glucose transport (by means of the insulin-dependent glucose transporter GLUT-4), phosphorylation (hexokinase II), and incorporation into glycogen (glycogen synthase). Decreased activity in any of these steps could be responsible for insulin resistance. UDP-glucose denotes uridine diphosphoglucose. they had a history of smoking or hypertension, were currently receiving drug therapy, were excessively sedentary, or participated in heavy physical activity. The subjects were instructed to consume an isocaloric diet (about 250 g of carbohydrate per day) for three days, after which peripheral insulin sensitivity was measured by the euglycemic hyperinsulinemic clamp technique.15 The mean glucose-infusion rate during the last hour of the clamp study served as a measure of the rate of whole-body glucose metabolism (the M value). Study Subjects On the basis of the results of the screening tests, the 10 offspring (3 subjects with both parents affected by NIDDM and 7 subjects with one) who had the greatest degree of insulin resistance were selected for the exercise studies. All eight of the normal subjects matched for age, weight, and activity were included in the exercise study. The thickness of subcutaneous fat in the calf was measured by magnetic resonance imaging and that at other sites by calipers. During the study, the subjects ate a weight-maintaining diet containing about 250 g of carbohydrate per day; they were weighed and their food consumption was monitored and adjusted every other day by a nutritionist. The screening and exercise-study protocols were approved by the Human Investiga The exercise program began one week after the base-line clamp study and the study of maximal aerobic capacity were completed. The first exercise session consisted of a 5-minute warm-up, followed by three 15-minute sets of stair-climbing exercise performed at 65 percent of maximal aerobic capacity on a stair-climbing machine (Aerostep, Temecula, Calif.), with 5-minute rest periods allowed between sets. The next day, the subjects were admitted to the Clinical Research Center of the Yale New Haven Hospital, where they ate a standardized meal and remained overnight. The NMR clamp study was repeated the next morning, 48 hours after the first exercise session. The exercise program consisted of six weeks of physical training, during which the subjects repeated the same exercise protocol they had followed during the first exercise session four times per week under medical supervision. As the physical conditioning of the subjects improved, the workloads increased to keep the pulse rates the same as during previous exercise sessions. NMR Measurements and Hyperglycemic Hyperinsulinemic Clamp Studies After the subjects fasted overnight, Teflon catheters were inserted into an antecubital vein in each arm for blood drawing and infusions. The subjects were placed in a 2.1-T NMR spectrometer (Biospec, Billerica, Mass.), and both 31P and 13C NMR spectra of the gastrocnemius muscle were acquired at 10-minute intervals to monitor intracellular glucose-6-phosphate and glycogen content, respectively.5,6 In addition, intracellular phosphate and phosphocreatine were measured. Five minutes before the insulin was administered, a somatostatin infusion (0.1 mg per kilogram of body weight per minute) was initiated and then was continued throughout the study to inhibit endogenous insulin secretion. Insulin (Humulin, Lilly, Indianapolis) was administered as a primed continuous infusion (6 pmol per kilogram per minute) along with a primed variable infusion of glucose (20 percent enriched with [1-13C]glucose), which was periodically adjusted to maintain the plasma glucose concentration at about 190 mg per deciliter (10.5 mmol per liter) for 145 minutes. Blood samples used for measuring insulin and enrichment with [13C]glucose were obtained every 15 minutes. The mean rate of glucose infusion minus the rate of urinary glucose excretion served as a measure of the rate of whole-body glucose metabolism.15 Insulin Secretion during Hyperglycemic Clamp Studies After the subjects fasted overnight, the plasma glucose concentration was rapidly raised and then maintained at 210 mg per deciliter (11.7 mmol per liter) for 150 minutes by a primed variable Oc to b er 3 1,

3 EXERCISE AND INSULIN SENSITIVITY TABLE 1. CLINICAL AND BIOCHEMICAL CHARACTERISTICS OF INSULIN-RESISTANT OFFSPRING OF PARENTS WITH NIDDM AND NORMAL SUBJECTS.* CHARACTERISTIC OFFSPRING NORMAL SUBJECTS P VALUE MALE FEMALE MALE FEMALE No Age (yr) Body weight (kg) Body-mass index Ideal body weight (%) Waist-to-hip ratio Subcutaneous adipose tissue (mm) Calf Biceps Suprailiac Fasting plasma glucose (mg/dl) Fasting plasma insulin (mu/ml) M value (mg/kg/min) Glycosylated hemoglobin (%) Maximal aerobic capacity (ml/kg/min) Physical-activity index** *Plus minus values are means SE. P values are for comparisons between the offspring and the normal subjects. Body-mass index is the weight in kilograms divided by the square of the height in meters. To convert values for plasma glucose to millimoles per liter, multiply by To convert values for plasma insulin to picomoles per liter, multiply by 6. M values were assessed with the use of a euglycemic hyperinsulinemic clamp test. To convert M values to micromoles per kilogram per minute, multiply by **The range of possible scores for the physical-activity index is 3 to 15; the lowest value corresponds to the level of physical activity of a clerical worker who plays a light sport (defined as one in which the energy expended is less than 0.76 MJ per hour e.g., bowling) and who participates in sedentary activities during leisure time. The highest value corresponds to the level of physical activity of a person who is very physically active at work (e.g., a construction worker), who plays heavy sports (in which the energy expended is at least 1.76 MJ per hour e.g., boxing, basketball, football, or rugby), and who is very physically active during leisure time (e.g., walking more than 1 hour per day or biking more than 45 minutes per day). infusion of glucose. Plasma glucose was measured every 5 minutes and plasma insulin every 2 minutes for the first 16 minutes of the clamp study, and at 10-minute intervals thereafter. Indirect Calorimetry Continuous indirect calorimetry was performed before and during hyperglycemic hyperinsulinemic clamping (at 120 to 140 minutes), as previously described. 5,15,16 Nonoxidative glucose metabolism was calculated by subtracting the amount of glucose oxidized from the total amount of glucose infused. Analytic Procedures Plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Instruments, Fullerton, Calif.). Plasma insulin was measured with a double-antibody radioimmunoassay technique (Diagnostic Systems Laboratories, Webster, Tex.). Glycosylated hemoglobin was measured by ion-exchange chromatography (Isolab, Akron, Ohio). The percent excess of the 13 C atom in plasma glucose was measured by gas chromatography mass spectrometry. 17 Statistical Analysis Increments in the muscle glycogen concentration measured after each 10-minute interval of each hyperglycemic hyperinsulinemic clamp study were calculated as previously described. 5 The rate of glycogen synthesis was calculated from the slope of the least-squares linear fit to the glycogen-concentration curve from 60 to 145 minutes. Paired two-tailed t-tests were used for comparisons within groups before and after the exercise training. Differences between groups were compared with the use of the unpaired two-tailed t-test and analysis of variance, with Scheffé s post hoc test used when appropriate. Subjects RESULTS Anthropometric measures, as well as work, sports, and leisure-time indexes of physical activity, were similar in the two groups (Table 1). The children of Volume 335 Number

4 diabetic parents were about one and a half times as insulin-resistant as the normal subjects, on the basis of the euglycemic hyperinsulinemic screening test (M value), and had a higher mean plasma insulin concentration after fasting. There was no change in any of the anthropometric data in either group after the six weeks of exercise training (data not shown), but the physical-activity index increased to a similar degree in both groups (P 0.05). Insulin Secretion Before exercise, insulin secretion during the first phase was similar in the two groups, whereas in the second phase it was significantly higher in the offspring (Table 2). Six weeks of exercise training had no detectable effect on either the first or second phase of insulin secretion in either group. Total Glucose Disposal There was a strong correlation (r 0.87, P 0.001) between the M values during the screening euglycemic hyperinsulinemic clamp study and those during the base-line hyperglycemic hyperinsulinemic clamp study in both the offspring and the normal subjects. During the base-line NMR study, the rate of wholebody glucose metabolism was lower in the offspring of diabetic parents (P 0.001) (Table 3). The rates of whole-body glucose metabolism increased by 22 percent in the offspring and 27 percent in the normal subjects after the first exercise session. The total increase from base line, after six weeks of exercise training, was 42 percent in the group of offspring and 38 percent in the normal group. TABLE 2. INSULIN SECRETION DURING A HYPERGLYCEMIC CLAMP STUDY BEFORE AND AFTER EXERCISE TRAINING IN OFFSPRING OF PARENTS WITH NIDDM AND IN NORMAL SUBJECTS. GROUP Insulin-resistant offspring Normal subjects BASAL PLASMA INSULIN CONCENTRATIONS* FIRST PHASE mu/ml SECOND PHASE *Plus minus values are means SE. To convert plasma insulin values to picomoles per liter, multiply by 6. First-phase values are the average values over a period of 2 to 16 minutes; second-phase values are the average values over a period of 60 to 150 minutes. P 0.02 for the comparison with normal subjects. P 0.05 for the comparison with normal subjects. Nonoxidative Glucose Disposal The base-line rates of nonoxidative glucose disposal were 58 percent lower in the offspring than in the normal subjects (Table 3). Nonoxidative glucose metabolism increased by 35 percent in the offspring and 41 percent in the normal group after the first exercise session., nonoxidative glucose metabolism increased further in both groups (offspring, 76 percent; normal subjects, 58 percent). Muscle Glycogen Content and Glycogen-Synthesis Rate The mean ( SE) base-line muscle glycogen concentration was similar in the offspring and the normal subjects ( vs mmol per liter). At base line, the insulin-stimulated rate of glycogen synthesis was 63 percent lower in the offspring than in the normal group (P 0.001) (Table 3). After the first exercise session, muscle glycogen synthesis in the offspring increased by 69 percent (P 0.04), and after six weeks of exercise training it increased further to 102 percent of the base-line value (P 0.02). The percentage increase was similar in the normal group (62 percent after the first exercise session and 97 percent after exercise training; P 0.04 and P for the comparisons with base line, respectively). Intracellular Glucose-6-Phosphate, the concentrations of glucose-6-phosphate, inorganic phosphate, and phosphocreatinine and the intracellular ph were similar in the two groups, and the values did not change after the first exercise session or after exercise training. During the base-line study, the increment in glucose-6-phosphate was 56 percent lower in the offspring than in the normal subjects (P 0.005), suggesting a defect in glucose transport phosphorylation (Table 3); however, this increment was normal after the first exercise session and after six weeks of exercise training. In contrast, the increment in glucose-6-phosphate in the normal group was similar during all three studies. DISCUSSION Insulin resistance in first-degree relatives of patients with NIDDM typically precedes the development of NIDDM by several decades, 2,18 and improving insulin sensitivity through exercise in these subjects might be helpful in preventing NIDDM. We undertook this study to determine the mechanism by which insulin sensitivity improves after exercise in young, sedentary subjects of normal weight and with normal glucose tolerance who are at high risk for diabetes because they have a strong family history of NIDDM and are insulin-resistant. 2,18 The exercise-training program improved wholebody insulin sensitivity by 40 percent and wholebody nonoxidative glucose metabolism by 60 to October 31, 1996

5 EXERCISE AND INSULIN SENSITIVITY TABLE 3. GLUCOSE AND GLYCOGEN METABOLISM IN OFFSPRING OF PARENTS WITH NIDDM AND IN NORMAL SUBJECTS.* WHOLE-BODY OXIDATIVE NONOXIDATIVE RATE OF MUSCLE INCREMENT IN GLUCOSE GLUCOSE GLUCOSE GLYCOGEN GLUCOSE-6-PHOSPHATE GROUP METABOLISM METABOLISM METABOLISM SYNTHESIS CONCENTRATION mg/kg/min mg/liter of muscle/min mm Offspring After the 1st exercise session Normal subjects After the 1st exercise session ** ** ** ** *Values are means SE. To convert values for glucose metabolism to micromoles per kilogram per minute, multiply by P for the comparison with normal subjects. P 0.01 for the comparison with normal subjects. P for the comparison with normal subjects. P 0.03 for the comparison with base line. P 0.05 for the comparison with base line. **P 0.04 for the comparison with base line. P 0.02 for the comparison with base line. P for the comparison with base line. P for the comparison with base line. percent in both the adult children of parents with NIDDM and normal subjects results consistent with those of previous exercise studies performed in normal subjects, 8 obese subjects, 9 and subjects with glucose intolerance or NIDDM. 19 The most striking finding was that exercise training resulted in a twofold increase in insulin-stimulated muscle glycogen synthesis in both groups. Because insulin resistance appears to be central to the pathogenesis of NIDDM, therapies that improve the action of insulin might be beneficial in preventing or delaying the onset of NIDDM. In this regard, we found that physical training increased insulin sensitivity by more (43 percent) than has been reported for metformin (16 to 25 percent) 20,21 or troglitazone (about 20 percent), 22 and exercise has the additional advantages of improving cardiovascular and respiratory performance and averting the possible side effects of long-term drug therapy. Furthermore, the finding that over 60 percent of the training effect on insulin-stimulated muscle glycogen synthesis was present 48 hours after the first exercise session suggests that similar results might be obtained with even fewer weekly exercise sessions. To understand the mechanism by which exercise improves insulin sensitivity in skeletal muscle, we used 31 P NMR spectroscopy to measure the intramuscular concentration of glucose-6-phosphate, which reflects the relative activities of glucose transport phosphorylation and glycogen synthase. In the base-line clamp study, glucose-6-phosphate was lower in the offspring than in the normal group; this is consistent with impaired glucose transport phosphorylation s being responsible for the reduced rate of muscle glycogen synthesis in these subjects. 7 Exercise training reversed this abnormality, as reflected by the normalization of the glucose-6-phosphate concentration during the hyperglycemic hyperinsulinemic clamp study after the first exercise session. Despite normalization of glucose-6-phosphate concentrations, however, rates of muscle glycogen synthesis were still lower than in the normal subjects, suggesting the existence of a defect in glycogen synthase in addition to the previously described defect in glucose transport phosphorylation, 6,7 which exercise was able to unmask. This finding is consistent with the observation that the activity of insulinstimulated glycogen synthase is reduced in skeletal muscle of nonobese first-degree relatives of patients with NIDDM. 23,24 With regard to the molecular mechanisms responsible for these observations, a single exercise session increases both the insulin-dependent activity and the number of GLUT-4 glucose transporters in the plasma membrane, 25,26 as well as the content and activity of hexokinase II messenger RNA. 27 The effects of exercise training could be explained in part by the residual effect of the last session of exercise, 28 but it could also be explained by long-term up-regulation, induced by training, of the number and function 29 of the glucose transporters; capillary proliferation 30 ; and the number of IIa (red glycolytic) fibers, which have a higher GLUT-4 protein content and are more insulin-responsive. 31,32 Volume 335 Number

6 Physical training increased whole-body insulin sensitivity similarly in both normal subjects and the offspring of diabetic parents, mostly through stimulation of insulin-mediated muscle glycogen synthesis. This improvement in insulin sensitivity in the offspring resulted from the reversal of a defect in insulin-stimulated glucose transport phosphorylation that was evident soon after the first exercise session. However, exercise training did not normalize rates of muscle glycogen synthesis in the offspring, reflecting an additional defect in glycogen synthase activity that may reflect a common abnormality in the insulin-signaling pathway between glucose transport phosphorylation and glycogen synthase. Supported by grants (R01 DK-49230, P30 DK-45735, M01 RR , and R29 NS-32126) from the Public Health Service. Dr. Perseghin was supported by a postdoctoral fellowship from the Juvenile Diabetes Foundation, Int., and by a research training award from the Istituto Scientifico San Raffaele (Stable Isotope Laboratory), University of Milan, Milan, Italy. Dr. Roden is the recipient of a Max Kade Foundation Fellowship Award. We are indebted to the staff of the Yale New Haven Hospital General Clinical Research Center and the staff of the Yale New Haven Hospital Pulmonary Laboratory for the assessment of maximal aerobic capacity; to Mr. Terry Nixon and Mr. Peter Brown for technical assistance with the NMR spectrometer; to Ms. Veronica Walton, Ms. Yvonne Milewski, Ms. Nicole Barucci, and Ms. Parveen Vohra for technical assistance with the studies; to Ms. Donna Casseria, M.S., R.D., for assistance with the diets; and to Ms. Ann DeCosta for assistance in the preparation of the manuscript. REFERENCES 1. Kobberling J. Studies on the genetic heterogeneity of diabetes mellitus. 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