Interaction Between Dietary Lipids and Physical Inactivity on Insulin Sensitivity and on Intramyocellular Lipids in Healthy Men Free

Eight healthy male volunteers (age 23 ± 1 years; weight 67.9 ± 2.9 kg, BMI 21.6 ± 0.8 kg/m², body fat 13.4 ± 1.7% [means ± SE]) participated in the study. All subjects were in good health, were nonsmokers, and were not taking any drugs. None had a personal or family history of diabetes or hypertension. The subjects did not regularly take part in strenuous physical activity. Body composition was estimated from subcutaneous skinfold thickness measurements according to Durnin and Womersley (19). The experimental protocol was approved by the ethical committees of the Lausanne University School of Medicine and of the Kanton of Bern. Before participating in the study, each subject was informed in detail about the protocol and signed a written informed consent form.

Subjects were studied on three occasions for 5 consecutive days spent in a metabolic unit (Fig. 1). During baseline (day −2 and day −1), volunteers consumed an isocaloric-equilibrated diet composed of 35% fat (∼50% saturated), 50% carbohydrates, and 15% protein corresponding to 1.6 × basal metabolic rate (BMR; previously estimated by indirect calorimetry); besides normal ambulatory activity, subjects performed two exercise bouts of 30 min at 75 W (10:00 a.m. and 2:30 p.m.) on a bicycle ergometer on day −2 . The effect of diet and physical inactivity was tested over the next 60 h (days 1, 2, and 3) with the following protocols: 1) bed rest associated with an isocaloric (1.2 × BMR) high-carbohydrate diet (BR-HCHO; 70% carbohydrates, 15% fat of which ∼35% was saturated fat, 15% protein), 2) bed rest associated with an isocaloric (1.2 × BMR) high-fat diet ([BR-HF]; 40% carbohydrate, 45% fat of which ∼60% was saturated fat, 15% protein), and 3) physical activity (2 × 30 min at 75 W on day 1 and 2) associated with an isocaloric (1.6 × BMR) high-fat diet (PA-HF; n = 6). The order of the two bed rest conditions was randomized with >9 days in between each, whereas the physical activity condition was always last after at least a 5-week washout period. Meals were provided at 8:00 a.m. (30% energy), 12:00 p.m. (35% energy), and 6:30 p.m. (35% energy) with no snacks in between. Heart rate was continuously monitored using a Polar cardiometer (Kempele, Finland) to control for physical inactivity compliance (2). To reduce the risk of deep venous thrombosis associated with bed rest, participants were given 100 mg acetylsalicylic acid on day 1 of each protocol.

On the morning of the clamp at 8:00 a.m., subjects received a breakfast containing 1.63 g carbohydrate/kg body wt (day −1), 1.77 g carbohydrate/kg (day 3 of high-carbohydrate diet), or 1.10 g carbohydrate/kg (day 3 of high-fat diet). Glucose infusion rate (GIR) was measured at least 5 h after breakfast (∼1:00–1:30 p.m.). On completion of the clamp procedure, subjects were transferred to the Magnetic Resonance Center at the University and Inselspital Bern, where IMCL concentrations were determined by localized ¹H-MRS of the vastus intermedius muscle of the right quadriceps. The IMCL measurements were realized 3 h after the completion of the clamp studies; the interval was kept constant between volunteers.

A 90-min hyperinsulinemic (1 mU insulin · kg⁻¹ · min⁻¹)-euglycemic (5.6 mmol/l) clamp (20) was performed between 12:00 p.m. and 1:30 p.m. Blood was obtained every 30 min for insulin and NEFA determination. During the last 30 min of the clamp, the GIR was used to evaluate insulin-mediated glucose disposal.

Magnetic resonance examinations of the right thigh included image acquisition and ¹H-MRS on a 1.5 Tesla whole-body system (SIGNA; General Electrics, Milwaukee, WI), using a ¹³C/¹H double-tuned flexible extremity coil (¹H-coil, Helmholtz design; Medical Advance, Milwaukee, WI). Inside the magnet, the volunteers were placed on a specially designed positioning aid to guarantee a reproducible fixation of the right leg for all six magnetic resonance sessions (four in two cases). Repositioning of the volunteer and placement of the coil were monitored on the localizer images.

It has been shown that the signals from IMCLs and extramyocellular lipids can be separated by single-voxel ¹H-MRS (21,22). The sensitive volume (18 × 11 × 12 mm³, inferior/superior × right/left × anterior/posterior) was positioned in the m. vastus intermedius close to the femoral bone. Care was taken that no sign of fatty infiltration was visible inside the sensitive volume, which could lead to contamination by the large extramyocellular lipid signals. Positioning as well as repositioning (sessions 2–6) of the sensitive volume was done in the localizer image series (fast spin echo sequence with a repetition time of t_r = 1,000 ms and an echo time of t_e = 17 ms). ¹H-MRSs were recorded using an optimized PRESS-sequence (t_r = 3 s, t_e = 20 ms, 128 acquisitions, 16 phase-rotating steps, 2,000 Hz, 1,024 pts) with water and additional outer-volume suppression. The spectra were quantified using the fully relaxed, unsuppressed water signal as an internal concentration standard. IMCL concentrations (in millimoles per kilogram wet weight) were evaluated as previously described (23).

Plasma glucose concentrations were measured by the glucose oxidase method using a Beckman glucose analyzer II (Beckman Instruments, Fullerton, CA). NEFA concentrations were measured with a commercial colorimetric method (NEFA C; Wako Chemicals, Freiburg, Germany). Radioimmunoassay kits were used for determination of plasma insulin and adiponectin (Linco Research, St. Charles, MO).

Data are expressed as means ± SE. Statistical significance was assessed using ANOVA with repeated measures and a post hoc Fisher’s test for comparison of the three test conditions or a paired t test for comparison between day 3 and day −1 within each condition.

Plasma glucose, insulin, NEFAs, and adiponectin concentrations are shown in Table 1. At baseline (day −1), there were no differences in plasma hormones, substrate concentrations, GIR, or IMCL levels between groups (Table 1 and Fig. 2).

On day 3, preclamp glucose concentrations were less after BR-HF (P < 0.05) and NEFA concentrations decreased after BR-HCHO (P = 0.021). Preclamp-insulin and -adiponectin concentrations remained unchanged. During the clamp (60–90 min), GIR decreased only in the BR-HF condition (−24 ± 6%, P = 0.007) (Fig. 2A). Compared with day −1, IMCL concentrations on day 3 increased significantly by 17 ± 8% with BR-HCHO (P = 0.037) and 32 ± 7% with BR-HF (P < 0.0001). After PA-HF, a 31 ± 19% increase in IMCL was observed but failed to reach statistical significance (P = 0.12) (Fig. 2B).

No correlations were observed between IMCL content and GIR on day 3 or between increments of IMCL and changes in GIR between day −1 and day 3. Similarly, no correlations were observed between IMCL and adiponectin concentrations or between GIR and adiponectin concentrations on day 3.

Compared with day −1, diurnal heart rate decreased during both bed rest conditions from 74 ± 3 to 64 ± 1 bpm (P < 0.001) and from 74 ± 2 to 64 ± 2 bpm (P = 0.003) during BR-HCHO and BR-HF, respectively. During the PA-HF condition, heart rate was not modified (77 ± 4 vs. 78 ± 3 bpm, NS).

The major observations in this prospective study are that 1) whole-body insulin sensitivity was decreased only in response to the combination of physical inactivity and high-fat diet; 2) IMCL concentrations increased with bed rest, whether associated with a high-carbohydrate or a high-fat diet, and a similar trend was observed with high-fat diet associated with physical activity; and 3) no correlations were observed during any of these three conditions between whole-body insulin sensitivity and IMCL concentration in the vastus intermedius muscle of the quadriceps; moreover, no correlations were noted between increments of IMCL levels and changes in whole-body insulin sensitivity between day −1 and day 3.

Insulin sensitivity was assessed as the average GIR during the last 30 min of the clamp. Since insulin concentrations achieved during the clamps are known to completely suppress hepatic glucose output (24), the difference most likely reflects extrahepatic (muscle) insulin resistance (25). These differences in insulin sensitivity were observed during hyperinsulinemic clamps performed between 12:00 p.m. and 1:30 p.m., 5 h after the last meal, i.e., breakfast at 8:00 a.m. Although one cannot completely exclude that dietary carbohydrate absorption was finished at this time, previous studies indicate that the bulk of carbohydrates are absorbed within 4 h of a liquid meal (26). Although absorption of a solid meal may take somewhat longer, it is very unlikely that differences in gastric emptying or rates of dietary carbohydrate absorption induced by high-fat feeding or physical inactivity were of sufficient magnitude to account for the reduction in GIR observed after BR-HF. We therefore feel confident that our observation reflects a decreased insulin sensitivity after BR-HF.

Numerous studies in humans and animals have investigated the acute effects of high concentrations of fatty acids on insulin sensitivity using intravenous triglyceride emulsion infusions. Increasing fatty acid concentrations has been shown on several occasions to reduce insulin-stimulated glucose uptake (4,6,27), but few experiments have focused on the effect of increased dietary fat on insulin sensitivity. Bachmann et al. (28) compared the effect of a high-fat diet (60% of energy from fat, predominantly saturated fat) with that of an acute intravenous lipid infusion. These authors reported that the decrease in GIR was less after 3 days of high-fat diet (−20%) than after triglyceride infusion (−40%). In our study, we did not observe a significant decrease in insulin sensitivity with the high-fat diet associated with physical activity. This discrepancy probably results from the higher proportion of fat (55–60 vs. 45%) and larger energy intake consumed by the subjects in Bachmann’s study or possibly by the smaller number of subjects in our study. In both our study and Bachmann et al.’s studies, the effect of short-term high-fat feeding on insulin sensitivity was small. This may be due to the short duration of dietary changes, since after extended exposure to dietary fat, rodents have been shown to develop larger changes in whole-body and muscle insulin sensitivity (29).

Even though some studies have also investigated the effect of physical inactivity on insulin sensitivity (30–35), none have combined physical inactivity and a high-fat diet. In 1972, Lipman et al. (31) reported a deleterious effect of 3 days of bed rest on glucose tolerance in healthy adults. However, in this study, no proper distinction could be made between the effects of the diet and physical inactivity on insulin sensitivity since the relative proportions of the macronutrients were not specified. More recently, Blanc et al. (34) reported a decrease in insulin sensitivity after 7 days of head-down bed rest in healthy men and women on a conventional diet. The authors suggested that this impairment occurred mainly in skeletal muscle and mostly in men. However, fluid redistribution during head-down tilt may cause insulin resistance by reducing muscle blood flow. Stuart et al. (35) studied the effect of 7 days of bed rest in healthy subjects on a controlled diet and showed that whole-body insulin-stimulated glucose uptake was impaired whereas hepatic glucose production was adequately inhibited by insulin. In the present study, 60 h of bed rest combined with a high-carbohydrate diet was not sufficient to alter insulin sensitivity. This difference might be explained by the shorter duration and lower proportion of fat in our study (15 vs. 31%).

To our knowledge, this is the first time that a relatively moderate increase in dietary fat (45% of total energy from fat, mainly saturated fat) and a short period of physical inactivity (60 h) have been shown to interact to decrease insulin sensitivity. The short duration of intervention was done to focus on acute metabolic effects rather than changes in body composition. Our experiment suggests an interaction between short-term bed rest and saturated fat, causing a significant ∼25% reduction in insulin sensitivity. In contrast, physical inactivity associated with high-carbohydrate diet or physical activity associated with high–saturated fat diet failed to lead to a similar deleterious effect. This indicates a protective effect of even a moderate amount of physical activity on fat-induced insulin resistance.

In the past decade, several studies have focused on the relation between IMCL content and insulin resistance (7–10). Most have shown, both in humans and in animals, a negative correlation between IMCL content and insulin sensitivity. Increased IMCL concentrations have been demonstrated in obesity and type 2 diabetes, and weight loss has been shown to decrease IMCL concentrations (36,37). In the current study, we observed an increase in IMCL concentrations of similar magnitude in the three conditions tested (although the increase after PA-HF fell short of statistical significance). Moreover, the increases observed after high-fat diet (with or without physical inactivity) tended to be larger than those observed after physical inactivity with high-carbohydrate intake. This appears to be consistent with the observation of Décombaz et al. (11,38) that IMCL stores are rapidly replenished by dietary fat after exercise-mediated depletion. However, the increased IMCL content after the BR-HCHO condition indicates that inactivity also exerts an effect on IMCL metabolism, presumably by decreasing muscle lipid oxidation.

Surprisingly, there was no correlation between IMCL content of the vastus intermedius muscle and insulin sensitivity in healthy subjects in these short-term conditions. It is not the first time that such a dissociation has been observed; it is well documented that endurance athletes have high insulin sensitivity despite increased IMCL levels (39). Along the same line, insulin sensitivity is improved in rats overexpressing hepatic malonyl-CoA decarboxylase by a mechanism that does not require lowering of muscle triglycerides or long-chain fatty acyl-CoA despite increased IMCL content (40). Furthermore, in a recent study, Pruchnic et al. (41) reported significant increases in both IMCL contents and muscle oxidative capacity in elderly volunteers who took part in a 12-week endurance training program. Together, these observations clearly indicate that high IMCL contents alone are not directly responsible for insulin resistance, but may rather indicate concomitant changes in intracellular lipid metabolites (42). It can be speculated that an imbalance between the release of fatty acid from IMCL and muscle fatty-acid oxidation during physical inactivity reduce insulin sensitivity through an accumulation of lipid metabolites but that such an imbalance is not reflected by IMCL concentrations.

It has also been proposed that both insulin sensitivity and IMCL concentrations are modulated by adiponectin. We therefore considered the possibility that adiponectin may mediate the effects of bed rest with a high-fat diet on insulin sensitivity. We did not, however, find any differences in preclamp adiponectinemia in the three studies. Moreover, no correlation was observed between preclamp adiponectinemia and IMCL or GIR. This is in accordance with a previous report by Thamer et al. (18) that indicated that adiponectinemia was not related to IMCL concentrations in soleus muscle of humans fed a high-fat diet for 3 days.

In summary, the present study demonstrated that high–saturated fat intake associated with physical inactivity after a period as short as 60 h is sufficient to significantly impair insulin sensitivity in healthy subjects. However, physical inactivity with a high-carbohydrate diet or physical activity with a high–saturated fat diet did not significantly impair insulin sensitivity. Thus, our results suggest that even mild physical activity may confer protection against the deleterious effects of a high-fat diet. The insulin resistance induced by short-term high-fat feeding and physical inactivity does not, however, appear to be directly related to changes in IMCLs.

This work was supported by grants 32-67787.02 (to L.T.) and 3100-065315.01 (to C.B.) from the Swiss National Science Foundation, and from la Fondation romande pour la recherché sur le diabète (to L.T).

We thank the staff of the Département de Physiologie de Lausanne and of the Magnetic Resonance Spectroscopy Unit of Bern for their excellent assistance. The authors are very indebted to Eric Ravussin for his critical review of this manuscript.

This study was presented as an abstract at the annual scientific meeting of the North American Association for the Study of Obesity, Las Vegas, Nevada, 14–18 November 2004.