Reduced Skeletal Muscle Oxygen Uptake and Reduced β-Cell Function: Two early abnormalities in normal glucose-tolerant offspring of patients with type 2 diabetes

Subjects were recruited from the ongoing Tübingen Family Study for Type 2 Diabetes (TÜF). The Tübingen Family Study recruits family members of subjects with type 2 diabetes. Over the years, subjects without any family history were asked to undergo metabolic checkup. This group was used as a control group in the present study. A total of 424 healthy subjects with normal glucose tolerance according to World Health Organization criteria were selected for analyses. In a total of 185 subjects (77 control subjects and 75 first-degree relatives [FDRs]), insulin sensitivity data from euglycemic-hyperinsulinemic clamp were available for analyses.

After the nature of the study was explained, all subjects gave informed written consent and underwent the standard procedures of the protocol, including medical history, physical examination, routine blood test, and oral glucose tolerance test (OGTT). The local ethics committee approved all protocols.

Blood glucose was measured using a bedside glucose analyzer (glucose oxidase method; Yellow Springs Instruments, Yellow Springs, CO). Serum insulin was determined with a micro particle enzyme immunoassay (Abbott, Wiesbaden, Germany). Serum C-peptide concentrations were determined by radioimmunoassay (Byk-Sangtec Diagnostika, Dietzenbach, Germany).

Lean body mass (kg) and total body fat (%) was determined by bioimpedance analysis (BIA-101; RJL Systems) following the guidelines of the user’s manual and the National Institutes of Health Consensus Conference on Bioelectric Impedance (16).

Family history status was inquired during medical history. Only subjects with profound knowledge of their family history status were included in this study.

All subjects completed a standardized self-administered and validated questionnaire to measure physical activity (17). Points are assigned for physical activity at work (work index), at sport during leisure time (sport index), and during leisure time excluding sport (leisure time index). The habitual physical activity (HPA) score was calculated as mean points of the three indexes.

All subjects underwent a continuous, incremental exercise test to volitional exhaustion using a cycle ergometer. The cycle ergometer test was performed on an electromagnetically braked cycle ergometer (Ergometrics 800 S; Ergoline, Bitz, Germany). Oxygen consumption was measured using a spiroergometer (MedGraphics System Breese Ex 3.02 A; MedGraphics). Subjects were asked to choose a pedaling rate of 60 rpm and to maintain that rate throughout the test. After a 2-min warm-up period at 0 W, the test was initiated at an initial power output of 20 W. Stepwise increments of 40 W were made every minute until exhaustion. Vo_2max is expressed as Vo₂ (ml/min) per kg lean body mass.

At approximately 7:00 a.m., after a 12-h overnight fast, an antecubital vein was cannulated for infusion of insulin and glucose. A dorsal hand vein of the contralateral arm was cannulated and placed under a heating device to allow sampling of arterialized blood. After basal blood was drawn, subjects received a primed insulin infusion at a rate of 1.0 mU · kg⁻¹ · min⁻¹ for 2 h. Blood was drawn every 5 min for determination of blood glucose, and a glucose infusion was adjusted appropriately to maintain the fasting glucose level. An insulin sensitivity index (ISI) (in μmol · kg⁻¹ · min⁻¹ · pmol⁻¹) for systemic glucose uptake was calculated as the mean infusion rate of glucose (GIR; in μmol · kg⁻¹ · min⁻¹) necessary to maintain euglycemia during the last 60 min of the euglycemic-hyperinsulinemic clamp divided by the steady-state serum insulin concentration.

Insulin sensitivity during OGTT was calculated by the formula of Matsuda and DeFronzo (18), as previously described.

First-phase insulin release during OGTT was calculated using the formula of Stumvoll et al. (19).

A disposition index was calculated by multiplying insulin sensitivity with insulin secretion. The importance of this index in assessing β-cell function was recently reviewed by Bergman et al. (20).

All data are given as mean ± SE unless otherwise stated. The statistical software package JMP (SAS Institute, Cary, NC) was used for statistical analyses. A P value <0.05 was considered statistically significant. Non-normally distributed parameters were log-transformed before statistical analyses. FDRs were compared with the control group using Student’s t test. Comparisons between all groups were performed by one-way ANOVA.

FDRs of type 2 diabetic patients were significantly older than the control group without FHD or second-degree relatives. The groups were comparable with respect to BMI, percent body fat, and WHR. Fasting and 2-h blood glucose levels during OGTT were significantly higher in the FDR group compared with the control group (Table 1). This effect was still significant after adjusting for age and percent body fat (P = 0.0045 for fasting and P = 0.0047 for 2-h glucose). Characteristics of the subjects who also underwent a euglycemic-hyperinsulinemic clamp were similar to those of the OGTT group.

Maximal oxygen consumption during incremental exercise on a bicycle ergometer was higher in men than in women (48.5 ± 0.9 vs. 39.3 ± 0.4 ml · kg lean body mass⁻¹ · min⁻¹, P < 0.001). Habitual physical activity was positively correlated to maximal oxygen consumption (r = 0.24, P < 0.001) and insulin sensitivity (clamp) (r = 0.23, P < 0.001). Furthermore, maximal oxygen consumption was correlated to insulin sensitivity (clamp) (r = 0.42, P < 0.001). Age (r = −0.29, P < 0.001), body fat content (r = −0.41, P < 0.001) and 2-h glucose (r = −0.18, P < 0.0001) were negatively correlated with maximal oxygen consumption (Fig. 1).

Maximal oxygen consumption was highest in the control group without family history of T2DM (45.2 ± 0.9 ml · kg lean body mass⁻¹ · min⁻¹) and significantly lower in second- and first-degree relatives of type 2 diabetic patients (42.7 ± 0.9 and 40.5 ± 0.6, P < 0.001, ANOVA). In contrast, habitual physical activity was comparable in all groups (8.2 ± 0.1 in control subjects versus 8.3 ± 0.1 in second-degree relatives versus 8.1 ± 0.1 in first-degree relatives, P = 0.27, ANOVA) (Fig. 2). In a multivariate regression analysis with maximal oxygen consumption as a dependent variable, the effect of family history status remained significant (P = 0.04) after adjusting for the effects of sex (P < 0.001), age (P = 0.11), BMI (P = 0.06), HPA (P < 0.001), and insulin sensitivity (clamp) (P = 0.01) (see Table 2). The results were similar using an insulin sensitivity estimate from the OGTT (P = 0.05 for the effect of family history status).

Insulin sensitivity (clamp) tended to be lower in FDRs compared with the control group (0.119 ± 0.107 vs. 0.100 ± 0.087 μmol · kg⁻¹ · min⁻¹ · pmol⁻¹, P = 0.051). This marginal effect of family history on insulin sensitivity disappeared completely after adjusting for sex, age, BMI, and HPA in a multivariate analysis (P = 0.87) (see Table 2). Similarly, family history status had no significant effect on insulin sensitivity estimated in the larger OGTT group after adjusting for the same covariates (P = 0.14). Moreover, no differences in first-phase insulin secretion between the control group and FDRs were observed (1,051 ± 43 vs. 992 ± 35, P = 0.28 and P = 0.30 after adjusting for sex, age, and BMI).

The calculated disposition index (ISI_clamp × Secretion_OGTT), however, was significantly higher in control subjects compared with FDRs (104 ± 6 vs. 87 ± 4 units, P = 0.02), indicating reduced insulin secretion relative to insulin sensitivity in FDR subjects. This relationship remained significant (P = 0.046) after adjusting for the effects of sex, age, and BMI (see Table 2).

The disposition index purely derived from OGTT was also significantly higher in control subjects than in FDRs (22,468 ± 883 vs. 18,291 ± 874, P < 0.001). This relationship remained significant (P = 0.02) after adjusting for sex, age, and BMI (Fig. 3).

The aim of the present study was to identify early metabolically relevant abnormalities predisposing to type 2 diabetes in FDRs of patients with the disease. There was a trend for lower insulin sensitivity in FDRs who also tended to be somewhat more obese and older. After adjusting, however, insulin sensitivity became comparable between the groups, indicating that most of the original trend was secondary to differences in fatness. Insulin secretion assessed by a validated index based on fasting and 30-min insulin and glucose concentrations during an OGTT was also comparable among the patients with FHD. However, insulin secretion relative to insulin sensitivity (expressed as the so-called disposition index) was significantly lower in FDR subjects. This is in line with results of previous studies identifying reduced β-cell compensation to insulin resistance in individuals with FHD (21–24).

An important finding of the present study is that maximal oxygen consumption was reduced in FDRs despite essentially identical physical activity scores (derived from a questionnaire). The fact that this difference was observed in the absence of any detectable difference in insulin sensitivity is the most interesting aspect but represents somewhat of a pathophysiological quandary. That is, how would an abnormality in oxidative muscle metabolism during exercise ultimately affect glucose metabolism if not via peripheral (i.e., muscle in the insulin-stimulated state) glucose uptake? There are two possible conjectures: first, our measurement of peripheral insulin sensitivity is too unphysiologic (steady-state insulin concentration during the clamp ∼400 pmol/l) to detect subtle abnormalities that translate into fasting or 2-h glucose concentrations manifest only at physiologic insulin levels. Second, our measurement of insulin sensitivity may be too crude and not sufficiently specific for skeletal muscle. Variability in insulin-suppression of endogenous glucose production, which is also measured by the glucose infusion rate during the clamp, may offset an isolated muscle abnormality on the whole-body level.

It is important to emphasize that although the study population had normal glucose tolerance, the abnormal subphenotypes somehow impacted fasting and 2-h glucose levels, which were significantly higher in FDRs. The lower disposition is probably the most relevant factor responsible for the differences in glycemia. Nevertheless, mechanism secondary to maximal oxygen consumption but outside the diagnostic scope of the euglycemic-hyperinsulinemic clamp may also be involved.

Previously, reduced maximal oxygen uptake was demonstrated in insulin-resistant relatives of patients with T2DM (12). In our study, maximal oxygen uptake was a strong determinant of insulin sensitivity but, in contrast to oxygen uptake, insulin sensitivity was not different between the patients with FHD. These findings are difficult to reconcile, but the observation that in patients with overt type 2 diabetes, resistance training improves insulin sensitivity without altering Vo_2max (25) in principle supports the possibility of a dissociation between effects of exercise and insulin sensitivity on skeletal muscle. Conceivably, reduced maximal oxygen consumption of skeletal muscle represents a primary defect predisposing skeletal muscle to insulin resistance when other confounding factors (e.g., physical inactivity) come into play.

The relative content of type 1 fibers in skeletal muscle was identified as a main determinant of skeletal muscle oxygen uptake during exercise (14,15). Type 1 muscle fibers are slow-twitch muscle fibers with preferentially oxidative metabolism rich in myoglobin, mitochondria, oxidative enzymes, and lipids (26). Therefore, the mechanism underlying our observation is likely to involve altered fiber composition of skeletal muscle, specifically a reduced number of type 1 fibers, which was found to be associated with adiposity and insulin resistance (27). Additionally, a high proportion of type 1 muscle fibers has been shown to protect against weight gain in response to a long-term positive energy balance (28). Moreover, fiber type proportions of skeletal muscle have been demonstrated to be genetically determined (29). In accordance with all these findings, the number of type 2 muscle fibers were found to be reduced in FDRs of patients with T2DM (13).

It is also necessary to point out that use of questionnaires in assessing habitual physical activity cannot be without problems. The same quantity or quality of physical exercise may seem more or less strenuous to different individuals. However, it is unlikely that the degree of underreporting is influenced by family history status. Therefore, and in view of the large number of subjects reported herein, our overall conclusions should remain valid.

Finally, this study is not merely another report of prediabetic subphenotypes in subjects with and without FHD. Instead, this study presents novel aspects that complement previously published work. First, by including every subject in the database and performing ANCOVA rather than matching and excluding “un-matchable” individuals, we largely avoided any selection bias. Second, we present Vo_2max data in addition to insulin sensitivity and demonstrate that there may be dissociation between skeletal muscle glucose and exercise metabolism, which implies the possibility that only in subgroups does skeletal muscle have a primary role in the pathogenesis of T2DM. Third, we found abnormalities in both β-cells and insulin sensitivity tissue but only conditionally, i.e., using the disposition index or measuring skeletal muscle oxidative capacity. This clearly demonstrates the subtlety of the influence of FHD on diabetes-relevant traits and may help to reconcile conflicting data and some ongoing debates.

In summary, we have demonstrated two metabolic abnormalities in a young and healthy group of subjects with a familial predisposition of type 2 diabetes: reduced insulin secretion relative to insulin sensitivity and reduced maximal oxygen consumption during exercise, which was not the result of lack of exercise.

This study was supported by a research grant from the Deutsche Forschungsgemeinschaft (Fr 1561/1–1) and the European Community (QLRT-1999–00674). M.S. is currently supported by a Heisenberg grant of the Deutsche Forschungsgemeinschaft.

We thank all the research volunteers for their participation and Elke Maerker, Anna Teigeler, and Heike Luz for their excellent technical assistance.