Endocrinology and Metabolism

Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss

David E. Kelley, Bret Goodpaster, Rena R. Wing, Jean-Aime Simoneau

Abstract

The current study was undertaken to investigate fatty acid metabolism by skeletal muscle to examine potential mechanisms that could lead to increased muscle triglyceride in obesity. Sixteen lean and 40 obese research volunteers had leg balance measurement of glucose and free fatty acid (FFA) uptake (fractional extraction of [9,103H]oleate) and indirect calorimetry across the leg to determine substrate oxidation during fasting and insulin-stimulated conditions. Muscle obtained by percutaneous biopsy had lower carnitine palmitoyl transferase (CPT) activity and oxidative enzyme activity in obesity (P < 0.05). During fasting conditions, obese subjects had an elevated leg respiratory quotient (RQ, 0.83 ± 0.02 vs. 0.90 ± 0.01;P < 0.01) and reduced fat oxidation but similar FFA uptake across the leg. During insulin infusions, fat oxidation by leg tissues was suppressed in lean but not obese subjects; rates of FFA uptake were similar. Fasting values for leg RQ correlated with insulin sensitivity (r = −0.57, P < 0.001). Thirty-two of the obese subjects were restudied after weight loss (WL, −14.0 ± 0.9 kg); insulin sensitivity and insulin suppression of fat oxidation improved (P < 0.01), but fasting leg RQ (0.90 ± 0.02 vs. 0.90 ± 0.02, pre-WL vs. post-WL) and muscle CPT activity did not change. The findings suggest that triglyceride accumulation in skeletal muscle in obesity derives from reduced capacity for fat oxidation and that inflexibility in regulating fat oxidation, more than fatty acid uptake, is related to insulin resistance.

  • arteriovenous balance
  • indirect calorimetry
  • fatty acid metabolism

skeletal muscle has an increased content of triglyceride in obesity (37, 38). The amount of triglyceride in skeletal muscle, although quite small relative to that in adipose tissue, is nevertheless significantly associated with insulin resistance (24, 38, 45, 53). However, the mechanisms that account for increased skeletal muscle lipid deposition in obesity are uncertain. Rates of de novo lipogenesis within skeletal muscle are low (49), and although muscle content of malonyl-CoA, a precursor for fatty acid synthesis, may be increased in obesity, this is thought to function more as a signal for fatty acid partitioning (44, 46). Accordingly, skeletal muscle accretion of triglyceride in obesity might result from either increased fatty acid uptake, out of proportion to normal rates of lipid oxidation, or, alternatively, from diminished fat oxidation. The primary objective of the current study was to test these alternative hypotheses.

It is now well established that during fasting conditions, skeletal muscle in lean, healthy individuals relies on lipid oxidation for the majority of resting energy production, as originally reported by Andres and colleagues (Andres and Cadar, Ref. 2, and Baltzan et al., Ref. 3) on the basis of regional indirect calorimetry across the forearm. During postabsorptive conditions, skeletal muscle has a high fractional extraction of fatty acids and thus is an important site for systemic utilization of fatty acids (55). Some of the fatty acids taken up by muscle enter tissue lipid pools of triglyceride or phospholipids (8,14, 45), although it is less clear whether fatty acids must initially enter tissue triglyceride before oxidization (11) or enter mitochondria directly, as recently suggested by the isotopic studies of Sidossis et al. (51). There are several biochemical determinants of the capacity for fatty acid utilization in skeletal muscle. In animal studies, the patterns of fatty acid utilization are related to fiber type, with slow-twitch oxidative muscle having a higher capacity for fatty acid uptake and lipid oxidation than fast-twitch glycolytic muscle fibers (8, 14, 37). In animal models of obesity, skeletal muscle content of malonyl-CoA, a potent allosteric inhibitor of carnitine palmitoyl transferase I (CPT I) in skeletal muscle, has been found to be increased (44, 47). Other potential determinants might be the patterns of expression of various fatty acid-binding proteins (6, 21, 57), although relatively little is known at present concerning human skeletal muscle and the effect of obesity. Intriguingly, some of these same skeletal muscle characteristics are associated with insulin resistance, specifically, a lower oxidative enzyme capacity (54), an increased proportion of glycolytic type II muscle fibers (31), and increased malonyl-CoA content (44). Moreover, defects in the activity of CPT, the enzyme complex regarded as rate limiting for oxidation of long-chain fatty acids (35), are associated with lipid accumulation within skeletal muscle (50).

On the basis of these considerations, it is biologically plausible that skeletal muscle patterns of fatty acid utilization during fasting conditions might associate with obesity-related insulin resistance, and the second objective of the current study was to explore this hypothesis. Whereas it is generally accepted that rates of lipid oxidation are increased in obesity during insulin-stimulated conditions, and, indeed, impaired suppression of lipid oxidation is a prominent manifestation of insulin resistance (16, 30), much less is known regarding a potential link between fasting patterns of muscle free fatty acid (FFA) utilization and insulin resistance. Another important consideration is whether potential impairments within the pathways of fatty acid utilization in skeletal muscle in obesity are primary defects or arise secondarily, after an individual has become obese. This is a difficult issue to effectively address by cross-sectional comparisons of lean and obese subjects. A prospective clinical study indicated that a decreased reliance on lipid oxidation is a risk factor for weight gain (59), and collateral analyses of skeletal muscle enzyme activities implicated skeletal muscle in impaired lipid oxidation (17, 60). A reduced reliance on lipid oxidation has also been identified as a risk factor for weight regain after weight loss (7). These data raise the possibility that a potential impairment in capacity for lipid oxidation might be a primary defect in obesity. Weight loss can substantially improve insulin-resistant glucose metabolism in skeletal muscle (20), indicating a substantial acquired or secondary component of obesity-related insulin-resistant glucose metabolism. Thus the third objective of the current study was to assess whether weight loss, undertaken by dietary restriction and without change in baseline levels of physical fitness, can also modulate patterns of skeletal muscle metabolism of fatty acids, and how this would compare with the effect on glucose metabolism.

METHODS

Subjects.

Sixteen lean (L) and forty obese (O) volunteers were recruited by advertisement in a community newspaper. All potential research volunteers underwent a thorough medical evaluation, including a 2-h 75-g oral glucose tolerance test, before participation. To be eligible, research volunteers were required to be within the ages of 20 and 45 yr, to have normal glucose tolerance, fasting triglyceride, and cholesterol values <300 mg/dl, and to be normotensive without anti-hypertensive medications. Women taking oral contraceptives were excluded, as were subjects taking lipid-lowering medication. Additional criteria were that neither L nor O volunteers engaged regularly in ≥2 exercise sessions weekly and that weight was stable (±2 kg) for ≥3 mo before enrollment. Informed, written consent was obtained from each subject. The research plan was reviewed and approved by the University of Pittsburgh Institutional Review Board.

Metabolic assessments.

Subjects were admitted to the University of Pittsburgh General Clinical Research Center 1 day before studies. Research subjects were instructed to ingest ≥200 g of carbohydrate and avoid strenuous exertion for 3 days preceding studies. On the evening before physiological investigations, subjects received a standard dinner (10 kcal/kg; 50% carbohydrate, 30% fat, 20% protein) and then fasted until completion of the metabolic measurements on the following day. In the morning, for the study of substrate exchange across the leg, catheters for blood sampling were inserted in a radial artery and femoral vein, as previously described (10, 25). To measure uptake of FFA across the leg, an infusion of [9,10-3H]oleate (New England Nuclear, Boston, MA), at 0.4 μCi/min, was begun in a forearm vein, with 1 h for isotope equilibration before arteriovenous sampling. During a 40-min period of basal (fasting) determinations, nine paired sets of arterial and femoral venous blood samples were obtained in iced, heparinized syringes for immediate measurement of blood O2 and CO2 content; each set was obtained at 5-min intervals. At 10-min intervals, arterial and venous blood was obtained for measurement of glucose, plasma FFA, and FFA specific activity and for determination of arterial insulin. Venous occlusion strain-gauge plethysmography was used to measure resting blood flow to the leg; the mean of five tracings was used to estimate resting limb blood flow. After completion of fasting measurements, a percutaneous muscle biopsy of the vastus lateralis muscle was performed. Muscle samples were immediately frozen in liquid N2 and kept at −80°C for later assay.

A 3-h continuous infusion of regular insulin (Humulin, Eli Lilly, Indianapolis, IN) at a rate of 40 mU ⋅ min−1 ⋅ m−2was started 30 min after completion of the muscle biopsy procedure. Blood glucose was measured every 5 min, and an infusion of 20% dextrose was adjusted to maintain euglycemia. During the final 40 min of insulin infusion, arterial and femoral venous blood sampling was repeated, with measurement of leg blood flow as described for the baseline period.

Weight loss intervention.

Subjects who completed the above metabolic assessments and who had a body mass index (BMI) ≥30 kg/m2were invited to participate in a 4-mo outpatient weight loss (WL) program. The goal of the WL intervention was to produce a WL of 10–15 kg and to do so by dietary restriction of calories and without an increase in patterns of physical activity. The WL program combined a very low calorie diet (VLCD) with an intensive program of behavioral intervention, followed by a period of weight stabilization, as previously described (58). The WL program was initiated within several days of the completion of baseline physiological assessments. Briefly, during the first 10 wk of this program, subjects were instructed to consume a VLCD (800 kcal/day), and this was followed by 2 wk of gradual refeeding up to isocaloric requirements and then 4 wk of weight stabilization. During the VLCD, subjects consumed a combination of liquid formula (Optifast, Novartis) and lean meat, fish, and fowl. During weeks 11–13, subjects were instructed to consume 1,200 kcal/day, with a gradual reintroduction of fruits, vegetables, and grains to the diet. Duringweeks 14–17, subjects were instructed to consume a weight-maintaining diet, with 30% of calories as fat, 15% as protein, and 55% as complex carbohydrates to avoid effects of acute fasting or calorie restriction on post-WL physiological assessments. Subjects were seen weekly for 16 wk by a nutritionist/behaviorist. To avoid potential confounding effects of exercise on muscle biochemistry and physiology, subjects were carefully instructed to maintain pre-WL levels of physical activity. Vitamin and mineral supplementation was provided during the VLCD. Serum potassium was monitored at 2 wk and then repeated at 4-wk intervals, with measurement of uric acid and other blood chemistries and periodic electrocardiograms. Metabolic reassessments, which entailed all of the parameters described previously, were repeated at the conclusion of the WL intervention and the 4-wk interval of weight stabilization.

Body composition and measurement of maximal aerobic power.

At baseline and after WL, muscle composition was assessed using cross-sectional computed tomography (CT) imaging of the mid-thigh. Systemic fat mass (FM) and fat-free mass (FFM) were assessed using dual-energy X-ray absorptiometry (Lunar model DPX-L, Madison, WI). Details for both methods have been previously described (24). To assess maximal aerobic power (V˙o 2 max), an incremental, modified Bruce protocol on an electronically braked cycle ergometer (Bosch ERG 601, Aachen, Germany) was used with continuous monitoring of heart rate. The test forV˙o 2 max was done ≥1 wk before studies of substrate metabolism (pre-WL and post-WL) were performed.

Analyses.

For analysis of FFA radioactivity, plasma FFA were extracted into isopropyl alcohol-heptane-1 NH2SO4(40:10:1), dried using vacuum evaporation (Savant Instruments, Farmingdale, NY), and reconstituted with scintillation fluid, and radioactivity was measured using liquid scintillation counting, as previously described (10, 12, 14). Before extraction, [14C]oleate was added to each sample as an internal standard, with a mean recovery of [14C]oleate >97%. Plasma FFA were measured using an enzymatic, colorimetric kit (Wako NEFA, Wako Chemical, Nuess, Germany). Samples for blood gas analysis were obtained in iced, heparinized syringes and immediately analyzed at the bedside using a blood gas analyzer to measure pH and plasma CO2 (IL BG3, Instruments Lab, Waltham, MA) and a co-oximeter (IL 482) to measure hemoglobin (Hb), Hb saturation, and blood O2 content. Blood CO2 content was calculated from measured plasma CO2 by use of a regression equation with measured values for Hb, Hb saturation, and pH (13). The mean of the nine arterial or femoral venous samples from basal and insulin-stimulated conditions was used for calculations. Blood glucose was measured, in duplicate with an automated glucose oxidase reaction (YSI 2300 Stat Plus glucose analyzer, Yellow Springs Instruments, Yellow Springs, OH), and the means of five basal and five insulin-stimulated samples were used for limb balance calculations. Serum insulin was determined using a commercially available radioimmunoassay kit (Pharmacia, Uppsala, Sweden).

For the determination of enzyme activity in skeletal muscle, small pieces of the muscle sample (∼10 mg) were homogenized in a glass-glass Duall homogenizer with 39 vol of ice-cold extracting medium (0.1 M Na+-K+-phosphate, 2 mM EDTA, pH = 7.2). Homogenate was transferred into 1.5-ml polypropylene tubes, and this suspension was magnetically stirred on ice for 15 min and sonicated six times for 5 s at 20 W, on ice, with pauses of 85 s between pulses. The resulting homogenate was used for determination of activity levels of cytochrome-coxidase (COX; EN 1.9.3.1) and carnitineO-palmitoyltransferase (CPT; EN 2.3.1.21). Spectrophotometric techniques were conducted at 30°C, according to methods previously used (10, 54). Values of these enzyme activities are expressed in units of micromoles of substrate per minute per gram of wet weight tissue (μM ⋅ min−1 ⋅ g−1).

Calculations.

Leg uptake of glucose was calculated as the product of the arteriovenous differences and blood flow. Uptake of FFA across the leg was calculated as the product of plasma flow, arterial FFA concentration, and the fractional extraction of [9,10-3H]oleate across the leg (53). Fractional extraction of [9,10-3H]oleate across the leg was calculated as the quotient of the arteriovenous difference (dpm/ml) divided by arterial radioactivity (dpm/ml). Plasma flow to the leg was calculated as blood flow × (1 − Hct). Uptake of O2 across the leg (V˙o 2) and production of CO2(V˙co 2) were calculated as the product of arteriovenous differences for O2 and CO2 and blood flow. Values of legV˙o 2 andV˙co 2 were used in indirect calorimetry equations to estimate glucose and lipid oxidation across the leg, as previously described (26).

Statistical analyses.

Data are presented as means ± SE, unless otherwise indicated. Two-way analysis of variance (ANOVA) was used to examine for effects of group (O vs. L) and gender with respect to parameters of leg balance studies, leg respiratory quotient (RQ), body composition, and insulin sensitivity. Repeated-measures ANOVA was used to examine for the effect of WL on these measures. Linear regression and step-wise multiple regression were used to examine for correlation among adiposity,V˙o 2 max, leg balance of substrates, and leg RQ. Statistics were performed using Sigma Stat 2.0 (Jandel Scientific Software, San Rafael, CA).

RESULTS

Skeletal muscle composition.

Body composition of lean and obese men and women are shown in Table1. As per study design, obese subjects had a greater mean weight and BMI than lean subjects and had greater FM and FFM. As measured by cross-sectional CT at the midthigh, obese subjects had more subcutaneous adipose tissue and a greater cross-sectional area of skeletal muscle (both P < 0.01). However, skeletal muscle in obese subjects had a lower attenuation value than that in lean subjects (39.2 ± 0.7 vs. 36.0 ± 0.8 Hounsfield units; P < 0.01). Based on the histogram distribution of these attenuation values, obese subjects had increased “low-density” skeletal muscle (i.e., Hounsfield units 0–30), as also shown in Table 1. Lean and obese subjects had similar values for aerobic fitness, with respective values for V˙o 2 max of 43 ± 0.9 vs. 39 ± 0.8 ml/kg FFM; values in each group are consistent with a sedentary status.

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Table 1.

Clinical characteristics of lean and obese men and women

Fasting metabolism measured by leg balance and enzyme activities.

Fasting values for plasma insulin were higher in obese subjects (46 ± 7 vs. 111 ± 16 pmol/l; P < 0.01), with similar arterial blood glucose (4.56 ± 0.11 vs. 4.68 ± 0.22 mmol/l). Fasting values for the arteriovenous difference of glucose across the leg were similar in lean and obese subjects, as shown in Table 2. Also, resting basal rates of blood flow in the leg were similar, and thus fasting rates of glucose uptake across the leg (LGU) were similar in lean and obese subjects. Fasting rates of glucose oxidation across the leg, measured by regional indirect calorimetry, were greater in obese compared with lean subjects (P < 0.01). In both lean and obese subjects, rates of glucose oxidation exceeded rates for uptake of blood glucose. The negative values for net storage are indicative of glycogenolysis during postabsorptive conditions. The negative values for net glucose storage were greater in obesity (−0.12 ± 0.10 vs. −0.35 ± 0.06 μmol ⋅ min−1 ⋅ 100 ml leg tissue−1;P < 0.05).

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Table 2.

Fasting metabolism across the leg

Data on fasting patterns of fatty acid uptake across the leg are also shown in Table 2. During fasting conditions, arterial concentrations of FFA were higher in obesity, but the group difference from lean subjects was not statistically significant. In both lean and obese subjects, femoral venous concentrations of plasma FFA were higher than arterial concentrations. The net FFA release across the leg during fasting conditions did not differ significantly in lean compared with obese subjects (−127 ± 32 vs. −149 ± 28 nmol ⋅ min−1 ⋅ 100 ml leg tissue−1; L and O, respectively). The constant infusion of labeled oleate permitted measurement of uptake of plasma FFA across the leg despite the negative net balance of plasma FFA. As shown in Table 2, there was robust fractional extraction (FEX) of labeled FFA across the leg, indicative of the uptake of plasma FFA by leg tissues during fasting conditions. The FEX was similar in lean and obese subjects and was ∼10- to 15-fold higher during fasting conditions than corresponding FEX for glucose. Rates of FFA uptake across the leg were similar in lean and obese subjects. However, rates of fat oxidation across the leg during fasting conditions were significantly greater in lean compared with obese subjects (P < 0.01). In both groups, rates of fatty acid uptake across the leg were greater than the corresponding rates of fat oxidation during fasting conditions. The difference (uptake − oxidation), or net storage of fatty acids taken up by leg tissues, was significantly greater in obese subjects (Δ188 ± 48 and Δ249 ± 26 nmol ⋅ min−1 ⋅ 100 ml leg tissue−1;P < 0.05, lean vs. O).

In vastus lateralis skeletal muscle, obese subjects had lower activity of COX, a marker enzyme of oxidative capacity (8.82 ± 0.55 vs. 7.43 ± 0.32 mmol substrate ⋅ min−1 ⋅ g wet weight muscle−1, L and O, P < 0.05). Skeletal muscle in obesity also had a lower activity of CPT, regarded as rate limiting for entry of long-chain fatty acyl-CoA esters into mitochondria (0.138 ± 0.007 vs. 0.109 ± 0.004 mmol substrate ⋅ min−1 ⋅ g wet weight muscle−1, L and O, P < 0.01). CPT activity in skeletal muscle was positively correlated with activity of COX (r = 0.60,P < 0.001).

Leg indirect calorimetry.

During both fasting and insulin-stimulated conditions, nine arterial and nine femoral venous blood gas determinations were obtained during 40 min of steady-state conditions. Arterial Hb saturation was similar in lean and obese subjects during basal conditions (0.96 ± 0.02 vs. 0.95 ± 0.02) and had a mean within-subject coefficient of variation (CV) of 0.5%. Femoral venous Hb saturation was similar in lean and obese subjects (0.72 ± 0.02 vs. 0.70 ± 0.01), although the within-subject CV was greater at 5.2%. Arterial and venous Hb saturation was not affected by insulin infusion. Femoral venous pH was significantly lower than arterial values (P < 0.001), and the mean within-subject CV for measurement of pH was below 0.5% for both arterial and venous samples. The within-subject CVs for measurement of arterial and femoral venous CO2were 1.9 and 1.7%, respectively, and did not change during insulin infusions compared with basal conditions.

During fasting conditions, the RQ across the leg was lower in lean compared with obese subjects (0.83 ± 0.02 vs. 0.90 ± 0.01,P < 0.01), as shown in Table3 and Fig. 1. On the basis of values for leg RQ, lean subjects derived a substantially greater percentage of energy expenditure (EE) from lipid oxidation (59 ± 7 vs. 35 ± 4%; L and O,P < 0.01), as shown in Fig. 1. Basal rates of EE across the leg did not differ between lean and obese subjects.

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Table 3.

Indirect calorimetry across the leg

Fig. 1.

A: fasting and insulin-stimulated values for respiratory quotient (RQ) across the leg in lean (L) and obese (O) subjects. In L but not in O subjects, there was a significant increase in leg RQ during insulin infusions (*P < 0.01). Fasting leg RQ was higher in O subjects (**P < 0.01), whereas insulin-stimulated leg RQ was higher in L subjects (**P < 0.01).B: relative contributions of lipid oxidation (filled bar) and carbohydrate oxidation (open bar) to resting energy expenditure. During fasting conditions, O subjects derived a lesser amount of energy expenditure from lipid oxidation (*P < 0.01); during insulin-stimulated conditions, L subjects had greater suppression of lipid oxidation (**P < 0.01).

In lean subjects, infusion of insulin stimulated a significant increase in leg RQ (P < 0.001), as shown in Fig. 1, whereas, in obese subjects, the insulin-stimulated values for leg RQ did not differ from fasting values of leg RQ. In lean subjects, infusion of insulin stimulated a significant increase in rates of EE across the leg (P < 0.01), as shown in Table 3, whereas in obese subjects, rates of EE across the leg were unchanged compared with fasting conditions. Insulin-stimulated values for leg RQ were significantly greater in lean compared with obese subjects (0.99 ± 0.03 vs. 0.91 ± 0.02;P < 0.01). During insulin infusions in lean individuals, glucose oxidation accounted for 87 ± 8% of EE, and lipid oxidation accounted for just 13 ± 8% of EE. In obese subjects, lipid oxidation accounted for 35 ± 4% of EE during insulin infusions; this value was unchanged from the basal condition, as was the absolute rate of fat oxidation across the leg. Thus, during insulin-stimulated conditions, obese subjects manifested a failure to suppress lipid oxidation, and rates of lipid oxidation were unchanged from fasting conditions.

Insulin-stimulated metabolism measured by leg balance.

Arterial glucose was maintained at euglycemic values during insulin infusions in lean and obese subjects (4.88 ± 0.08 vs. 5.03 ± 0.06 mmol/l). During insulin infusions, arterial insulin was higher in obese subjects (483 ± 18 vs. 596 ± 25 pmol/l;P < 0.01), consistent with reduced insulin clearance in obesity. During steady-state insulin-stimulated conditions, obese subjects manifested systemic insulin resistance (9.6 ± 0.6 vs. 6.0 ± 0.4 mg ⋅ min−1 ⋅ kg FFM−1;P < 0.001) and insulin-resistant glucose metabolism across the leg (P< 0.001). Values for leg glucose uptake, oxidation, and nonoxidative glucose metabolism are shown in Table 4. During insulin infusions, lean subjects had twofold greater arteriovenous differences for glucose compared with obese subjects and slightly higher, although not significantly greater, blood flow than obese subjects. Rates of leg glucose uptake in lean subjects were twice the value in obese subjects. During insulin infusions, lean subjects had significantly higher rates of glucose oxidation than obese subjects (P < 0.01). Rates of glucose uptake exceeded rates of glucose oxidation in both groups. In lean subjects, rates of glucose oxidation accounted for 40 ± 5% of glucose uptake, whereas in obese subjects the percentage of glucose uptake accounted for by glucose oxidation was 55 ± 5%. Nonoxidative glucose metabolism across the leg during insulin infusion was significantly higher in lean subjects (2.03 ± 0.32 vs. 0.78 ± 0.15 μmol ⋅ min−1 ⋅ 100 ml leg tissue−1;P < 0.001). Insulin-stimulated LGU had a positive correlation with systemic glucose metabolism (r = 0.65;P < 0.001) and a negative correlation with BMI (r = −0.53;P < 0.001).

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Table 4.

Insulin-stimulated metabolism across the leg

The effect of insulin to augment LGU (clamp minus basal rates) was at least twofold greater in lean subjects (changes of: Δ3.11 ± 0.31 vs. Δ1.20 ± 0.18 μmol ⋅ min−1 ⋅ 100 ml tissue−1;P < 0.001). The insulin-induced change in leg blood flow also tended to be higher in lean subjects, although this difference was not significant (changes of Δ0.56 ± 0.21 vs. Δ0.21 ± 0.17 ml ⋅ min−1 ⋅ 100 ml leg tissue−1,P = 0.17). The insulin-induced change in glucose oxidation across the leg was greater in lean subjects (0.66 ± 0.16 vs. 0.08 ± 0.11 μmol ⋅ min−1 ⋅ 100 ml tissue−1, L and O,P < 0.001), indicating the lack of responsiveness to modulation of glucose oxidation in obesity-related insulin resistance.

During insulin infusions, plasma FFA fell considerably compared with fasting conditions in both lean and obese subjects (P < 0.01), and steady-state values were slightly but not significantly higher in obesity, as shown in Table 4. Femoral venous FFA also decreased substantially. The net balance for plasma FFA across the leg, which had been strongly negative during fasting conditions, narrowed to values close to or slightly above zero but were not significantly different in lean and obese subjects (37 ± 18 vs. 1 ± 5 nmol ⋅ min−1 ⋅ 100 ml leg tissue−1). As during the basal condition, FEX of labeled oleate continued to be robust during insulin infusions. However, rates of FFA uptake across the leg during insulin-stimulated conditions were substantially less than during fasting conditions (P < 0.001). Rates of FFA uptake across the leg were similar in lean and obese subjects during insulin infusion. During insulin infusions in lean subjects, rates of fat oxidation across the leg were lower than during the basal condition (P < 0.01). In obesity, rates of fat oxidation across the leg were higher than in lean subjects (P < 0.01) and were unchanged from basal rates. In lean subjects, there was a small positive difference between rates of fatty acid uptake and rates of lipid oxidation during insulin-stimulated conditions (uptake − oxidation: Δ42 ± 12 nmol ⋅ min−1 ⋅ 100 ml leg tissues−1), whereas in obese subjects, there was a negative balance (Δ−33 ± 12 nmol ⋅ min−1 ⋅ 100 ml leg tissues−1). The group difference in net fatty acid storage across the leg was significant (P = 0.02). The negative value in obesity would be consistent with utilization of muscle triglyceride and, hence, incomplete suppression of lipolysis within muscle during insulin infusions.

Association between insulin sensitivity and fasting metabolism.

The second objective of this study was to address the potential relation between insulin-resistant glucose metabolism and fasting or insulin-stimulated patterns of fatty acid uptake and oxidation. A decreased reliance on lipid oxidation during fasting conditions was associated with resistance to insulin stimulation of glucose metabolism; as shown in Fig. 2, fasting values for leg RQ were negatively correlated with insulin sensitivity (r = −0.57;P < 0.001). Thus, in addition to group differences in fasting leg RQ, there was significant correlation between the individual variation in the severity of obesity-related insulin resistance and fasting patterns of glucose and lipid oxidation in leg tissues. However, fasting levels of fatty acids and fasting rates of fatty acid uptake across the leg did not correlate significantly with insulin resistance.

Fig. 2.

Fasting values of leg RQ are plotted against insulin-stimulated glucose utilization in L and O subjects. The correlation was significant:r = −0.57,P < 0.001.

During insulin infusions, rates of leg lipid oxidation were negatively correlated to insulin sensitivity (r = −0.45, P < 0.001). However, the correlation between insulin-stimulated LGU and rates of FFA uptake across the leg during the clamp was not significant. Obese subjects had less change in leg RQ in response to insulin infusion than did lean subjects. Across the entire cohort, the amplitude of insulin-stimulated change in leg RQ (Δleg RQ: insulin-stimulated leg RQ − fasting leg RQ) correlated significantly with insulin-stimulated increases in LGU (r= 0.54, P < 0.001), as well as with systemic insulin sensitivity (r = 0.66, P < 0.001). This indicates that response to insulin modulation of leg RQ was related to stimulation of glucose uptake.

Patterns of fat oxidation and insulin-resistant glucose metabolism also correlated significantly with the biophysical characteristics of skeletal muscle composition determined by CT. Skeletal muscle attenuation values correlated with insulin sensitivity (r = 0.48,P < 0.01). In stepwise regression analysis, fasting leg RQ and the cross-sectional area of low-density muscle contributed independently to predict 48% of the within-group variance in insulin sensitivity of LGU. Greater amounts of low-density muscle measured by CT imaging also correlated with higher rates of leg lipid oxidation during the clamp (r = 0.43, P < 0.01). Also, rates of net fatty acid storage during the clamp (which were negative in obesity and indicative of nonsuppressed lipolysis) correlated negatively with the amount of low-density muscle (r = −0.45; P < 0.05). Values of leg RQ or LGU did not correlate with the amount of subcutaneous adipose tissue in the thigh.

Effect of WL on muscle FFA metabolism.

The third objective of the current project was to examine the effect of WL on fasting patterns of fatty acid metabolism by leg muscle and on insulin-stimulated patterns of glucose metabolism. The time course of the WL is shown in Fig. 3; as shown, 4 wk of weight stabilization preceded reassessments of metabolism. Thirty-two subjects completed the WL intervention. Mean WL was 14.0 ± 0.9 kg, of which 10.3 ± 0.8 kg were loss of FM. Detailed studies of changes in body composition and in relation to changes in insulin-stimulated glucose metabolism have been previously reported (24). After WL, measurements ofV˙o 2 max were repeated and were unchanged compared with pre-WL values (39 ± 0.8 vs. 40 ± 1.1 ml/kg FFM).

Fig. 3.

Weight loss (WL) progression during 12 wk of caloric restriction and 4 wk of weight stabilization.

There were a number of systemic metabolic changes after WL. Fasting plasma insulin decreased (117 ± 5 vs. 62 ± 5 pmol/l;P < 0.01), as did plasma leptin (from 39 ± 2 to 21 ± 2 ng/ml in women and from 16 ± 3 to 6 ± 2 ng/ml in men). Also, as previously reported (24), there were reductions in fasting plasma triglycerides, apoB, and total cholesterol. In these glucose-tolerant subjects, WL did not significantly change fasting plasma glucose or plasma FFA (535 ± 42 vs. 499 ± 26 pmol/l, pre-WL vs. post-WL).

In the leg balance studies, as shown in Table5, there were no significant changes in fasting rates of glucose uptake. There was a modest but statistically significant decrease in resting blood flow. Resting EE across the leg was reduced after WL (P < 0.01). Values for leg RQ during fasting conditions were unchanged after WL (0.90 ± 0.02 vs. 0.90 ± 0.02, pre-WL and post-WL, respectively), and the slight decreases in rates of basal oxidation of glucose and fat measured across the leg were not statistically significant compared with respective pre-WL values. The FEX of plasma FFA across the leg decreased after WL (39 ± 2 vs. 34 ± 2%, pre-WL vs. post-WL, P < 0.05), and rates of FFA uptake across the leg were lower after WL (P < 0.05). Rates of FFA uptake continued to exceed corresponding rates of lipid oxidation across leg tissues (uptake − oxidation: Δ253 ± 37 vs. Δ174 ± 26 nmol ⋅ min−1 ⋅ 100 ml leg tissue−1, pre-WL and post-WL, respectively), and the net storage of FFA across the leg declined significantly after WL (P < 0.05). With respect to muscle composition, there was a decrease in cross-sectional area of thigh muscle, and this was entirely due to decrease in the area of low-density muscle, because the area of normal-density muscle did not change. The activity of the oxidative enzyme COX decreased significantly (7.82 ± 0.93 vs. 6.72 ± 0.38 mmol substrate ⋅ min−1 ⋅ g wet weight muscle−1;P < 0.05). Activity of muscle CPT did not change after WL (0.120 ± 0.005 vs. 0.124 ± 0.005 mmol substrate ⋅ min−1 ⋅ g wet weight muscle−1, not significant).

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Table 5.

Fasting metabolism across the leg after weight loss

Data on post-WL metabolism during insulin infusions are shown in Table6. Arterial glucose was maintained at similar values during pre-WL and post-WL clamp studies (5.03 ± 0.06 vs. 5.00 ± 0.04 mmol/l). Despite equivalent insulin infusions, arterial insulin was lower after WL (596 ± 25 vs. 497 ± 20 pmol/l; P < 0.01), indicative of improved insulin clearance. Systemic insulin-stimulated glucose metabolism increased after WL (5.9 ± 0.4 vs. 7.5 ± 0.5 mg/kg FFM; P < 0.001). Leg balance data during insulin-stimulated conditions are shown in Table 6. The lower values for leg blood flow observed after WL during basal conditions were also observed during insulin infusions (P < 0.001). Thus, despite a twofold increase in arteriovenous differences for glucose after WL (P < 0.001), the increase in insulin-stimulated values for LGU did not achieve statistical significance (P = 0.10). Rates of insulin-stimulated glucose oxidation across the leg were not significantly changed after WL, but rates of nonoxidative glucose metabolism across the leg were significantly increased (0.71 ± 0.16 vs. 1.14 ± 0.18 μmol ⋅ min−1 ⋅ 100 ml leg tissue−1, pre-WL and post-WL, P < 0.01).

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Table 6.

Insulin-stimulated metabolism across the leg after weight loss

During insulin infusions, arterial FFA were lower after WL (94 ± 13 vs. 54 ± 5 μmol/l, P < 0.001). Uptake of plasma FFA across the leg was lower after WL (69 ± 12 vs. 36 ± 5 nmol ⋅ min−1 ⋅ 100 ml leg tissues−1;P < 0.001). After WL, insulin infusions significantly increased leg RQ (P < 0.01), as shown in Fig.4, and significantly suppressed fat oxidation by leg tissues (P< 0.01), whereas these effects were markedly blunted before WL. As previously described, during insulin infusions before WL, obese subjects had a higher rate of lipid oxidation than corresponding rates of fatty acid uptake (Δ−26 ± 12 nmol ⋅ min−1 ⋅ 100 ml leg tissues−1), indicative of incomplete suppression of lipolysis within leg tissues. However, after WL, rates of FFA uptake and rates of lipid oxidation during insulin infusions were similar, and the rate of net FFA storage was not significantly different from zero (Δ2 ± 12 nmol ⋅ min−1 ⋅ 100 ml leg tissues−1). This indicates more effective insulin suppression of lipolysis in leg tissues after WL and similar to the pattern observed in lean subjects. The change in rates of lipid oxidation after WL (lower) were correlated with the change in nonoxidative glucose metabolism (r = −0.36,P < 0.05).

Fig. 4.

A: fasting and insulin-stimulated leg RQ in O subjects before and after WL. Fasting leg RQ did not change after WL, but insulin-stimulated values increased (*P < 0.01).B: relative contributions of lipid oxidation (filled bar) and carbohydrate oxidation (open bar) to resting energy expenditure. During fasting conditions, the proportion of energy derived from lipid oxidation remained similar, whereas during insulin infusions, there was a greater suppression of lipid oxidation after WL (*P < 0.01).

DISCUSSION

One of the striking physiological characteristics of skeletal muscle is metabolic flexibility (48), expressed by a high capacity to modulate rates of energy production, blood flow, and substrate utilization. Part of this metabolic plasticity of skeletal muscle is the capacity to utilize either carbohydrate or fatty acids. In obesity, there is a well-characterized impairment in the capacity of skeletal muscle to modulate glucose uptake in response to insulin (15). The present studies were undertaken to address whether another aspect of obesity-related “insulin resistance” could be perturbations of fatty acid metabolism during fasting conditions. The chief impetus for this query derives from the emerging concept that fat deposition within skeletal muscle is increased in obesity and is associated with the severity of insulin resistance (23, 38, 53). These findings raise the question of how lipid accumulates within skeletal muscle in obesity. During resting postabsorptive conditions, as after an overnight fast, it is generally considered that the predominant substrate oxidized by skeletal muscle is lipid, provided substantially by a high rate of uptake of plasma FFA (2, 55).

In the current study, during fasting conditions, these patterns were reaffirmed in lean subjects. However, in obese subjects, despite rates of fatty acid uptake that were equivalent to those of lean subjects, fasting rates of fatty acid oxidation by leg tissues were significantly lower. The values for the RQ across the leg denoted a reduced reliance on lipid oxidation in obesity, such that only one-third of energy production was accounted for by fat oxidation, whereas nearly twice this proportion was found in muscle of lean volunteers. Skeletal muscle in the obese subjects had lower attenuation value on CT, a biophysical characteristic suggestive of increased lipid content (9). Fasting rates of fatty acid uptake across the leg were greater than fasting rates of lipid oxidation, indicating a modest net surplus of fatty acid uptake, as has been well described in animal studies (8, 14). These rates of “net storage” of fatty acids were greater in obesity. Thus the paradigm we would propose on the basis of these novel physiological observations is that, in obesity, skeletal muscle accrues triglyceride due to a reduced rate of lipid oxidation in the face of equivalent fatty acid uptake. Furthermore, fasting patterns of fatty acid metabolism by muscle correlated with the severity of insulin-resistant glucose metabolism and, in obesity, fat oxidation in muscle is resistant to modulation by physiological hyperinsulinemia. It was also observed that weight loss only partially rectifies these patterns, with lesser effects on fatty acid metabolism compared with glucose metabolism, raising the provocative question as to whether abnormal fatty acid metabolism by muscle is a primary rather than acquired metabolic impairment in obesity or obesity-prone individuals.

A decreased reliance on lipid oxidation has previously been identified as a metabolic risk factor for weight gain (59), and this was related to a decreased oxidative enzyme capacity of skeletal muscle (17, 60). The findings of the current study provide further support that decreased oxidative enzyme capacity of skeletal muscle and reduced activity of CPT may be a mechanism responsible for reduced fasting rates of fatty acid oxidation in obesity, despite more than sufficient uptake of plasma fatty acids and despite an expanded tissue triglyceride content. Lower oxidative enzyme capacity in muscle in obesity has been previously reported (32), as has lower CPT activity (10), and in prior studies, we had reported a positive correlation between oxidative enzyme capacity of muscle and insulin-stimulated glucose metabolism (10, 54); this has been confirmed by others (28). In studies with rat skeletal muscle, it has been well established that oxidative muscle fibers have an increased capacity for the uptake and oxidation of fatty acids compared with glycolytic muscle fibers (8, 14,37). Marked defects of CPT in skeletal muscle can produce clinically symptomatic impairment of fat oxidation, manifest especially during fasting and exercise, as well as lipid accumulation within muscle (50). However, the severity of the impairment in CPT found among obese subjects in the current study is not this extreme but nevertheless might impede fatty acid oxidation.

The concept that perturbed skeletal muscle fatty acid metabolism may contribute to skeletal muscle insulin resistance in certainly not new (34, 40), although the hypothesis that insulin resistance is associated with decreased fasting fatty acid oxidation is a novel reformulation. It has been well established, including work from our laboratory, that experimental infusions of fatty acids can induce insulin resistance (5,25). However, several recent studies indicate that glucose inhibits fat oxidation (33, 52), a so-called “reverse Randle cycle,” which could be pertinent to the observation that insulin-resistant skeletal muscle in animal models of obesity has increased malonyl-CoA (44, 46,47). Under the stimulation of insulin, utilization of fatty acids by skeletal muscle is normally suppressed (25, 26). In obesity, this effect of insulin to suppress lipid oxidation is blunted (16, 30), which does fit the classic concept of fatty acid-induced insulin resistance (40). Incomplete suppression of lipid oxidation during insulin stimulation was observed among the obese volunteers in the current study, and rates of muscle lipid oxidation during insulin infusion correlated with the severity of insulin-resistant glucose metabolism. Yet, these observations do not indicate that fatty acid oxidation within insulin-resistant muscle is persistently “increased.” Although insulin infusion did not suppress muscle lipid oxidation in obesity (compared with strong suppression in lean individuals), a salient point to emphasize is that, in obesity, rates of fat oxidation in skeletal muscle during insulin infusion were unchanged from the fasting condition and were lower than in lean individuals during fasting. Rates of fatty acid uptake during insulin infusions did not have a significant association with insulin resistance, whereas, in stepwise regression analysis, greater amounts of low-density muscle and an elevated fasting leg RQ contributed independently to account for nearly one-half of the variability in insulin resistance. The amount of low-density muscle also correlated (negatively) with the suppression of fat oxidation by insulin. The tissue composition data reinforce the emerging concept that muscle triglyceride in obesity is reflective of the severity of insulin resistance and may be a link between perturbed metabolism of fatty acids and insulin-resistant glucose metabolism. Thus, in regard to the nature of substrate competition, muscle in obesity most plainly manifested a severe inflexibility in the modulation of fatty acid oxidation, with neither suppression by insulin infusion nor an appropriate enhancement in response to an overnight fast.

Another important component of the metabolic inflexibility and perturbed patterns of fatty acid oxidation in obesity was the observation that a poor reliance on fatty acid oxidation by skeletal muscle during fasting conditions significantly predicted the severity of insulin-resistant glucose metabolism, as was shown in Fig. 1. To our knowledge, this is the first report of a correlation between fasting patterns of substrate oxidation in muscle and insulin-resistant glucose metabolism. The observation, although only associative in nature rather than truly mechanistic, is important because it extends the “phenotype of insulin resistance” in skeletal muscle beyond defects of insulin-stimulated metabolism to a broader concept of an organ system poorly performing one of its key homeostatic functions of substrate utilization. This concept may be central to understanding the role of skeletal muscle in the pathogenesis of obesity and obesity-related comorbid conditions, such as type II diabetes mellitus.

The third objective of the current study was to examine the effect of weight loss, and more narrowly defined, weight loss achieved through restricted calorie intake and without changes in physical activity patterns or maximal aerobic capacity. A substantial weight loss was achieved and then was followed by a 1-mo period of careful weight stabilization before reassessments of metabolism. The positive effects of this weight loss on insulin-resistant glucose metabolism, and on reductions in visceral adiposity, muscle adiposity, and metabolic factors such as hyperinsulinemia, lipoproteins, and blood pressure have been previously reported (24). The data of the current study concern primarily the effects on patterns of fatty acid uptake and oxidation in skeletal muscle. With respect to fasting patterns of skeletal muscle fatty acid metabolism, the effect of weight loss was relatively modest. Skeletal muscle oxidative enzyme activity was slightly diminished, and activity of CPT was unchanged (and remained lower than in lean subjects). Not unexpectedly (1, 29), rates of resting energy expenditure measured by limb balance were lower than in the obese state. The rates of fatty acid uptake were slightly reduced. However, the leg RQ was unchanged from pre-WL values, and thus reliance of skeletal muscle upon fat oxidation during fasting conditions was not improved. Persistent impairment of fatty acid metabolism has been previously reported after weight loss (4, 7, 41). These data would suggest that these defects could be primary impairments, leading to obesity rather than merely resulting from obesity. Furthermore, it is interesting to speculate on what might have happened had the intervention included aerobic exercise. In lean sedentary subjects, aerobic exercise training promotes an increase in oxidative enzyme capacity, and this is accompanied by an increased rate of fatty acid oxidation during exercise conditions (22, 56).

Two changes that did occur in muscle fatty acid metabolism after weight loss were that insulin infusion stimulated a significant rise in leg RQ (compared with fasting values) and a significant suppression of fat oxidation, and there was an overall increase in muscle attenuation values on CT imaging. Furthermore, after weight loss, rates of fatty acid uptake during insulin-stimulated conditions were nearly equivalent to rates of fat oxidation, whereas the rate of fat oxidation had exceeded fatty acid uptake during insulin infusions before weight loss. This change in insulin-stimulated patterns of muscle fatty acid metabolism is indicative of more complete suppression of lipolysis of muscle triglyceride.

Many of the observations of the current study are based on limb balance methods, including the use of regional indirect calorimetry, and it is important to review some of the limitations of these methods. One of the advantages of limb balance is to “isolate” peripheral tissue metabolism from systemic patterns, which is of particular value for studies of fasting metabolism wherein the contribution of skeletal muscle is relatively modest. The original observations of a high reliance on fatty acid oxidation by human skeletal muscle in lean, healthy individuals were made by Andres and colleagues (Andres and Cadar, Ref. 2, and Baltzan et al., Ref. 3) by use of the limb balance method; the current data on lean subjects closely conform to the patterns presented in those previous studies. Furthermore, the mean rates of lipid and glucose oxidation across the leg found in the current study are highly consistent with several prior reports from our laboratory published over the past five years (10, 27, 33). Nevertheless, it is important, as previously suggested by Frayn et al. (19), that data on regional indirect calorimetry be interpreted within the context of substrate exchange. This was comprehensively followed in the current study, in that values for leg RQ were one aspect of an integrated physiological context of fatty acid and glucose uptake, tissue composition, and muscle biochemistry. In this context, an elevated fasting value for leg RQ and a resistance to modulate values for leg RQ by insulin infusion were significantly correlated with obesity, with altered muscle composition in obesity (in a pattern denoting increased lipid content), and with insulin-resistant patterns of glucose uptake. The tissues of the leg that could be responsible for the leg RQ patterns include skeletal muscle, adipose tissue, skin, and bone. Adipose tissue has a relatively high value for RQ, of ∼1.0, (18), and conceivably the increased adiposity of the leg in obese subjects caused the higher values for leg RQ. However, adipose tissue has a substantially lower rate of oxygen consumption than skeletal muscle, being estimated at approximately one-tenth of the rate of oxygen consumption by resting skeletal muscle. In the current study, rates of energy expenditure across the leg were similar in lean and obese subjects despite the greater adiposity in obese subjects, and there was no significant correlation between the amount of thigh adiposity and rates of energy expenditure across the leg. Furthermore, there was no significant correlation between leg adipose tissue and values for leg RQ, whereas the values for leg RQ did relate to several independently measured parameters of leg glucose metabolism, and these are widely accepted as predominantly reflective of muscle metabolism. Moreover, the enzyme activities and determinations of muscle composition are independent markers that skeletal muscle has a reduced efficiency for the oxidation of fat in obesity. Another potential consideration concerns the determinations of blood flow, or more precisely the tissue distribution of blood flow, which is important both for calculation of limb balance exchanges of substrate and for interpolation of respective tissue contributions (e.g., muscle and adipose tissue) to metabolism. Venous occlusion strain-gauge plethysmography, as used in the current study, provides a measurement of flow to all tissue of the extremity and therefore does not solely represent skeletal muscle blood flow (43).

In summary, the current study indicates that skeletal muscle in obesity is more disposed to partition fatty acids toward esterification during fasting conditions and manifests a lesser reliance on fatty acid oxidation compared with skeletal muscle of lean individuals. Biochemical markers are consistent with these findings, as reduced oxidative enzyme capacity and a diminished activity of CPT were found in obesity. The diminished capacity for fatty acid oxidation in obesity may contribute more directly to accretion of triglyceride within muscle than fatty acid uptake per se, because rates of the latter were not increased in obesity. Also, fasting patterns of fatty acid oxidation by muscle in obesity were found to associate with insulin-resistant glucose metabolism and yet, unlike glucose metabolism, did not improve substantially after weight loss, although improvement in insulin suppression of muscle lipid oxidation was observed. These intervention data suggest that an important area of future research should be to better determine whether the fasting impairment of fat oxidation in skeletal muscle is indeed a primary metabolic defect disposing to obesity and to weight regain after weight loss. In conclusion, the current study supports the concept that perturbations of fatty acid metabolism in skeletal muscle are integral components of the phenotype of obesity-related insulin resistance.

Acknowledgments

We are grateful for the participation of the research volunteers and acknowledge the help and cooperation of the nursing and nutritional staffs at the General Clinical Research Center and the Obesity and Nutrition Research Center of the University of Pittsburgh. We acknowledge specifically the special efforts of Nancy Mazzei, Amy Meier, Susan Andreko, Dr. Monica Nuturajhan, and Freddy Troost. We are also grateful for the support of Novartis Pharmaceuticals in providing Optifast for the weight loss intervention.

Footnotes

  •  Deceased 27 August 1999.

  • This research was supported by funding from National Institutes of Health (NIH) R01 DK-49200–02, General Clinical Research Center Grant 5M01RR-00056, and Obesity and Nutrition Research Center Grant 1P30DK-46204. B. Goodpaster was supported by NIH National Research Service Award DK-07052–23.

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

REFERENCES

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