To elucidate the molecular mechanisms behind physical inactivity–induced insulin resistance in skeletal muscle, 12 young, healthy male subjects completed 7 days of bed rest with vastus lateralis muscle biopsies obtained before and after. In six of the subjects, muscle biopsies were taken from both legs before and after a 3-h hyperinsulinemic euglycemic clamp performed 3 h after a 45-min, one-legged exercise. Blood samples were obtained from one femoral artery and both femoral veins before and during the clamp. Glucose infusion rate and leg glucose extraction during the clamp were lower after than before bed rest. This bed rest–induced insulin resistance occurred together with reduced muscle GLUT4, hexokinase II, protein kinase B/Akt1, and Akt2 protein level, and a tendency for reduced 3-hydroxyacyl-CoA dehydrogenase activity. The ability of insulin to phosphorylate Akt and activate glycogen synthase (GS) was reduced with normal GS site 3 but abnormal GS site 2+2a phosphorylation after bed rest. Exercise enhanced insulin-stimulated leg glucose extraction both before and after bed rest, which was accompanied by higher GS activity in the prior-exercised leg than the rested leg. The present findings demonstrate that physical inactivity–induced insulin resistance in muscle is associated with lower content/activity of key proteins in glucose transport/phosphorylation and storage.

Lifestyle-related diseases like type 2 diabetes are rapidly increasing worldwide, and there is strong evidence that physical inactivity contributes to this development (1). The prediabetic and diabetic states are characterized by peripheral insulin resistance (2), and due to the large mass of skeletal muscle, the insulin action in this tissue has major influence on whole-body insulin sensitivity, especially in lean adults (3). Insulin sensitivity in skeletal muscle is to a large extent a dynamic parameter that can be enhanced by exercise training and decreased by physical inactivity (47). Thus, research attempting to combat diseases related to insulin resistance has focused on mechanisms by which the sensitivity of the glucose uptake to insulin is regulated.

Type 2 diabetes patients with peripheral insulin resistance have impaired GLUT4 translocation in skeletal muscle (8). Moreover, muscle insulin–resistant individuals and type 2 diabetes patients have impaired insulin-stimulated Akt Thr308 and Ser473 phosphorylation (911), lower insulin-stimulated Akt substrate of 160 kDa (AS160/TBC1D4) phosphorylation on multiple sites (7,911), and impaired insulin-stimulated glycogen synthase (GS) activity (9). The impaired GS activity in type 2 diabetes patients is associated with hyperphosphorylation of GS site 2+2a during insulin stimulation, whereas GS site 3a phosphorylation appears to be unaffected (9,12), indicating that GS phosphorylation downstream of Akt is maintained despite lower Akt phosphorylation. Exercise training has been shown to enhance the capacity for glucose transport and glycogen storage in skeletal muscle by increasing GLUT4, hexokinase II (HKII), and TBC1D4 protein content as well as GS activity in both healthy and type 2 diabetic patients, and these adaptations may contribute to the training-induced improvements in insulin sensitivity (1315). In addition, the improved insulin sensitivity observed after exercise training has been reported to be associated with improved, and in fact normalized, insulin-induced phosphorylation of TBC1D4 in type 2 diabetes (7). In line with the changes observed with training, physical inactivity has been demonstrated to induce insulin resistance (5,16). However, to the best of our knowledge, no study has reported the effects of physical inactivity on key proteins in glucose metabolism in human skeletal muscle.

Muscle insulin sensitivity has been shown to be increased in the period after a single bout of exercise (17). Thus, enhanced insulin-mediated glucose clearance is observed for up to 48 h after a single exercise bout (1719). It has been shown in rat skeletal muscle that the increased insulin sensitivity to glucose uptake is associated with an increased GLUT4 translocation to the plasma membrane (20). Moreover, multiple studies have documented that insulin-mediated activation of the most proximal insulin signaling elements (e.g., insulin receptor, insulin receptor substrate, phosphatidylinositol-3 kinase, and Akt) is not enhanced after a single bout of exercise (2123). Interestingly, recent studies have revealed that basal and insulin-induced TBC1D4 phosphorylation are increased in the period after a single bout of exercise (24,25), although unchanged TBC1D4 phosphorylation has also been reported, but in a study not detecting increased glucose uptake after exercise (26). Studies have also reported an increased GS activity 3–4 h after exercise (27), and thus a combination of increased phosphorylation of TBC1D4 (regulating glucose transport) and increased GS activity (regulating glycogen synthesis) may explain the increased sensitivity of insulin to stimulate glucose uptake 3–4 h after a single bout of exercise.

Knowing the underlying molecular mechanisms behind physical inactivity–induced insulin resistance and the impact of physical inactivity on exercise-induced insulin sensitivity may provide the basis for developing pharmaceutical agents and interventions that can counteract the development of insulin resistance in skeletal muscle during bed rest after surgery/illness as well as induced by a physically inactive lifestyle. The aim of the current study was to test the hypotheses that 1) physical inactivity–induced insulin resistance in human skeletal muscle is due to dysregulation of both GS and TBC1D4 and 2) exercise-induced enhancement of insulin sensitivity is lost with physical inactivity.

Subjects.

Twelve young, healthy male subjects participated in the study (Table 1). The subjects were physically active, as they all walked or bicycled for >1 h/day. The subjects were given both oral and written information about the experimental protocol and procedures as well as the discomfort they might experience during the experiment. After having received this information, the subjects gave their written consent. The study was performed according to the Declaration of Helsinki and was approved by the Copenhagen and Frederiksberg Ethics Committee in Denmark (H-C-2007-0085).

TABLE 1

Subject characteristics before and after bed rest

Subject characteristics before and after bed rest
Subject characteristics before and after bed rest

Bed rest.

The subjects were placed in bed for 7 days and were given information about the importance of not moving their legs. All transport of subjects took place in wheelchairs and subjects were told to sit down during showers. The subjects were encouraged to sit in the wheelchair for up to 5 h per day to avoid vascular complications. The daily energy intake during the bed rest came from meals containing 10–20% protein, 50–60% carbohydrates, and 25–35% fat. The meals were served ad libitum.

Physical performance and body composition.

Twelve subjects were included in the bed rest part (Table 1). Six of these subjects were chosen randomly to follow a study protocol involving one-legged exercise and a hyperinsulinemic euglycemic clamp. The characteristics of these subjects are given in brackets in Table 1.

Approximately 2 weeks before the bed rest, maximal oxygen uptake (Vo2max) was determined by an incremental test on a cycle ergometer (Monarch Ergomedic) for all 12 subjects. Body composition and body weight were determined by dual-emission X-ray absorptiometry scanning (GE Healthcare). The Vo2max test and the dual-emission X-ray absorptiometry scan were repeated at the end of the bed rest period. In addition, the six subjects participating in the exercise and clamp protocol performed an incremental one-legged knee extensor exercise test to exhaustion on a modified cycle ergometer bike (Monarch Ergomedic) to determine Wattmax as previously described (28).

Experimental protocol.

The day before the experimental day (both before and after the bed rest period), the subjects consumed a prepackaged, standardized meal and evening snack regulated for body weight (14.3 kcal/kg and 2.9 kcal/kg, respectively). On the experimental day, the subjects arrived to the laboratory in the morning. Catheters were placed in the femoral vein of each leg and in one femoral artery. Three and a half hours after consuming a standardized breakfast (2.9 kcal/kg), the subjects completed 45 min of one-legged knee extensor exercise at 60% of Wattmax. This relatively low intensity was chosen to ensure that the subjects would be able to complete the exercise bout after the bed rest. Three hours into recovery, a 3-h euglycemic hyperinsulinemic clamp with a constant rate of insulin infusion (50 mU/[min · m2]) (Actrapid; Novo Nordisk, Bagsvaerd, Denmark) was performed. Arterial blood samples were obtained every 5–10 min for immediate determination of plasma glucose concentration (Radiometer ABL 725 series Acid-Base Analyser). Euglycemia was maintained by subsequent adjustment of the glucose infusion rate (GIR). The duration of 3 h was chosen to approach clamp steady-state conditions at the sampling time points.

Muscle biopsies were obtained from the vastus lateralis of both the previously exercised (Ex leg) and the rested leg (Rest leg) both before (B) and after (I) the clamp using the needle biopsy technique (29) with suction. Incisions for biopsies were made under local anesthesia (lidocaine; AstraZeneca, Södertälje, Sweden) and individual incisions were used for each biopsy. The biopsies were quickly frozen in liquid nitrogen (within 10–20 s) after removing visual blood and connective tissue.

Before as well as at 2, 2.5, and 3 h of the insulin clamp, blood samples were drawn from the three femoral catheters and placed in EDTA-treated tubes. Plasma was collected by centrifugation and stored at −80°C.

Plasma analyses.

The femoral arterial and venous plasma glucose concentrations were measured using an acid-base analyzer (Radiometer ABL 725 series Acid-Base Analyser), and arterial insulin concentration was measured by ELISA (K6219; DAKO, Glostrup, Denmark). Glucose extraction across the leg was calculated as the difference between the glucose concentration in the arterial and venous plasma samples. The plasma palmitate concentration was determined by gas chromatography (30)

Muscle samples were freeze dried for >48 h. Visible blood, fat, and connective tissue were removed under a microscope.

Glycogen was measured as glycosyl units after acid hydrolysis using an automatic spectrophotometer as previously described (31,32).

Enzyme activities.

The activity of 3-hydroxyacyl-CoA dehydrogenase (HAD) and citrate synthase (CS) was analyzed spectrophotometrically as previously described (32). The GS activity was determined in muscle homogenates as previously described (22,33).

SDS-PAGE and Western blotting.

Muscle homogenate, muscle lysate, and protein concentration determination were prepared as previously described (25,34).

The protein or phosphorylation of β-actin, HKII, GLUT4, Akt, GS kinase 3β (GSK-3β), and TBC1D4 in muscle lysates and GS protein and protein phosphorylation in muscle homogenates were measured by SDS-PAGE and Western blotting (25,34). Primary polyclonal antibodies against β-actin (4967; Cell Signaling Technology, Beverly, MA), HKII (2867; Cell Signaling Technology), GLUT4 (ABR-PAI1065; Thermo Scientific, Golden, CO), Akt1 and Akt2 (2967 and 3063, respectively; Cell Signaling Technology), GSK-3α and -3β (9338 and 9315, respectively; Cell Signaling Technology), and TBC1D4 protein (ABCAM, Cambridge, U.K.) as well as against protein phosphorylation of Akt Thr308 (05-669; Upstate Biotechnology, Lake Placid, NY) and Ser473 (05-802; Millipore, Bedford, MA), GSK-3α Ser21 and GSK-3β Ser9 (9331 and 9323, respectively; Cell Signaling Technology), TBC1D4 Ser588 and Thr642 (3028-P2 and 3028-P1, respectively; Symansis, Auckland, N.Z.), and TBC1D4 Ser751 (provided by Graham Hardie, Dundee University, Dundee, U.K.) were used. Primary polyclonal antibodies against GS protein (provided by Oluf Pedersen, University of Copenhagen) and phosphospecific antibodies against 2+2a, 1b (provided by Graham Hardie), and 3a (3891; Cell Signaling Technology) were also used. Membranes probed with phosphospecific antibodies were stripped to allow for determination of the level of the corresponding protein as previously described (25).

Statistics.

Values presented are means ± SE. Two-way ANOVA with repeated measures was applied to evaluate the effect of exercise and insulin before as well as after bed rest. Also, a two-way ANOVA with repeated measures was used to evaluate the effect of bed rest and insulin within the rested leg as well as within the exercised leg. The data were log transformed if normality or equal variance tests failed. If significant main effects were found, the Student-Newman-Keuls post hoc test was used to locate differences. Differences were considered significant at P < 0.05. A tendency is reported for 0.05 ≤ P < 0.1. Statistical calculations were performed using SigmaStat version 3.1.

Physical characteristics and performance.

Total body weight and BMI were unaffected by 7 days of bed rest (Table 1). Leg muscle mass was reduced 3 ± 0.7% (P < 0.05), whereas whole body fat did not change significantly with bed rest (Table 1). Seven days of bed rest reduced whole body Vo2max 5 ± 1.4% (P < 0.05) (Table 1).

Protein content and enzyme activity.

Skeletal muscle HAD activity tended to decrease 8 ± 4.7% (0.05 ≤ P < 0.1) by bed rest, whereas CS activity did not change significantly (Table 2). The protein content of HKII (66 ± 15%), GLUT4 (74 ± 14%), Akt1 (56 ± 6%), and Akt2 (68 ± 7%) was reduced after bed rest relative to before (P < 0.05), whereas the protein level of TBC1D4 and GS and GS total activity were unchanged with bed rest (Fig. 1 and Table 2).

TABLE 2

Muscle protein content and activity before and after bed rest

Muscle protein content and activity before and after bed rest
Muscle protein content and activity before and after bed rest
FIG. 1.

Representative blots showing GLUT4, HKII, Akt1, Akt2, TBC1D4, and GS protein before and after 7 days of bed rest in rested (Rest) and prior-exercised (Ex) leg for the same subject. Although the average Akt2 protein content of all subjects is reduced with bed rest (Table 2), it may be noted that the abundance of Akt2 is not reduced with bed rest in the subject used for the representative blots. kD, kilodaltons.

FIG. 1.

Representative blots showing GLUT4, HKII, Akt1, Akt2, TBC1D4, and GS protein before and after 7 days of bed rest in rested (Rest) and prior-exercised (Ex) leg for the same subject. Although the average Akt2 protein content of all subjects is reduced with bed rest (Table 2), it may be noted that the abundance of Akt2 is not reduced with bed rest in the subject used for the representative blots. kD, kilodaltons.

Plasma insulin.

The average plasma insulin concentration before exercise, immediately after, and 3 h into recovery was not significantly different before and after bed rest, with an average of 32 ± 15 mmol/L before and 74 ± 20 mmol/L after bed rest. However, during the last hour of the clamp, plasma insulin concentration tended to be higher after (452 ± 66 pmol/L) than before (341 ± 30 pmol/L) bed rest (0.05 ≤ P < 0.1) (Table 3).

TABLE 3

Plasma concentrations before and after bed rest

Plasma concentrations before and after bed rest
Plasma concentrations before and after bed rest

Plasma palmitate.

The plasma palmitate concentration was unaffected by bed rest both before and after the insulin clamp. Insulin reduced the plasma palmitate concentration both before and after bed rest (P < 0.05).

Plasma glucose and GIR.

The arterial plasma glucose concentration during the clamp was similar, 4.9 ± 0.1 mmol/L before and after bed rest. The GIR did not reach a steady state even after 180 min (Fig. 2A). At all time points after 50 min, GIR was higher before than after bed rest (P < 0.05). During the last hour of the clamp, GIR after bed rest was 75 ± 12% of the level before bed rest (P < 0.05) (Fig. 2A).

FIG. 2.

A: GIR during 3 h of hyperinsulinemic euglycemic clamp initiated 3 h after 45 min of one-legged knee extensor exercise before and after 7 days' bed rest. B: Arterio-venous (a-v) glucose difference across rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis during 3 h of hyperinsulinemic euglycemic clamp performed 3 h after 45 min of one-legged knee extensor exercise before and after bed rest. Samples were obtained before (B) as well as during (I) the clamp, presented here by the accumulated data during the last hour of the clamp (2–3 h). C: Muscle glycogen concentration (mmol/kg dry weight [dw]) in rested and prior-exercised leg before (B) and after (I) 3 h of hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after bed rest. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. (†)Tendency, 0.05 ≤ P < 0.1.

FIG. 2.

A: GIR during 3 h of hyperinsulinemic euglycemic clamp initiated 3 h after 45 min of one-legged knee extensor exercise before and after 7 days' bed rest. B: Arterio-venous (a-v) glucose difference across rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis during 3 h of hyperinsulinemic euglycemic clamp performed 3 h after 45 min of one-legged knee extensor exercise before and after bed rest. Samples were obtained before (B) as well as during (I) the clamp, presented here by the accumulated data during the last hour of the clamp (2–3 h). C: Muscle glycogen concentration (mmol/kg dry weight [dw]) in rested and prior-exercised leg before (B) and after (I) 3 h of hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after bed rest. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. (†)Tendency, 0.05 ≤ P < 0.1.

Glucose extraction.

Glucose extraction was evaluated during the last hour as the arterio-venous concentration difference across the leg. It has previously been reported that glucose extraction increased only in the exercising leg during the one-legged exercise (35). Three hours after exercise, glucose extraction across the leg was similar in the exercised and rested leg, reflecting that the acute exercise effect on glucose extraction was fully diminished at 3 h of recovery from exercise (corresponding to pre-insulin) (Fig. 2B). This was seen both before and after bed rest. Insulin infusion increased glucose extraction both in the prior-exercised and the rested leg, both before and after bed rest (Fig. 2B). During the last hour of the clamp before bed rest, glucose extraction in the rested leg was 11-fold (P < 0.05), and the prior-exercised leg sixfold (P < 0.05), higher after insulin than at basal. After bed rest, glucose extraction was six- and sevenfold (P < 0.05) higher after insulin than at basal in the rested and prior-exercised leg, respectively. The insulin-stimulated glucose extraction in the rested leg after bed rest was 78 ± 10% (P < 0.05) of the level before bed rest. Also, insulin-stimulated glucose extraction before bed rest was 1.6 ± 0.2-fold (P < 0.05), and after bed rest 1.9 ± 0.2-fold (P < 0.05), higher in the prior-exercised than in the rested leg (Fig. 2B).

Muscle glycogen.

Three hours after exercise, muscle glycogen content was ∼20% (P < 0.05) lower in the prior-exercised than the rested leg. This was observed both before and after bed rest. There was no change in the muscle glycogen level in response to insulin infusion. Bed rest per se tended to enhance the muscle glycogen content ∼20% (0.05 ≤ P < 0.1) in the rested leg (Fig. 2C).

Muscle signaling

Akt phosphorylation.

Akt Thr308 phosphorylation was similar in the rested and exercised leg at 3 h of recovery both before and after bed rest. Insulin infusion increased Akt Thr308 phosphorylation similarly four- to fivefold in both legs before and after bed rest. No effect of prior exercise was observed on insulin-induced Akt phosphorylation. However, bed rest decreased Akt Thr308 phosphorylation 20–30% (P < 0.05) after the clamp (Fig. 3A and C). This decrease was observed in both the prior-exercised and rested leg. A similar phosphorylation pattern was observed for Akt Ser473 (data not shown). Thus, the insulin resistance induced by bed rest was associated with decreased signaling through Akt, even after accounting for decreased Akt2 protein expression (Table 2).

FIG. 3.

Akt Thr308 phosphorylation (A), GSK-3β Ser9 phosphorylation (B), and representative blots (C) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. (#)Tendency, 0.05 ≤ P < 0.1.

FIG. 3.

Akt Thr308 phosphorylation (A), GSK-3β Ser9 phosphorylation (B), and representative blots (C) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. (#)Tendency, 0.05 ≤ P < 0.1.

GSK-3 phosphorylation.

The phosphorylation pattern of Akt target sites (GSK-3α Ser21 and GSK-3β Ser9) did not reflect the 20–30% lower Akt phosphorylation after bed rest. Phosphorylation of GSK-3β Ser9 was however 10–20% (P < 0.05) lower in the prior-exercised than the rested leg at 3 h recovery both before and after bed rest, but the ability of insulin to induce phosphorylation was apparently not significantly changed by either bed rest or prior exercise (Fig. 3B and C). Effects of insulin on GSK-3α Ser21 phosphorylation were too small to be detected with the antibody applied (data not shown).

TBC1D4 phosphorylation.

Being a direct target of Akt TBC1D4 phosphorylation was evaluated by the use of phosphospecific antibodies toward three phosphorylation sites (Ser588, Thr642, and Ser751). The general phosphorylation pattern revealed by these antibodies was similar (Fig. 4). Thus, both before and after bed rest, TBC1D4 phosphorylation 3 h after exercise was similar in the rested and prior-exercised leg. Insulin stimulation increased the TBC1D4 phosphorylation 1.5- to 3-fold (P < 0.05) in both legs before and after bed rest, and there was no change in the insulin-stimulated TBC1D4 phosphorylation with bed rest (Fig. 4).

FIG. 4.

TBC1D4 Ser588 phosphorylation (A), TBC1D4 Ser751 phosphorylation (B), TBC1D4 Thr642 phosphorylation (C), and representative blots (D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05.

FIG. 4.

TBC1D4 Ser588 phosphorylation (A), TBC1D4 Ser751 phosphorylation (B), TBC1D4 Thr642 phosphorylation (C), and representative blots (D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05.

Glycogen synthase activity and phosphorylation

GS activity.

The GS activity was evaluated both as %I-form (not shown) and percent fractional velocity (%FV). The two measures of GS activity revealed the same pattern of regulation. GS %FV activity was enhanced (P < 0.05) in the prior-exercised leg relative to the rested leg 3 h after exercise both before (P < 0.05) and after bed rest (0.05 ≤ P < 0.1). Insulin increased GS activity in both legs before and after bed rest and with a similar magnitude (P < 0.05). However, in both legs, the level of GS activity tended to be lower after bed rest than before (0.05 ≤ P < 0.1) (Fig. 5A).

FIG. 5.

Glycogen synthase (GS) activity (%FV) (A), representative blots (B), GS site 3a phosphorylation (C), and GS site 2+2a phosphorylation (D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. Symbols within parentheses indicate tendency, 0.05 ≤ P < 0.1.

FIG. 5.

Glycogen synthase (GS) activity (%FV) (A), representative blots (B), GS site 3a phosphorylation (C), and GS site 2+2a phosphorylation (D) in rested (Rest leg) and prior-exercised (Ex leg) vastus lateralis before (B) and after (I) 3-h hyperinsulinemic euglycemic clamp performed 3 h after one-legged knee extensor exercise before and after 7 days of bed rest. AU, arbitrary units; kD, kilodaltons. Values are means ± SE; n = 6. *Significantly different from B, P < 0.05. †Significantly different from before bed rest, P < 0.05. #Significantly different from Rest leg at given time point, P < 0.05. Symbols within parentheses indicate tendency, 0.05 ≤ P < 0.1.

GS phosphorylation.

GS phosphorylation at site 2+2a was, in general, elevated after bed rest relative to before (P < 0.05). Site 2+2a phosphorylation was 30% (P < 0.05) lower in the prior-exercised leg than in the rested leg 3 h after exercise only before bed rest (Fig. 5B and C). Insulin stimulation decreased site 2+2a phosphorylation by 70% (P < 0.05) before but not after bed rest.

GS site 3a phosphorylation was reduced 40% (P < 0.05) by insulin in the rested leg after bed rest and was reduced 45% in the prior-exercised leg (P < 0.05) before and tended to be reduced 35% (0.05 ≤ P < 0.1) after bed rest in response to insulin. Bed rest resulted in an apparent (P < 0.05) 1.2-fold higher GS site 3a phosphorylation in both legs 3 h after exercise (Fig. 5B and D). GS phosphorylation at site 1b was not different between legs and was not affected by insulin, exercise, or bed rest (data not shown).

The main findings of the current study are that bed rest–induced insulin resistance is associated with reduced insulin-stimulated muscular GS activity and Akt signaling as well as decreased protein level of HKII and GLUT4. Thus, this study may indicate that decreased glucose extraction with bed rest occurs as a consequence of both decreased glucose transport/phosphorylation as well as decreased nonoxidative glucose metabolism in skeletal muscle. In addition, the ability of acute exercise to increase insulin-stimulated glucose extraction is well maintained even after 7 days of bed rest. However, acute exercise after bed rest does not fully normalize the ability of skeletal muscle to extract glucose to the level seen when exercise is performed before bed rest.

The observation that 7 days of bed rest resulted in lower insulin-stimulated glucose extraction across the leg is in accordance with previous bed rest studies (5,16). Unfortunately, we are not able to evaluate whether this relates to changes in blood flow. However, based on previous observations, insulin-stimulated blood flow is expected to decrease with bed rest (5,36). Thus, it seems unlikely that the decreased glucose extraction observed after bed rest is a compensation for an increased blood flow. Thus, we are confident that the decreased leg glucose extraction observed reflects decreased leg glucose uptake after bed rest.

The current study provides some indications for the molecular mechanisms behind the observed bed rest–induced insulin resistance. Although it is assumed that the observed molecular changes are due to the physical inactivity in itself, a contribution from a potential positive energy balance cannot be ruled out (37). But the finding that the body weight was maintained by the ad libitum food intake during the bed rest period indicates that the subjects were in energy balance. Central to the observed bed rest–induced insulin resistance is the finding that both basal and insulin-stimulated GS activity was lower after bed rest than before. This impairment is likely one explanatory factor for the decreased leg glucose extraction. The observed lower GLUT4 protein content after bed rest is similar to the previously reported decrease in GLUT4 protein after a period of physical inactivity in trained humans (38) and rats (39). This suggests a similar effect of bed rest and detraining on the regulation of GLUT4 expression. Although our data suggest that physical inactivity–induced insulin resistance is associated with reduced GLUT4 abundance, it is notable that other studies have found no relationship between GLUT4 protein content and insulin action (40). Furthermore, the lower HKII and GLUT4 protein levels after bed rest likely decrease glucose transport and phosphorylation in skeletal muscle. These modifications are likely additional explanatory factors. Although we can only speculate as to the mechanism for the latter observations, it is tempting to suggest that the absence of muscle use during bed rest leads to lack of stimulation of transcriptional/translational processes normally seen in contracting skeletal muscle (41,42).

The current study does not provide insight as to whether translocation of GLUT4 to the plasma membrane is also compromised in bed rest–induced insulin resistance as seen in, for example, insulin-resistant muscle (40). But the 20–30% decreased protein and phosphorylation level of Akt after bed rest, similar to what has recently been observed with reduced levels of physical activity (43), may suggest such a mechanism because impaired Akt phosphorylation is seen in skeletal muscle of type 2 diabetes subjects. A possible upstream regulator responsible for the observed decreased Akt phosphorylation could be phosphatidylinositol-3 kinase, but due to lack of sufficient tissue, this cannot be elucidated further in the current study. However, the impaired phosphorylation on both of the key regulatory sites for Akt activity did not translate to two endogenous substrates of Akt. Thus, in contrast to insulin resistance in type 2 diabetes subjects (7), basal and insulin-induced phosphorylation of TBC1D4 was fully normal on multiple proposed Akt target sites after bed rest. In addition, the present observations do not indicate that reduced TBC1D4 phosphorylation is responsible for muscle insulin resistance after bed rest. However, other mechanisms involved in the GLUT4 recruitment to the plasma membrane, dependent on Akt, may be impaired (e.g., cytoskeleton rearrangements) (44).

Interestingly, the impaired activity and activation of GS after bed rest might be linked to impaired covalent regulation of the enzyme. Thus, whereas GS site 3a dephosphorylation was not compromised after bed rest, the ability of insulin to induce dephosphorylation on site 2+2a was lost. Furthermore, the general level of site 2+2a phosphorylation was increased and potentially contributing to the suppressed GS activity after bed rest. Such a pattern of dysregulation of site 2+2a rather than site 3a is in agreement with observations in muscle insulin resistance related to obesity and type 2 diabetes (9,12). Site 2+2a is apparently not a direct target of either GSK-3 or Akt. Yet it is uncertain whether the impaired Akt signaling indirectly relates to the lack of site 2+2a dephosphorylation seen after bed rest. Site 2+2a is directly targeted by multiple kinases, but so far we have not been able to find indications that these kinases are dysregulated with bed rest. Thus, the fully nonresponsive site 1b phosphorylation indicates that neither calcium calmodulin–dependent kinase nor cAMP-dependent protein kinase is regulated under these conditions (45). Also, AMP-activated protein kinase activity/phosphorylation is not regulated by either insulin or bed rest (data not shown). In addition, both GS site 3 and site 2+2a are dephosphorylated by the multisubstrate phosphatase (PP1) (46), and a dysregulation of PP1 activity would be expected to affect phosphorylation at both sites, and not only site 2+2a as seen in the current study.

Previous studies have suggested that GS site 2+2a phosphorylation is affected by muscle glycogen levels, and that GS site 2+2a phosphorylation is a possible link between the inverse relationship between muscle glycogen and GS activity (47,48). Thus, although mechanistically unresolved, the lower GS activity and higher site 2+2a phosphorylation after bed rest might be related to the higher muscle glycogen level after bed rest. Of notice is, however, that the differences in muscle glycogen concentration eliciting changes in GS activity in previous studies (4750) are larger than the 20% difference seen in the current study. This makes it less likely that the enhanced glycogen level is the only reason for the differences observed in the current study. The findings that resting muscle glycogen was elevated despite impaired insulin-induced glucose extraction and reduced GS activity after bed rest may at first seem contradictory. However, the impact of the dysregulated GS activation on the glycogen level likely first becomes evident when a significant amount of glycogen has to be synthesized (e.g., in the period after glycogen-depleting exercise).

The finding that insulin-stimulated GS activity in the prior-exercised leg was higher than in the rested leg both before and after bed rest is in line with previous studies (14,22) and indicates that activation of GS may play a role in the exercise-induced enhanced glucose uptake several hours after exercise both before and after the inactivity period. The similar insulin-mediated regulation of GS site 3a and Akt phosphorylation in the rested and prior-exercised leg both before and after bed rest is also in accordance with earlier studies (22,48) and suggests that changes in GS site 3a and Akt phosphorylation do not contribute to the observed exercise-induced effects on GS activity. Previous findings show that reduced muscle glycogen is associated with dephosphorylation of GS site 2+2a (34), and thus the more marked dephosphorylation of GS site 2+2a could be a link between the reduced muscle glycogen and the increased GS activity in the prior-exercised leg. Previous studies showing that prior exercise increased TBC1D4 phosphorylation both before and after insulin stimulation (25) suggest a role of TBC1D4 in the enhanced glucose clearance in a prior-exercised muscle. However, the current study does not confirm these observations, although it cannot be excluded that a more intense or more prolonged bout of exercise would elicit such changes. Thus, the analyses performed in this study do not bring further insights to the mechanisms behind exercise-induced enhanced insulin sensitivity but do provide the important observation that the mechanisms still seem fully functional in bed rest–induced insulin-resistant muscle.

We are well aware of the limitation in the study by having investigated the effect of acute exercise in only six subjects, leaving us in some cases with near-significant trends. Still we are able to draw some conclusions to potential mechanisms involved in bed rest–induced insulin resistance in skeletal muscle. Thus bed rest–induced insulin resistance in skeletal muscle relates to decreased expression and impaired activation of central players regulating glucose uptake and storage in skeletal muscle, together with a possible impaired insulin-stimulated blood flow. In addition, the similar enhancement of glucose removal by prior exercise before and after bed rest demonstrates that the ability of exercise to increase skeletal muscle insulin sensitivity is maintained after 7 days of physical inactivity.

B.G. and J.A.L.C. were visiting Copenhagen Muscle Research Centre, Rigshospitalet 7652, during this study.

This study was supported by grants from the Lundbeck Foundation, Denmark; The Danish Medical Research Council, Denmark; and the Novo Nordisk Foundation, Denmark. The Centre of Inflammation and Metabolism (CIM) is supported by a grant from the Danish National Research Foundation (02-512-55). The Copenhagen Muscle Research Centre is supported by a grant from the Capital Region of Denmark. CIM and the Molecular Physiology Group (Department of Exercise and Sport Sciences) are part of UNIK: Food, Fitness & Pharma for Health and Disease, supported by the Danish Ministry of Science, Technology, and Innovation. The experimental part was funded by a grant to B.S., while laboratory analyses were funded by grants to H.P. and J.F.P.W.

No potential conflicts of interest relevant to this article were reported.

R.S.B. designed the study, took part in conducting the human experiments, performed the laboratory analyses, wrote the manuscript, and commented on the manuscript. S.R. designed the study, took part in conducting the human experiments, performed the laboratory analyses, and commented on the manuscript. K.K., C.L., and P.P. designed the study, took part in conducting the human experiments, and commented on the manuscript. N.-J.A.-A., R.K.-M., B.G., G.v.H., and J.A.L.C. took part in conducting the human experiments and commented on the manuscript. J.T.T. provided valuable guidance for specific laboratory analyses and commented on the manuscript. B.S. designed the study and commented on the manuscript. H.P. and J.F.P.W. designed the study, took part in conducting the human experiments, assisted with writing the manuscript, and commented on the manuscript. J.F.P.W. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Parts of this study were presented in poster form at the 70th Scientific Sessions of the American Diabetes Association, Orlando, Florida, 25–29 June 2010.

The authors would like to thank the subjects for their extraordinary effort during the study. In addition, the authors have sincere appreciation for the whole Copenhagen 2008 Bed Rest Team for a fantastic collaboration. The authors are grateful to Oluf Pedersen (University of Copenhagen), Graham Hardie, and Carol Mackintosh (Dundee University) for donating antibodies essential for this study.

1.
Venables
MC
,
Jeukendrup
AE
.
Physical inactivity and obesity: links with insulin resistance and type 2 diabetes mellitus
.
Diabetes Metab Res Rev
2009
;
25
(
Suppl. 1
):
S18
S23
[PubMed]
2.
Chen
YD
,
Golay
A
,
Swislocki
AL
,
Reaven
GM
.
Resistance to insulin suppression of plasma free fatty acid concentrations and insulin stimulation of glucose uptake in noninsulin-dependent diabetes mellitus
.
J Clin Endocrinol Metab
1987
;
64
:
17
21
[PubMed]
3.
DeFronzo
RA
,
Ferrannini
E
,
Sato
Y
,
Felig
P
,
Wahren
J
.
Synergistic interaction between exercise and insulin on peripheral glucose uptake
.
J Clin Invest
1981
;
68
:
1468
1474
[PubMed]
4.
King
DS
,
Dalsky
GP
,
Clutter
WE
, et al
.
Effects of exercise and lack of exercise on insulin sensitivity and responsiveness
.
J Appl Physiol
1988
;
64
:
1942
1946
[PubMed]
5.
Mikines
KJ
,
Richter
EA
,
Dela
F
,
Galbo
H
.
Seven days of bed rest decrease insulin action on glucose uptake in leg and whole body
.
J Appl Physiol
1991
;
70
:
1245
1254
[PubMed]
6.
Richter
EA
,
Kiens
B
,
Mizuno
M
,
Strange
S
.
Insulin action in human thighs after one-legged immobilization
.
J Appl Physiol
1989
;
67
:
19
23
[PubMed]
7.
Vind
BF
,
Pehmoller
C
,
Treebak
JT
, et al
.
Impaired insulin-induced site-specific phosphorylation of TBC1 domain family, member 4 (TBC1D4) in skeletal muscle of type 2 diabetes patients is restored by endurance exercise-training
.
Diabetologia
2010
;
54
:
157
167
8.
Zierath
JR
,
He
L
,
Gumà
A
,
Odegoard Wahlström
E
,
Klip
A
,
Wallberg-Henriksson
H
.
Insulin action on glucose transport and plasma membrane GLUT4 content in skeletal muscle from patients with NIDDM
.
Diabetologia
1996
;
39
:
1180
1189
[PubMed]
9.
Højlund
K
,
Staehr
P
,
Hansen
BF
, et al
.
Increased phosphorylation of skeletal muscle glycogen synthase at NH2-terminal sites during physiological hyperinsulinemia in type 2 diabetes
.
Diabetes
2003
;
52
:
1393
1402
[PubMed]
10.
Højlund
K
,
Glintborg
D
,
Andersen
NR
, et al
.
Impaired insulin-stimulated phosphorylation of Akt and AS160 in skeletal muscle of women with polycystic ovary syndrome is reversed by pioglitazone treatment
.
Diabetes
2008
;
57
:
357
366
[PubMed]
11.
Karlsson
HK
,
Zierath
JR
,
Kane
S
,
Krook
A
,
Lienhard
GE
,
Wallberg-Henriksson
H
.
Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects
.
Diabetes
2005
;
54
:
1692
1697
[PubMed]
12.
Højlund
K
,
Birk
JB
,
Klein
DK
, et al
.
Dysregulation of glycogen synthase COOH- and NH2-terminal phosphorylation by insulin in obesity and type 2 diabetes mellitus
.
J Clin Endocrinol Metab
2009
;
94
:
4547
4556
[PubMed]
13.
Christ-Roberts
CY
,
Pratipanawatr
T
,
Pratipanawatr
W
, et al
.
Exercise training increases glycogen synthase activity and GLUT4 expression but not insulin signaling in overweight nondiabetic and type 2 diabetic subjects
.
Metabolism
2004
;
53
:
1233
1242
[PubMed]
14.
Frøsig
C
,
Rose
AJ
,
Treebak
JT
,
Kiens
B
,
Richter
EA
,
Wojtaszewski
JF
.
Effects of endurance exercise training on insulin signaling in human skeletal muscle: interactions at the level of phosphatidylinositol 3-kinase, Akt, and AS160
.
Diabetes
2007
;
56
:
2093
2102
[PubMed]
15.
O’Gorman
DJ
,
Karlsson
HK
,
McQuaid
S
, et al
.
Exercise training increases insulin-stimulated glucose disposal and GLUT4 (SLC2A4) protein content in patients with type 2 diabetes
.
Diabetologia
2006
;
49
:
2983
2992
[PubMed]
16.
Mikines
KJ
,
Dela
F
,
Tronier
B
,
Galbo
H
.
Effect of 7 days of bed rest on dose-response relation between plasma glucose and insulin secretion
.
Am J Physiol
1989
;
257
:
E43
E48
[PubMed]
17.
Richter
EA
,
Mikines
KJ
,
Galbo
H
,
Kiens
B
.
Effect of exercise on insulin action in human skeletal muscle
.
J Appl Physiol
1989
;
66
:
876
885
[PubMed]
18.
Dela
F
,
Mikines
KJ
,
von Linstow
M
,
Secher
NH
,
Galbo
H
.
Effect of training on insulin-mediated glucose uptake in human muscle
.
Am J Physiol
1992
;
263
:
E1134
E1143
[PubMed]
19.
Mikines
KJ
,
Sonne
B
,
Farrell
PA
,
Tronier
B
,
Galbo
H
.
Effect of physical exercise on sensitivity and responsiveness to insulin in humans
.
Am J Physiol
1988
;
254
:
E248
E259
[PubMed]
20.
Hansen
PA
,
Nolte
LA
,
Chen
MM
,
Holloszy
JO
.
Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise
.
J Appl Physiol
1998
;
85
:
1218
1222
[PubMed]
21.
Holloszy
JO
.
Exercise-induced increase in muscle insulin sensitivity
.
J Appl Physiol
2005
;
99
:
338
343
[PubMed]
22.
Wojtaszewski
JF
,
Hansen
BF
,
Gade
, et al
.
Insulin signaling and insulin sensitivity after exercise in human skeletal muscle
.
Diabetes
2000
;
49
:
325
331
[PubMed]
23.
Wojtaszewski
JF
,
Hansen
BF
,
Kiens
B
,
Richter
EA
.
Insulin signaling in human skeletal muscle: time course and effect of exercise
.
Diabetes
1997
;
46
:
1775
1781
[PubMed]
24.
Arias
EB
,
Kim
J
,
Funai
K
,
Cartee
GD
.
Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle
.
Am J Physiol Endocrinol Metab
2007
;
292
:
E1191
E1200
[PubMed]
25.
Treebak
JT
,
Frøsig
C
,
Pehmøller
C
, et al
.
Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle
.
Diabetologia
2009
;
52
:
891
900
[PubMed]
26.
Howlett
KF
,
Mathews
A
,
Garnham
A
,
Sakamoto
K
.
The effect of exercise and insulin on AS160 phosphorylation and 14-3-3 binding capacity in human skeletal muscle
.
Am J Physiol Endocrinol Metab
2008
;
294
:
E401
E407
[PubMed]
27.
Wojtaszewski
JF
,
Nielsen
P
,
Kiens
B
,
Richter
EA
.
Regulation of glycogen synthase kinase-3 in human skeletal muscle: effects of food intake and bicycle exercise
.
Diabetes
2001
;
50
:
265
269
[PubMed]
28.
Pilegaard
H
,
Saltin
B
,
Neufer
PD
.
Exercise induces transient transcriptional activation of the PGC-1alpha gene in human skeletal muscle
.
J Physiol
2003
;
546
:
851
858
[PubMed]
29.
Bergstrom
J
.
Muscle electrolytes in man
.
Scand J Clin Lab Invest
1962
;
14
(
Suppl. 68
):
1
110
30.
Van Hall
G
,
Bülow
J
,
Sacchetti
M
,
Al Mulla
N
,
Lyngso
D
,
Simonsen
L
.
Regional fat metabolism in human splanchnic and adipose tissues; the effect of exercise
.
J Physiol
2002
;
543
:
1033
1046
[PubMed]
31.
Passonneau
JV
,
Lauderdale
VR
.
A comparison of three methods of glycogen measurement in tissues
.
Anal Biochem
1974
;
60
:
405
412
[PubMed]
32.
Lowry
OH
,
Passonneau
JV
.
A Flexible System of Enzymatic Analysis
.
New York, Academic Press
,
1972
33.
Thomas
JA
,
Schlender
KK
,
Larner
J
.
A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose
.
Anal Biochem
1968
;
25
:
486
499
[PubMed]
34.
Prats
C
,
Helge
JW
,
Nordby
P
, et al
.
Dual regulation of muscle glycogen synthase during exercise by activation and compartmentalization
.
J Biol Chem
2009
;
284
:
15692
15700
[PubMed]
35.
Kiilerich
K
,
Ringholm
S
,
Bienso
RS
.
Exercise-induced pyruvate dehydrogenase activation is not affected by 7 days of bed rest
.
J Appl Physiol
2011
;
111
:
751
757
36.
Sonne
MP
,
Alibegovic
AC
,
Højbjerre
L
,
Vaag
A
,
Stallknecht
B
,
Dela
F
.
Effect of 10 days of bed rest on metabolic and vascular insulin action: a study in individuals at risk for type 2 diabetes
.
J Appl Physiol
2010
;
108
:
830
837
[PubMed]
37.
Newsom
SA
,
Schenk
S
,
Thomas
KM
, et al
.
Energy deficit after exercise augments lipid mobilization but does not contribute to the exercise-induced increase in insulin sensitivity
.
J Appl Physiol
2010
;
108
:
554
560
[PubMed]
38.
Vukovich
MD
,
Arciero
PJ
,
Kohrt
WM
,
Racette
SB
,
Hansen
PA
,
Holloszy
JO
.
Changes in insulin action and GLUT-4 with 6 days of inactivity in endurance runners
.
J Appl Physiol
1996
;
80
:
240
244
[PubMed]
39.
Host
HH
,
Hansen
PA
,
Nolte
LA
,
Chen
MM
,
Holloszy
JO
.
Rapid reversal of adaptive increases in muscle GLUT-4 and glucose transport capacity after training cessation
.
J Appl Physiol
1998
;
84
:
798
802
[PubMed]
40.
Garvey
WT
,
Maianu
L
,
Zhu
JH
,
Brechtel-Hook
G
,
Wallace
P
,
Baron
AD
.
Evidence for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal muscle as a cause of human insulin resistance
.
J Clin Invest
1998
;
101
:
2377
2386
[PubMed]
41.
Pilegaard
H
,
Ordway
GA
,
Saltin
B
,
Neufer
PD
.
Transcriptional regulation of gene expression in human skeletal muscle during recovery from exercise
.
Am J Physiol Endocrinol Metab
2000
;
279
:
E806
E814
[PubMed]
42.
Williams
RS
,
Neufer
PD
.
Regulation of gene expression in skeletal muscle by contractile activity
. In
The Handbook of Physiology. Exercise: Regulation and Integration of Multiple Systems
.
Bethesda, MD
,
American Physiological Society
,
1996
, p.
1124
1150
43.
Krogh-Madsen
R
,
Thyfault
JP
,
Broholm
C
, et al
.
A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity
.
J Appl Physiol
2010
;
108
:
1034
1040
[PubMed]
44.
Thong
FS
,
Dugani
CB
,
Klip
A
.
Turning signals on and off: GLUT4 traffic in the insulin-signaling highway
.
Physiology (Bethesda)
2005
;
20
:
271
284
[PubMed]
45.
Chasiotis
D
,
Sahlin
K
,
Hultman
E
.
Regulation of glycogenolysis in human muscle in response to epinephrine infusion
.
J Appl Physiol
1983
;
54
:
45
50
[PubMed]
46.
Cohen
P
.
The structure and regulation of protein phosphatases
.
Annu Rev Biochem
1989
;
58
:
453
508
[PubMed]
47.
Nielsen
JN
,
Wojtaszewski
JF
.
Regulation of glycogen synthase activity and phosphorylation by exercise
.
Proc Nutr Soc
2004
;
63
:
233
237
[PubMed]
48.
Jensen
J
,
Jebens
E
,
Brennesvik
EO
, et al
.
Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling
.
Am J Physiol Endocrinol Metab
2006
;
290
:
E154
E162
[PubMed]
49.
Nielsen
JN
,
Derave
W
,
Kristiansen
S
,
Ralston
E
,
Ploug
T
,
Richter
EA
.
Glycogen synthase localization and activity in rat skeletal muscle is strongly dependent on glycogen content
.
J Physiol
2001
;
531
:
757
769
[PubMed]
50.
Danforth
WH
.
Glycogen synthetase activity in skeletal muscle. Interconversion of two forms and control of glycogen synthesis
.
J Biol Chem
1965
;
240
:
588
593
[PubMed]