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Original Article |
Divisions of Medicine and Molecular Medicine, Departments of Emergency and Cardiovascular Medicine (P.N.B.), Endocrinology (S.E., J.K.), and Anesthesiology (J.P.), Karolinska Hospital and Institute, S-17176 Stockholm, Sweden; and Diabetes and Metabolism Unit (N.B.R., A.K.S.), Boston University Hospital, Boston, Massachusetts 02118
Address all correspondence and requests for reprints to: Dr. Peter Båvenholm, Department of Emergency and Cardiovascular Medicine, Division of Internal Medicine, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: peter.bavenholm{at}ks.se.
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Abstract |
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In conclusion, the results confirm that insulin sensitivity is decreased in slightly overweight men with mild type 2 diabetes and that this correlates closely with an increase in truncal fat mass and a decrease in physical fitness. Whether the unexpectedly high levels of malonyl CoA in muscle, together with the diminished suppression of plasma free fatty acid, explains the insulin resistance of the diabetic patients during the clamp remains to be determined.
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Introduction |
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In regard to molecular mechanisms responsible for insulin resistance, it has been demonstrated in rodents that a sustained high level of malonyl coenzyme A (CoA) in muscle is associated with decreases in the activity of carnitine palmitoyl transferase, the enzyme that regulates the transport of long chain fatty acyl (LCFA) CoA into the mitochondria where they are oxidized (7). This results in a decreased rate of fatty acid oxidation and increases in muscle diacylglycerol (DAG) concentration and triglyceride mass (8). Augmented levels of DAG activate protein kinase C (PKC), and this could decrease insulin sensitivity in muscle. Decreases in fatty acid oxidation during refeeding after a fast have been attributed not only to decreases in free fatty acid (FFA), but also to increases in tissue malonyl CoA levels (9). After exercise, insulin sensitivity in muscle is improved, and the concentration of malonyl CoA in muscle is decreased (10). Recently, we have demonstrated in skeletal muscle of healthy humans an increase in malonyl CoA concentration during a hyperinsulinemic euglycemic clamp that is associated with decreases in plasma FFA and fat oxidation, as well as increases in tissue citrate and malate levels (11).
The present study was undertaken to examine the impact of both regional adiposity and physical fitness on insulin resistance in a well defined population of slightly overweight, diet-treated male type 2 diabetic patients and control subjects matched by sex, age, and body mass index (BMI; kilograms per square meter). For this purpose, we measured lean body mass (LBM), total and truncal fat mass by dual-energy x-ray absorptiometry (DEXA), intra-abdominal and sc fat areas by single-slice computer tomography at the L4–L5 level, and maximal oxygen uptake (VO2 max). In addition, we studied possible metabolic mechanisms responsible for insulin resistance with a special effort to assess the hypothesis that malonyl CoA levels are increased in relation to citrate and malate levels in the muscle of patients with type 2 diabetes compared with healthy controls. For this purpose, we determined plasma FFA levels, glucose and fatty acid oxidation, and muscle citrate, malate, and malonyl CoA levels before and during a hyperinsulinemic euglycemic clamp.
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Subjects and Methods |
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A total of 30 Swedish men were invited to participate in the study. Thirteen of them had mild type 2 diabetes and were recruited from our own clinic; the first 17 subjects from a large population without diabetes who fulfilled the inclusion criteria served as controls. Duration of diabetes was not more than 2 yr, and all patients were on diet treatment alone. Oral glucose tolerance was assessed using World Health Organization criteria (12). The two study groups were strictly matched by age, height, and BMI. Individuals who had a BMI between 23 and 32 kg/m2 were considered eligible for the study. None of the study subjects participated in a regular exercise program, and the two groups reported similar physical activity levels. Only three individuals had a BMI greater than 28 kg/m2. None of the subjects had chronic diseases (except diabetes); none were on lipid-lowering, antidiabetic, or antihypertensive medications; and none had a resting blood pressure in excess of 160/95 mm Hg. None of the control subjects had a family history of diabetes. All diabetic patients had been given dietary information before the studies. Both groups received written and oral information about the nature and potential risks of the study and gave their informed consent. The experimental protocol was approved by the Ethical Committee at the Karolinska Hospital.
Metabolic investigations were performed after a 12-h fast and started at 0800 h. All subjects were free of symptoms of infectious disease during the preceding 4 wk. The participants were asked to maintain their normal physical activity and diet during the 3 d before the study. All clinical tests and metabolic investigations in each subject were generally performed within a 4- to 6-wk period and were evenly distributed over the year. They were never performed the first day after a weekend or holiday.
Oral glucose tolerance test (OGTT)
Oral glucose tolerance and plasma insulin were assessed over 120 min during a 75-g OGTT (12). The insulin response was calculated as the incremental insulin area (above fasting insulin concentration) between 0–30 and 0–120 min.
Hyperinsulinemic euglycemic clamp
Sequential two-step hyperinsulinemic euglycemic clamps were performed after an overnight fast. At the onset of the experiment, a basilic vein of each arm was cannulated, one for sampling and the other for infusion. Both arms were put into a heated (50 C) sleeve. A third catheter was introduced into the cephalic vein of the arm used for infusions, for continuous sampling of arterialized blood for glucose measurement by a Biostator (Glucose-Controlled Insulin Infusion System, Miles Laboratories, Inc., Life Science Instruments, Elkhart, IN). The two levels of hyperinsulinemia of 150-min duration each were induced by iv infusion of 0.25 and 1.0 mU/kg body weight·min human rapid-acting insulin (Actrapid, Novo Nordisk A/S, Bagsværd, Denmark; 0.2 IU/ml with 4 mg/ml of human albumin in saline). Euglycemia was maintained by using a Biostator, which calculated glucose infusion rate from the reading of blood glucose measurements during the previous 4 min according to an algorithm (14). Potassium (0.15 mmol/g of glucose) was added to the infusate.
Indirect calorimetry
The Deltatrac II Metabolic Monitor (Datex-Ohmeda, Helsinki, Finland) was used to measure oxygen consumption and carbon dioxide production and from this to calculate the rate of fatty acid oxidation (for review, see Ref.15). For this purpose, 45 min before the two insulin infusion periods, a transparent plastic hood was placed over the subject’s head for 30 min to determine O2 consumption and CO2 production. Timed samples of urine were collected for analysis of urinary urea excretion, and from this, changes in urea pool size were calculated to correct for amino acid oxidation (16).
Body composition and physical fitness
LBM, total fat, and total truncal (abdominal and thorax regions) fat mass were calculated using DEXA (Lunar DPX-L x-ray bone densitometer, version 1.3Z, Lunar Corp., Madison, WI). Computerized tomography was used to determine intra-abdominal fat mass. All subjects were examined in the morning before the insulin clamp test. Siemens Somatom Plus (Siemens Corp., New York, NY) equipment was used. One 10-mm slice was exposed and examined at the level of the upper part of the iliac crest, which coincided with the disc between the fourth and fifth lumbar vertebrae and the umbilicus. A density range between -150 and -40 Houndsfield units was used to define fat mass. Total abdominal adipose tissue (sc plus intra-abdominal fat mass, measured in square centimeters) in this interval was calculated by the computer. The intra-abdominal cavity, including the retroperitoneal space, was outlined, and the area was estimated separately. Subcutaneous fat mass was calculated as the difference between total and intra-abdominal fat mass area. Total adipose tissue in this interval was calculated by the computer.
Physical fitness was determined as the VO2 max during an exercise test performed on an electrically braked cycloergometer. After a short period of exercise at 30 W, the load was increased in a stepwise manner by 30 W every minute until exhaustion or dyspnea. A 12-lead electrocardiogram was recorded during the test. Expired gases were measured for oxygen content, and calculations were performed by a Jaeger Oxycon 
computer software program (Jaeger, Hoechberg, Germany).
Muscle biopsies
Muscle biopsies (75–100 mg) were obtained from the vastus lateralis portion of the quadriceps femoris muscle using a Weil-Blakesley’s conchotome (17). Three biopsies were taken from each individual from the same area of the vastus lateralis: one at the end of the equilibration period, and the others at the end of the low-dose and high-dose insulin infusions. First, anesthetic [Xylocaine (lidocaine) 10 mg/ml] was given; then a small incision (1 cm) was made in the skin using a scalpel. A small cut was done in the muscle fascia to perform the biopsy without interfering with the fascia. All tissues were immediately frozen (within seconds) in liquid nitrogen and stored at -70 C for subsequent analysis. A trained nurse and a physician (P.N.B.) were present throughout the experiments.
Assays
Muscle was homogenized and deproteinized with 10% perchloric acid, and the filtrate was neutralized as described previously (18). Data were expressed per gram of muscle wet weight. Malonyl CoA was determined radioenzymatically in the neutralized filtrate by the method of McGarry et al. (7), citrate and malate were determined by standard spectrophotometric methods (19), plasma glucose was determined by the glucose oxidase method using a glucose analyzer (YSI, Inc., Yellow Springs, OH), and plasma insulin (20) and C-peptide (21) were determined by RIA using an antibody developed in this laboratory and a commercial kit (Novo Nordisk A/S, Bagsværd, Denmark), respectively. Interassay and intra-assay coefficients of variation were, respectively, less than 3.9% and less than 3.1% for insulin, and 4.5% and 3% for C-peptide. Cross-reactivity with proinsulin was 100% in the insulin assay and approximately 80% in the C-peptide assay. Very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein were determined by a combination of preparative ultracentrifugation and precipitation (22). Plasma nonesterified fatty acids (i.e. FFAs) were determined using a commercially available kit (Wako Pure Chemical Industries Ltd., Richmond, VA).
Statistical analyses
All values are presented as means ± SEM. Logarithmic transformation was performed on all skewed variables to obtain a normal distribution before statistical computations and significance testing were undertaken. When two sets of data were compared, a Student’s unpaired t test was used to evaluate statistical differences. A paired t test was used to determine the significance of the stepwise increase in malonyl CoA, citrate, and malate levels during clamps, and repeated measurements ANOVA was used to assess the significance of the increase in carbohydrate oxidation and the decrease in fatty acid oxidation. Scheffé test was used as the post hoc test. Regression analysis was performed to identify variables that correlated with M-values or VO2 max (adjusted for age). Multiple stepwise linear regression analysis was used to study independent determinants of M-values. The models have been checked by inspection of residuals. Partial correlation coefficients were calculated using age as a forced variable in the equations.
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Results |
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As indicated in Table 1
, the two groups were very closely matched by age and BMI. However, individuals with type 2 diabetes had an increased waist/hip ratio and total fat mass, the latter mainly due to increased truncal obesity. VO2 max per total body weight was decreased in diabetics compared with that in control subjects; however, the difference was not significant when VO2 max was calculated against kilograms of LBM. Plasma triglyceride concentration was increased in the patients compared with controls (2.0 ± 0.2 vs. 1.3 ± 0.2 mM; P = 0.02), but lipid content in lipoprotein subfractions obtained after ultracentrifugation was similar in the two groups (data not shown). The patients were mildly hyperglycemic and had fasting hyperinsulinemia. The early insulin response (0–30 min) and total incremental insulin release during an OGTT tended to be lower in the diabetic patients despite greater increases in plasma glucose concentration.
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Plasma insulin levels and rates of glucose infusion during the various stages of the hyperinsulinemic clamps are shown in Table 2. To maintain normoglycemia (plasma glucose, 5.1 mM) during the low-dose insulin clamp (0.25 mU/kg body weight·min), glucose had to be infused at a higher rate in the control than in the diabetic subjects (2.0 ± 0.2 vs. 0.7 ± 0.2 mU/kg body weight·min; P < 0.001; M-value). Corresponding M-values during the high-dose insulin infusion rate (1.0 mU/kg·min) were 8.3 ± 0.7 vs. 4.6 ± 0.4 mU/kg body weight·min (P < 0.001). These differences remained significant when calculated against kilograms of LBM.
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Plasma FFA levels and substrate oxidation rates
Under basal conditions, substrate oxidation rates and plasma FFA levels were similar in patients and control subjects; however, during the high-dose insulin clamp, fat oxidation rates and plasma FFA levels were significantly higher in the diabetic patients, and glucose oxidation rates were lower (Table 2
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Malonyl CoA
In rat muscle, insulin and glucose have been shown to increase the concentration of malonyl CoA by increasing the cytosolic concentration of citrate, an allosteric activator of acetyl CoA carboxylase (ACC), and the precursor of its substrate, cytosolic acetyl CoA (18). In this study, the concentrations of citrate, malate, and malonyl CoA were similar in the muscle of patients and control individuals under basal conditions (Table 3). During the high-dose insulin clamp, the increases in muscle citrate [controls, 31 nmol/g (P < 0.001) vs. patients, 15 nmol/g (P < 0.05)], malate [controls, 47 nmol/g (P < 0.001) vs. patients, 24 nmol/g (P < 0.001)], and the sum of citrate and malate [i.e. a presumed index of cytosolic citrate; controls, 77 ± 10 nmol/g (P < 0.001) vs. patients, 39 ± 10 nmol/g (P < 0.001)] in muscle were all less in the patients than in control subjects (Table 3). However, the increase in malonyl CoA concentration during the high-dose insulin clamp was nearly the same in the two groups [controls, 0.043 nmol/g (P < 0.001) vs. patients, 0.039 nmol/g (P = 0.02)]. Whole-body fatty acid oxidation rates during the clamps correlated inversely with malonyl CoA levels (r = -0.27; P < 0.001) and the sum of citrate plus malate (r = -0.59; P < 0.001) in all subjects (Fig. 1).
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Discussion |
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Obesity, in particular upper-body obesity, is a common finding in insulin-resistant states such as type 2 diabetes (23). Originally, it was proposed that this association was due to increases in visceral (intra-abdominal) adipose tissue (24, 25); however, others have suggested that it reflects more closely increases in upper-body sc adipose tissue (26, 27). In the present study, we demonstrate that truncal fat, representing both sc and intra-abdominal fat depots, was the strongest determinant of insulin sensitivity (M-value; r = -0.63; P < 0.001) and that, together with VO2 max (r = 0.58; P < 0.001), it explained 52% of the variation in M-values. Clausen et al. (28) found that estimates of body fat (measured with an impedance technique) and of maximal aerobic capacity explained approximately one third of the variation in an insulin sensitivity index in 380 population-based healthy Caucasian men. Our results do not allow us to evaluate the impact of truncal sc vs. intra-abdominal fat mass on insulin sensitivity.
Decreased physical fitness in patients with type 2 diabetes has been attributed to both genetic and environmental factors (29). Schneider et al. (5) demonstrated that people with type 2 diabetes have a decreased VO2 max compared with sedentary control subjects and that after 3 months of physical training their VO2 max remained lower than that of the control subjects who also trained for this period. In addition, Nyholm et al. (30) have shown that healthy first-degree relatives of type 2 diabetic patients have a decreased sensitivity to insulin and, on average, a 15% lower VO2 max than age- and BMI-matched control subjects with similar physical activity habits (assessed by questionnaire). They pointed out, as others had previously (31), that an alteration in skeletal muscle morphology and capillary density correlated with differences in insulin sensitivity between the two groups. These observations suggest that a low level of physical fitness antedates overt type 2 diabetes and could contribute to its development. VO2 max may be an insensitive indicator of physical activity in a free-living individual, however. Thus, Dvorak et al. (32) recently reported that young normal-weight and insulin-resistant women have increased total and visceral fat, compared with control women of similar weight and BMI but normal insulin sensitivity. They found no difference in VO2 max between the two groups; however, the insulin-resistant women had a 40% lower physical activity energy expenditure in their free-living time.
The cellular and molecular mechanisms responsible for insulin resistance in muscle are incompletely understood. It has been proposed that the combination of an increased level of FFA in plasma and malonyl CoA in muscle could play a causal role in a wide variety of situations, including obesity, type 2 diabetes and inactivity (33). An impaired ability of insulin and glucose to decrease plasma FFA levels such as described here (Table 2) has been described by Reaven (34) and his coworkers in insulin-resistant individuals. Malonyl CoA, by virtue of its inhibitory effect on carnitine palmitoyl transferase 1 activity, regulates the transfer of LCFA CoA into mitochondria where they are oxidized (35). Increases in the concentration of LCFA CoA in the cytosol of the muscle cell may thus be due to increases in malonyl CoA, an elevated plasma FFA level, or the two in combination. LCFA CoAs that are not oxidized to generate ATP may be incorporated in DAG and other glycerolipids. It has been proposed that this could lead to insulin resistance through activation of PKC isoforms and, subsequent to this, inhibition of insulin signaling and glycogen synthase (for review, see Ref.35). In support of this hypothesis are studies in animal models of type 2 diabetes in which increases in malonyl CoA and LCFA CoA levels and altered PKC distribution have been shown to parallel decreases in insulin action in muscle (8). In addition, Itani et al. (36) have recently reported that the insulin resistance produced in muscle of normal humans by increasing plasma FFA levels (by fat infusion plus heparin) during an euglycemic hyperinsulinemic clamp is accompanied temporally by increases in DAG mass and membrane-associated PKC activity. As already noted, malonyl CoA levels are increased in normal humans during such a clamp (11) and this, together with an increase in glycerol-3-phosphate, would favor fatty acid esterification at the expense of oxidation. A similar imbalance between fatty acid supply and oxidation, which leads to an increase in the de novo synthesis of DAG, has also been associated with the development of insulin resistance in cultured human umbilical vein endothelial cells (37).
We have recently demonstrated in normal men that an increase in the concentration of citrate plus malate in skeletal muscle during an insulin and glucose infusion (euglycemic-hyperinsulinemic clamp) is associated with a simultaneous increase in malonyl CoA concentration and a decrease in fatty acid oxidation (11). This strongly suggests that the malonyl CoA fuel sensing and signaling mechanism described in rats also operates in humans. The patients and control subjects in the present study had similar increases in the concentrations of malonyl CoA in muscle during the glucose and insulin infusion, despite an almost 2-fold greater increase in citrate plus malate in the control group (Table 3). Despite these comparable increases in malonyl CoA, the oxidation of FFAs was significantly greater in type 2 diabetic patients, a finding attributable to their higher plasma FFA levels during the clamps. As already noted, it has been proposed that this combination of increased plasma FFA and muscle malonyl CoA levels enhances intracellular fatty acid esterification, leading to increases in DAG content, protein kinase C activity, triglyceride accumulation, and insulin resistance (35). In the present study, intramyocellular lipids were not determined; however, there is ample evidence of a strong correlation between intramyocellular lipid accumulation and insulin resistance (38).
The reason for a higher than expected increase in malonyl CoA in muscle during the hyperinsulinemic clamp in the diabetic subjects is not clear. The formation of malonyl CoA is acutely regulated by AMP kinase (AMPK), which phosphorylates and inhibits the muscle isoform of ACC (ACC-ß or ACC2). Exercise has been shown to activate AMPK and diminish ACC-ß activity in human muscle (39, 40), and in the rat it appears to increase the activity of malonyl CoA decarboxylase (41). By both of these effects, exercise would decrease the concentration of malonyl CoA. It remains to be determined whether the relative inactivity of the diabetic patients in the present study contributed to their inappropriately high malonyl CoA levels during the clamp by causing AMPK activity to be diminished. Recent studies clearly demonstrate that pharmacological activation of AMPK by AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside; Ref.42), metformin (43), and rosiglitazone (44), improve insulin sensitivity. In addition, leptin was recently identified as a stimulator of fatty acid oxidation mediated by activation of AMPK and suppression of the activity of ACC in muscle (45).
In conclusion, insulin resistance in skeletal muscle of slightly overweight individuals with type 2 diabetes is strongly associated with decreased physical fitness and truncal obesity; however, the cellular mechanisms responsible for this association are not fully understood. The results of the present study suggest that a failure of insulin and glucose to suppress plasma FFA levels, together with an inappropriately high concentration of malonyl CoA in muscle, may be pathogenetic factors.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACC, Acetyl CoA carboxylase; AMPK, AMP kinase; BMI, body mass index; CoA, coenzyme A; DAG, diacylglycerol; DEXA, dual-energy x-ray absorptiometry; FFA, free fatty acid; LBM, lean body mass; LCFA, long chain fatty acyl; OGTT, oral glucose tolerance test; PKC, protein kinase C; VO2 max, maximal oxygen uptake.
Received March 4, 2002.
Accepted September 12, 2002.
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