INTRODUCTION

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THERE IS SOME EVIDENCE that the morphology of skeletal muscle is altered in the insulin-resistant state. Several research groups have observed a higher percentage of glycolytic, fast-twitch, white (type IIb) muscle fibers in individuals with non-insulin-dependent diabetes mellitus (NIDDM) compared with lean controls (9, 18). An increased percentage of type IIb muscle fibers was also evident in insulin-resistant, first-degree relatives of NIDDM patients (22). Other work indicates an increased expression of type IIb muscle fibers in conditions where insulin resistance is prevalent such as obesity, chronic heart failure (CHF), and NIDDM (2, 13, 14, 16, 17, 19, 31, 33, 35, 36). Together, these data suggest a relationship between the expression of type IIb muscle fibers and the insulin-resistant state.

In support of this relationship, 7 days of hyperinsulinemia increased the concentration of type IIb muscle fibers in the oxidative, slow-twitch, red (type I) soleus in rats (10). This finding, coupled with the observation that fasting and/or postprandial hyperinsulinemia is prevalent with insulin resistance (4), suggests that hyperinsulinemia may regulate the expression of type IIb muscle fibers. To test this hypothesis, we determined whether hyperinsulinemia (hyperinsulinemic-euglycemic clamp) influences myosin heavy chain gene expression (MHC type I, IIa, IIx mRNA) in human skeletal muscle. Hyperinsulinemia was induced with a 3-h hyperinsulinemic-euglycemic clamp, and the mRNA responses of the MHC isoforms were measured as an index of gene expression in muscle samples from healthy volunteers.

	METHODS

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Experimental design. Subjects were initially screened and tested for anthropometric characteristics to ensure that nonobese, healthy individuals were studied. Subjects refrained from exercise and alcohol consumption and consumed a diet containing a minimum of 250 g of carbohydrate/day in the 48 h preceding testing. Subjects reported at 0630 after a 12-h fast and rested in the supine position for 60 min. A muscle sample was obtained with the needle-biopsy technique from the vastus lateralis. Approximately 20 min after the biopsy, a 3-h euglycemic, hyperinsulinemic glucose clamp was initiated. After 3 h of hyperinsulinemia, muscle tissue was extracted from the contralateral leg. Muscle was subsequently analyzed for MHC mRNA isoform content (I, IIa, IIx).

Subjects. Subjects were 10 young [age 24.6 ± 1.0 (SE) yr] men (height 179.2 ± 1.9 cm, weight, 82.8 ± 3.8 kg). Inclusion criteria were no medications that could affect metabolism and normative body composition. Body composition was determined from seven skinfold sites (11) (11.9 ± 1.7% body fat) and body mass index (26.1 ± 1.1 kg/m²).

Hyperinsulinemic-euglycemic clamp. The hyperinsulinemic-euglycemic clamp was performed according to a modification of the method of DeFronzo et al. (5). An intravenous catheter was placed in an antecubital vein for infusion of insulin and glucose. Another catheter was placed retrograde in a dorsal hand vein for blood sampling. This hand was kept in a warming box at 70°C. Four samples were obtained at 10-min increments to determine fasting glucose and insulin concentration. A primed continuous infusion of insulin (Humulin, Eli Lily, Indianapolis, IN) at a dosage of 600 pmol · m² · min¹ was used; 4-min after the start of insulin infusion, a variable 20% glucose infusion was initiated. Blood samples were obtained every 5 min, centrifuged, and autoanalyzed for serum glucose (Glucose II Analyzer, Beckman, Fullerton, CA). Samples for insulin were obtained every 10 min and centrifuged, and plasma was stored at 70°C for subsequent analysis. Insulin concentration was determined by microparticle enzyme immunoassay (IMx, Abbott Laboratories, Abbott Park, IL). An M value, which is equal to the rate of whole body glucose disposal at the given insulin concentration, was calculated from the final 60 min of the clamp (5). A higher M value is indicative of enhanced insulin action, mainly in skeletal muscle (5). Because of a computer malfunction in storing the data, an M value was obtainable in 9 of the 10 subjects.

Muscle analysis. After the biopsy, muscle specimens were quick frozen and stored in liquid nitrogen. Total RNA was subsequently isolated by using TRIzol reagent (GIBCO-BRL, Gaithersburg, MD). Briefly, the tissue was homogenized in 1 ml of TRIzol. Chloroform (200 µl) was added, and the sample was vortexed vigorously for 15 s and incubated at room temperature for 5 min. Samples were microfuged for 15 min (12,000 g) at 4°C, and 400 µl of the top aqueous layer were transferred to a fresh microfuge tube. RNA was precipitated by the addition of equal volumes of isopropanol and incubated at room temperature for 10 min. Samples were microfuged for 10 min (12,000 g) at 4°C. RNA pellets were washed with 1 ml of 70% ethanol. After a brief air dry, pellets were resuspended in 50 µl of diethyl pyrocarbonate-treated water.

Statistics. Variables were compared with repeated measures analysis of variance at the P < 0.05 level. Post hoc comparisons were performed by using a Fisher's protected least significant differences test. Single-order Pearson product-moment correlations (P < 0.05) were used to examine relationships between given variables.

	RESULTS

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Euglycemic clamp. Fasting glucose and insulin concentrations were 5.1 ± 0.1 mmol/l and 39.0 ± 8.4 pmol/l, respectively. Mean glucose and insulin concentrations for minutes 20-180 of the clamp were 4.9 ± 0.1 mmol/l and 1,065.7 ± 9.8 pmol/l, respectively. Mean M value calculated from the final 60 min of the clamp was 52.2 ± 5.0 µmol · m² · min¹.

Skeletal muscle. As presented in Figs. 1 and 2, hyperinsulinemia produced a significant (P < 0.05) increase in type IIx MHC mRNA concentration in the vastus lateralis. When fasting MHC mRNA was assigned a value of 1.0 (Fig. 2), the relative change in type IIx MHC mRNA (1.44 ± 0.23) with hyperinsulinemia was significantly (P < 0.05) greater than for the other mRNA (GAPDH, 0.96 ± 0.09; MHC I, 0.92 ± 0.14; type IIa MHC, 1.08 ± 0.09). This difference could not be attributed to sampling from contralateral legs, becaused we have observed that type IIx MHC mRNA varied by only 6% between legs in similar subjects (n = 10) with no consistent pattern between dominant and nondominant limbs (data not shown). In contrast to studies in adipocyte and hepatoma cell lines (1, 20, 23), we observed no increase in GAPDH mRNA (0.28 ± 0.03 vs. 0.27 ± 0.01 arbitrary units, test-retest correlation of r = 0.76, P < 0.05) in human skeletal muscle with insulin. When normalized to GAPDH, hyperinsulinemia increased MHC IIx mRNA by 25% from a fasting value of 0.17 ± 0.02 to 0.22 ± 0.03 arbitrary units (P < 0.05). There were no changes in type I MHC (0.33 ± 0.02 vs. 0.30 ± 0.05 arbitrary units) and type IIa MHC (0.19 ± 0.01 vs. 0.20 ± 0.01 arbitrary units) with this normalization strategy. Type IIa MHC IIx mRNA increased in 7 of the 10 subjects with either normalization technique.

View larger version (83K): [in this window] [in a new window]   Fig. 1.   Detection and measurement of type IIx myosin heavy chain (MHC) mRNA in human skeletal muscle. Presented is a representative ribonuclease protection assay with 32P-labeled riboprobes, yielding a 122-bp protected fragment for human type IIx MHC mRNA and a 315-bp fragment for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Two micrograms of total RNA from each individual sample were incubated with GAPDH and type IIx MHC riboprobes, digested with ribonuclease, resolved on polyacrylamide gels, and identified by phosphorimager. Presented is a typical ribonuclease protection assay of type IIx MHC mRNA in the fasted, control state (lane C) and after 3 h of hyperinsulinemia (lane I) in 3 subjects. Type IIx MHC mRNA increased in 7 of the 10 subjects.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   mRNA for MHC isoforms in vastus lateralis in fasted state and after 3 h of hyperinsulinemia. A: normalized to GAPDH. B: relative change with fasting mRNA for each subject assigned a value of 1.0. * P < 0.05 compared with fasting value. ** P < 0.05 compared with relative changes for GAPDH and other MHC isoforms.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Relationships between rate of whole body glucose disposal at the given insulin concentration derived from clamp (M value) with relative percentage of type IIb muscle fibers (A) and type IIx MHC mRNA concentration (B) in vastus lateralis.

	DISCUSSION

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The main finding of the present study was that hyperinsulinemia rapidly increased the mRNA of the type IIx MHC isoform in human skeletal muscle. Because type IIx MHC mRNA is the predominant isoform in human type IIb muscle fibers (30, 32), our data support the findings of Holmang et al. (10) that 7 days of hyperinsulinemia increased the relative percentage of type IIb muscle fibers in rat soleus. The present data extend this finding (10) by demonstrating that MHC IIx mRNA increases with only 3 h of hyperinsulinemia in human skeletal muscle.

The MHC isoforms are thought to be primarily transcriptionally controlled (12, 25, 29, 30). Insulin can directly affect the rate of gene transcription and/or mRNA stability through various signaling mechanisms or indirectly control transcription through the suppression of plasma free fatty acid concentration or other factors (15, 20, 23). The cellular mechanism by which insulin could modify MHC mRNA content is not evident from our findings. Regardless, the physiological situation studied closely replicates the cellular milieu that the skeletal muscle cell is exposed to with hyperinsulinemia. Insulin may thus exert a rapid regulation at the transcriptional level for the type IIx MHC isoform, as with other genes (1, 20, 23). On the other hand, no changes in the type I or IIa MHC isoforms were apparent with this relatively acute stimulus (Fig. 2); the impact of more chronic hyperinsulinemia on these isoforms is not evident from the present findings.

Although some of the variance in fiber type between individuals can be genetically explained, the majority has been attributed to environmental factors (25, 29, 30). Data from the present study indicate that hyperinsulinemia provides, at least in part, a potential explanation for the increase of type IIb muscle fibers in conditions where either a fasting or postprandial hyperinsulinemia is present. This relationship has been proposed by others (10, 18, 21) but has not been directly tested in human skeletal muscle.

In the present study, MHC protein distribution was not measured because it was doubtful that a change would occur in such an acute period of time, due to the relatively long half-life of the MHC proteins (12, 25). In support of an eventual regulation at the protien level, an increased concentration of type IIb muscle fibers, which predominantly express MHC type IIx mRNA (32), has been reported in human skeletal muscle in the insulin-resistant states of obesity and NIDDM (2, 13, 14, 16, 17, 19, 31, 35, 36). An increase in type IIb fibers has also been reported with hyperinsulinemia in rodents (10). As more direct evidence, an increase in the proportion of type IIx MHC fibers has been reported in skeletal muscle (vastus lateralis) obtained from patients with CHF (33). Pronounced insulin resistance and hyperinsulinemia are evident with CHF (24, 34). Insulin has also been demonstrated to exert a regulatory role in controlling MHC isoform expression at the mRNA and protein levels in cardiac muscle (8).

Despite such evidence, we cannot definitely conclude from our data that muscle morphology is altered at the MHC protein level with hyperinsulinemia. For example, significant and rapid increases in GLUT-4 mRNA in human skeletal muscle were evident during insulin infusion (15, 28). GLUT-4 protein levels were not, however, altered in insulin-resistant/hyperinsulinemic conditions such as obesity or NIDDM (7). This indicates that protein expression is controlled at multiple levels with insulin exposure (20, 23), which may indeed also be the case with the type IIx MHC isoform. The present results are still, however, important in that they indicate that hyperinsulinemia exerts a rapid influence at the transcriptional level for the type IIx MHC protein. This effect, in combination with other mechanisms, would ultimately determine MHC IIx protein expression in human skeletal muscle.

The regulation of the MHC IIx isoform is potentially important because an increased expression could have an impact on functional capacity in insulin-resistant individuals. The contractile characteristics of the type IIb and IIx MHC protein are not energetically efficient and not conducive toward sustaining contractile activity (30). An increased expression of IIb and IIx fibers could thus be at least partially responsible for the reduced exercise tolerance reported in male and female NIDDM patients and their insulin-resistant relatives (21, 27).

In conclusion, these data indicate that hyperinsulinemia rapidly increases type IIx MHC mRNA in human skeletal muscle. Insulin may thus contribute, at least in part, to regulating MHC gene expression.