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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Obesity (Silver Spring). Author manuscript; available in PMC 2011 December 5.
Published in final edited form as:
PMCID: PMC3230250
NIHMSID: NIHMS329920

Altered Intramuscular Lipid Metabolism Relates to Diminished Insulin Action in Men, but Not Women, in Progression to Diabetes

Abstract

Whether sex differences in intramuscular triglyceride (IMTG) metabolism underlie sex differences in the progression to diabetes are unknown. Therefore, the current study examined IMTG concentration and fractional synthesis rate (FSR) in obese men and women with normal glucose tolerance (NGT) vs. those with prediabetes (PD). PD (n = 13 men and 7 women) and NGT (n = 7 men and 12 women) groups were matched for age and anthropometry. Insulin action was quantified using a hyperinsulinemic–euglycemic clamp with infusion of [6,6-2H2]-glucose. IMTG concentration was measured by gas chromatography/mass spectrometry (GC/MS) and FSR by GC/combustion isotope ratio MS (C-IRMS), from muscle biopsies taken after infusion of [U-13C]palmitate during 4 h of rest. In PD men, the metabolic clearance rate (MCR) of glucose was lower during the clamp (4.71 ± 0.77 vs. 8.62 ± 1.26 ml/kg fat-free mass (FFM)/min, P = 0.04; with a trend for lower glucose rate of disappearance (Rd), P = 0.07), in addition to higher IMTG concentration (41.2 ± 5.0 vs. 21.2 ± 3.4 μg/mg dry weight, P ≤ 0.01), lower FSR (0.21 ± 0.03 vs. 0.42 ± 0.06 %/h, P ≤ 0.01), and lower oxidative capacity (P = 0.03) compared to NGT men. In contrast, no difference in Rd, IMTG concentration, or FSR was seen in PD vs. NGT women. Surprisingly, glucose Rd during the clamp was not different between NGT men and women (P = 0.25) despite IMTG concentration being higher (42.6 ± 6.1 vs. 21.2 ± 3.4 μg/mg dry weight, P = 0.03) and FSR being lower (0.23 ± 0.04 vs. 0.42 ± 0.06 %/h, P = 0.02) in women. Alterations in IMTG metabolism relate to diminished insulin action in men, but not women, in the progression toward diabetes.

INTRODUCTION

Increasing evidence suggests that the development of type 2 diabetes may be different in men vs. women. Repeated observations show sex differences in the “prediabetic” (also known as high-risk) phase with more women having isolated impaired glucose tolerance and more men having isolated impaired fasting glucose (1,2). As a result, efficacy of preventive interventions appears to differ by sex (3). Sex itself (beyond sex steroids) has been suggested to influence the development of diabetes (1), but the mechanisms underlying its effect have gone largely unexplored.

There is reason to believe that lean, healthy women may be more insulin sensitive than men when matched on important confounders, such as age, ethnicity, BMI, and physical activity level (4,5). As men and women gain fat mass, the distribution generally differs by sex, with more visceral fat deposited in men and more gluteal fat deposited in women (6). Untoward metabolic effects of visceral fat have been well studied, as have those of fat deposited in liver and skeletal muscle (7). Less often discussed, however, is that young women tend to store more fat in skeletal muscle (intramuscular triglyceride (IMTG)) than men (8) without a decrement in muscle insulin action.

The role of IMTG in the pathogenesis of skeletal muscle insulin resistance has received considerable attention as of late. Repeated observations using different techniques have noted a positive linear relationship between IMTG concentration and insulin resistance (912). Nevertheless, a direct effect of IMTG on insulin action is unsupported. Rather, IMTG metabolites, such as diacylglycerol (DAG) (13), ceramide (14), and long-chain acyl-CoA (15), or saturated fats therein (16), may accumulate in a stagnant pool and inhibit insulin signaling. Thus, the relative toxicity of IMTG may be more related to its turnover than absolute pool size.

Some (8,17), but not all (18), studies show that young, healthy women use more IMTG during a bout of exercise than men, highlighting their ability to turn over the IMTG pool. Whether this metabolic adaptation (which may relate to skeletal muscle insulin sensitivity) is lost in women in the development of diabetes is unknown. Therefore, the aims of this study were as follows: (i) to measure IMTG concentration and fractional synthesis rate (FSR), as well as (ii) to measure skeletal muscle insulin action, in obese men and women with and without prediabetes (PD) and (iii) to examine possible sex differences in intramuscular lipid metabolism in the progression toward diabetes. We hypothesized that (i) women with normal glucose tolerance (NGT) would be more insulin sensitive, despite higher IMTG concentration, than men with NGT, but would have a higher FSR, (ii) PD women would have similar IMTG concentration as NGT women, but a lower FSR associated with lower insulin sensitivity, and (iii) IMTG concentration would be higher, whereas FSR and insulin sensitivity would be lower, in men with PD vs. NGT.

METHODS AND PROCEDURES

Subjects

Thirty-nine healthy, sedentary (<90 min/week planned activity), non-smoking men and postmenopausal women between the ages of 45 and 70 were placed into one of the two groups based on two 2-h 75 g oral glucose tolerance tests, separated by 1 week: a control group with NGT (n = 7 men and 12 women; fasting glucose <5.6 mmol/l and 2-h oral glucose tolerance test <7.8 mmol/l), or a group with PD (n = 13 men and 7 women; fasting glucose 5.6–6.9 mmol/l; and/or 2-h oral glucose tolerance test 7.8–11.1 mmol/l) (19). Approval for this study was obtained by the Colorado Multiple Institutional Review Board prior to its commencement.

Prestudy diet control and assessments

Subjects were fed a standardized isocaloric control diet for 3 days prior to admission to the General Clinical Research Center (GCRC) for each of the two separate study days: assessment of (i) insulin action and (ii) intramuscular lipid metabolism. Body composition was estimated from dual-energy X-ray absorptiometry (20).

Subjects were fasted overnight and admitted to the GCRC on the morning of the study days. Upon admission, an intravenous catheter was placed in an antecubital vein for infusion, and sampling catheter was placed in a dorsal hand vein of the contralateral arm. For all blood samples, the heated hand technique was used to arterialize the blood. Background sampling began 30 min after sampling catheters had been placed. A baseline blood sample was drawn for determination of circulating hormone and substrate concentrations (glucose, insulin, C-peptide, free fatty acids (FFAs), glycerol, and lactate).

Assessment of insulin action

For the measurement of glucose turnover, a primed (3.5 mg/kg) constant (0.04 mg/kg/min) infusion of [6,6-2H2]glucose was initiated and continued through the end of the hyperinsulinemic–euglycemic clamp. Resting blood measurements were made over the final 30 min of the 120-min basal infusion to allow for equilibration of the tracer in the glucose pool. Blood samples for tracer, hormone, and substrate concentrations (see above) were taken at the same time points. Indirect calorimetry was performed before starting resting blood sampling using a respiratory canopy (SensorMedics 2900; SensorMedics, Yorba Linda, CA).

A two-stage hyperinsulinemic–euglycemic clamp was then initiated and continued for the next 3 h using the method of DeFronzo et al. (21). A low-dose insulin infusion was used to examine adipose tissue and hepatic insulin sensitivity, whereas the high dose was intended to determine skeletal muscle insulin sensitivity, to better delineate possible sex differences in tissue-specific insulin action. Briefly, a primed continuous infusion of insulin was infused at 4 mU/m2/min for 1.5 h, and then increased to 40 mU/m2/min for the final 1.5 h. A variable infusion of 20% dextrose was infused to maintain blood glucose ~90 mg/dl. Blood was sampled every 5 min to determine glucose concentration and the dextrose infusion adjusted as necessary. The dextrose was “spiked” with 15 mg/ml [6,6-2H2]glucose to minimize changes in isotope enrichment. Blood samples were taken over the final 30 min of both stages of the clamp for measurement of glucose kinetics, as well as hormone and substrate concentrations (see above). Immediately prior to blood sampling during each stage, measurement of respiratory gas exchange was made.

Assessment of intramuscular lipid metabolism

The combination of stable isotope methodology and skeletal muscle biopsy was used to examine intramuscular lipid metabolism. Following the baseline blood draw, a continuous infusion of [U-13C]palmitate (Isotec, Miamisburg, OH) bound to human albumin was initiated at 0.0174 mmol/kg/min and continued throughout the study. Subjects remained semirecumbent for 4 h to allow for tracer incorporation into the intramuscular lipid pools. Blood samples were taken for hormone and substrate concentrations (as above) during the final 30 min of the 4-h rest period. Following the rest period, a vastus lateralis skeletal muscle biopsy was performed using the Bergstrom technique (22). Muscle was immediately flash-frozen in liquid nitrogen and stored at −80 °C until dissection and analysis.

Methods

Circulating hormone and substrate concentrations

All samples were stored at −80 °C until analysis. Radioimmunoassay was used to determine insulin (Linco Research, St Louis, MO) and C-peptide (gamma counter; Diagnostic Products, Los Angeles, CA) concentrations. Standard enzymatic assays were used to measure glucose (COBA-Mira Plus; Roche Diagnostics, Mannheim, Germany), lactate (Sigma kit no. 826; Sigma, St Louis, MO), glycerol (Boehringer Mannheim Diagnostics, Mannheim, Germany), and FFA (NEFA kit; Wako Diagnostics, Richmond, VA). Measures of estradiol and testosterone (Calbiotech, Spring Valley, CA), C-reactive protein (Chemicon International, Temecula, CA), sex hormone–binding globulin (Alpha Diagnostics, San Antonio, TX), leptin (Signosis, Sunnyvale, CA), and adiponectin (Assaypro, St Charles, MO) were made using ELISA.

Whole-body substrate oxidation

Whole-body substrate oxidation was measured using indirect calorimetry. Oxygen consumption and carbon dioxide production were used to calculate metabolic rate, as well as the oxidation of carbohydrate and fat using standard equations (23).

Glucose turnover during the clamp

Glucose isotopic enrichment was measured using gas chromatography/mass spectrometry (GCMS; GC model 6890 series II and MS model 5973A; Hewlett-Packard, Palo Alto, CA) using published methods (24).

Muscle lipid analysis

Skeletal muscle lipid extraction, isolation, and analysis were performed as previously described by our laboratory (25). Briefly, skeletal muscle samples were dissected free of extra muscular fat on ice, lyophilized, added to 1 ml iced MeOH along with internal standards of tripentadecanoic acid and dipentadecanoic acid, and homogenized (Omni TH; Omni International, Marietta, GA). Total lipids were extracted (26) and then added to solid-phase extraction columns (Supelclean LC-NH2, 3 ml; Supelco Analytical, Bellefonte, PA) to isolate FFAs, IMTG, and DAG. IMTG, DAG, and FFA fractions were converted to fatty-acid methyl esters, and the stable isotope ratios of 13C in fatty-acid methyl esters were measured using a gas chromatography-combustion isotope ratio mass spectrometer (GC/C-IRMS) system (Thermo Electron, Bremen, Germany). Concentration and composition analysis were performed on an HP 6890 GC with a 30m DB-23 capillary column, connected to a HP 5973 MS. Peak identities were determined by retention time and mass spectra compared to standards of known composition.

Whole-body palmitate oxidation

Two milliliters of breath CO2 were transferred into a 20 ml exetainer for the measurement of 13CO2/12CO2 with continuous flow isotope ratio mass spectrometry (IRMS) (Delta V; Thermo Electron, Bremen, Germany). Each sample was injected (1.2 μl per injection) in duplicate for isotope ratio analyses, with an average standard deviation for all injections of 0.0001 atom percent.

Plasma palmitate isotope analysis

Methylation and extraction of plasma palmitate were performed as previously described (27). Samples were run on an HP 6890 GC with a 30m DB-23 capillary column, connected to a HP 5973 MS. Enrichments were calculated based on a standard curve of known enrichments and corrected for variations in abundance (28). Peak identities were determined by retention time and mass spectra compared to standards of known composition.

Western blotting

Frozen skeletal muscle samples were weighed, and homogenized on ice using a Kontes glass homogenizer (Kimble/Kontes, Vineland, NJ) in buffer (25). Protein was extracted, concentration measured (Calbiochem, San Diego, CA), and 40 μg of sample protein and an internal standard were run on an SDS-PAGE 8% Bis-Tris gel (Invitrogen, Carlsbad, CA), using standard methods previously described (25). Antihuman myosin A4.840 and A4.74 antibodies were purchased from the University of Iowa Hybridoma Bank (Iowa City, IA), anti-rabbit succinate dehydrogenase and PPAR-α (Santa Cruz Biotechnology, Santa Cruz, CA), MAP4K4 (Abgent, San Diego, CA), IRS-1ser636 and IRS-1 total (Cell Signaling Technology, Danvers, MA), PKC-ε (Cell Signaling Technology, Beverly, MA), and CPT-1 (Alpha Diagnostics International, San Antonio, TX) antibodies were commercially available. The rabbit anti-4-HNE antibody was a generous gift from Dennis Peterson (University of Colorado, Denver, CO). Secondary antibodies were from Bio-Rad (Bio-Rad, Hercules, CA).

Calculations

Rates of glucose appearance (Ra), disappearance (Rd), and metabolic clearance rate (MCR) before the clamp were calculated using a modified Steele equation as described by Wolfe (29). Equations described by Finegood et al. (30) were used to account for the tracer in the “spiked” dextrose solution during the insulin clamp. Nonoxidative glucose disposal was calculated by subtracting carbohydrate oxidation from glucose Rd. IMTG and DAG FSRs were calculated as previously described by our laboratory (25).

%SaturationofIMTGandDAG=laurate+myristate+palmitate+stearateFFAspecies×100

where FFA represents concentration of individual FFA species in IMTG and DAG after transmethylation.

Palmitate Rd and palmitate rate of oxidation were calculated using steady-state kinetics and a whole-body estimate of carbon label retention as previously described (29). Calculation of palmitate oxidation rates was made using published values for the acetate recovery factor in obese humans at rest (31). Palmitate incorporation rate into IMTG was calculated as the product of IMTG FSR and palmitate pool size in IMTG. Fat-free mass (FFM) was used to extrapolate skeletal muscle palmitate IMTG storage to the whole body. Tissue-specific fat oxidation is calculated as the difference between whole-body fat oxidation and whole-body palmitate oxidation.

Statistical analysis

Testing of the data revealed a non-normal distribution; therefore, analyses were conducted on log-transformed values. Comparisons between groups were made using ANOVA for continuous variables and χ2 (Fisher’s exact where appropriate) for categorical variables (SPSS, Chicago, IL). All data are presented as mean ± s.e.m. Overall significance was set at P ≤ 0.05.

RESULTS

Subject demographics

Baseline subject demographics by sex are summarized in Table 1. Briefly, men and women were of similar age and BMI regardless of glucose tolerance group. Likewise, women had greater percent body fat, less FFM, and a consequently lower resting metabolic rate compared to men regardless of group (P < 0.05 for all). No differences in baseline demographics (age, BMI, % fat, FFM, or resting metabolic rate) were observed between groups for the same sex.

Table 1
Subject demographics

Insulin action

Hepatic insulin action: glucose Ra

Basal glucose Ra was similar between PD (men vs. women, P = 0.57) and NGT (men vs. women, P = 0.76) (Figure 1a). Glucose Ra was not different in absolute terms or in its suppressibility during the low (4 μU/m2/min) or high (40 μU/m2/min) dose insulin infusion in men vs. women with NGT. In contrast, men with PD had a higher (P = 0.02), less suppressible (P = 0.03) glucose Ra than women with PD during the low-dose clamp, with a similar trend seen during the high-dose clamp (P = 0.06 for both). No differences in glucose Ra or its suppressibility by insulin were observed between groups for the same sex.

Figure 1
Glucose rate of (a) appearance and (b) disappearance, (c) metabolic clearance rate, (d) nonoxidative and (e) oxidative glucose disposal in men and women with normal glucose tolerance (NGT) and prediabetes (PD) during a low-dose (4 mU/m2/min) and high-dose ...

Muscle insulin action: glucose Rd

A nonsignificant trend for higher glucose Rd was noted during the low-dose clamp comparing women vs. men with NGT (P = 0.06; Figure 1b), which became significant after adjusting for small differences in circulating glucose concentrations (MCR; P = 0.03; Figure 1c). However, in men vs. women with NGT, no such difference was seen for Rd (P = 0.25) or MCR (P = 0.28) during the high-dose clamp, or for oxidative (Rd Ox) or nonoxidative glucose disposal at any time during the clamp (Figure 1d,e). Conversely, Rd (P = 0.01), Rd Ox (P = 0.02), and MCR (P = 0.03) were higher in women than men with PD during the high-dose clamp, with lesser dissimilarity seen during the low-dose clamp (Rd, P = 0.57; Rd Ox, P = 0.63; MCR, P = 0.06). Again, no differences in Rd Ox or nonoxidative glucose disposal were observed in men vs. women with PD. Interestingly, a higher MCR (P = 0.04; and trend for higher Rd, P = 0.07) was appreciated in men with NGT vs. men with PD during the high-dose insulin infusion, whereas no such differences were observed comparing Rd or MCR in women with NGT vs. women with PD.

Hormone and substrate concentrations

Fasting and 2-h glucose concentrations were higher in PD vs. NGT, by study design (Table 2). Fasting glucose was also higher in NGT men vs. NGT women (P < 0.05). Otherwise, insulin, C-peptide, FFA, glycerol, and lactate concentrations were similar between groups and between the sexes at baseline (Table 2). Concentration of baseline sex steroids and selected metabolic markers are shown in Table 3. Briefly, testosterone was higher in men and leptin higher in women (P < 0.05 for both), regardless of group, with no differences seen in estradiol or sex hormone–binding globulin. Adiponectin was higher in NGT women vs. NGT men (P = 0.003) with no sex difference observed within the PD group (P = 0.16). Glucose concentration remained higher in PD men vs. NGT men, and also vs. PD women, during the low-dose insulin infusion (P < 0.05 for both). The C-peptide concentration followed the same pattern albeit without any group differences in circulating insulin concentration. During the high-dose clamp, FFAs and glycerol levels were higher in PD men compared to NGT men and PD women (P < 0.05, Table 2). Hormone and substrate concentrations drawn on the intramuscular lipid metabolism study day were virtually identical to those at baseline on the insulin action study day (data not shown).

Table 2
Hormone and substrate concentrations at baseline and during the clamp
Table 3
Concentrations of sex steroids and selected metabolic markers

Intramuscular lipid concentration, saturation, and turnover

Contrary to our hypotheses, greater IMTG concentration (42.6 ± 6.1 vs. 21.2 ± 3.4 μg/mg dry weight, P = 0.03) and lower FSR (0.23 ± 0.04 vs. 0.42 ± 0.06 %/h, P = 0.02) were observed in NGT women vs. NGT men despite their similarity in insulin action. Conversely, no difference in IMTG concentration (41.2 ± 5.0 vs. 46.4 ± 14.3 μg/mg dry weight, P = 0.70) or FSR (0.21 ± 0.03 vs. 0.29 ± 0.10 %/h, P = 0.60) was noted when comparing PD men and PD women (Figure 2a,b). PD men did, however, have a lower DAG FSR than PD women (0.54 ± 0.05 vs. 1.01 ± 0.23 %/h, P = 0.05) (Figure 3a,b). Nevertheless, NGT and PD women, who were of comparable insulin sensitivity, also had comparable IMTG concentration (P = 0.84) and FSR (P = 0.56). In contrast, PD men had higher IMTG concentration (P = 0.01) and lower FSR (P < 0.01) than NGT men (Figure 2a,b). No sex or group differences were observed with respect to IMTG percent saturation (35 ± 2.1% in NGT men, 31 ± 1.2% in NGT women, 32 ± 1.3% in PD men, and 31 ± 0.8% in PD women), DAG concentration (Figure 3a), or DAG percent saturation (46 ± 4.5% in NGT men, 52 ± 4.6% in NGT women, 52 ± 3.2% in PD men, and 40 ± 5.2% in PD women). More linoleate was noted in the IMTG (P < 0.01) and DAG (P = 0.04) of NGT men vs. PD men; otherwise, no differences in fatty-acid composition were appreciated (data not shown). Palmitate incorporation into IMTG, Rd, and oxidation are depicted in Figure 4a–c. No differences were seen between men and women in the same group or for the same sex between groups in any of these measures with the exception of a higher palmitate Rd in NGT women vs. PD women (P = 0.001).

Figure 2
(a) Intramuscular triglyceride concentration, (b) fractional synthesis rate, and (c) nonplasma-derived fat oxidation in men and women with normal glucose tolerance (NGT) and prediabetes (PD). *P < 0.05 men vs. women (same group); §P < ...
Figure 3
(a) Intramuscular diacylglyceride concentration and (b) fractional synthesis rate in men and women with normal glucose tolerance (NGT) and prediabetes (PD). *P < 0.05 men vs. women (same group).
Figure 4
(a) Palmitate rate of disappearance, (b) incorporation, and (c) palmitate oxidation rate in men and women with normal glucose tolerance (NGT) and prediabetes (PD). §P < 0.05 PD vs. NGT (same sex).

Western blots

Processes affecting or affected by IMTG were examined by western blot. Skeletal muscle oxidative capacity, assessed by succinate dehydrogenase, was lower in PD men (0.79 ± 0.06 western units) compared to both PD women (1.29 ± 0.19 western units, P = 0.01) and NGT men (1.12 ± 0.14 western units, P = 0.03). Protein abundance for myosin A4.840 (type 1 muscle fibers), MAP4K4, 4-HNE, CPT-1, IRS-1ser636, ratio of IRS-1ser636/IRS-1 total, PPAR-α, and PCK-e were not different between groups (data not shown).

DISCUSSION

Emerging evidence suggests that diabetes may develop differently in men vs. women, yet little is known about how this occurs. The current study examined the role of intramuscular lipid concentration and FSR as potentially distinguishing features between men and women in the development of diabetes. Major findings from this study demonstrated that alterations in intramuscular lipid metabolism related to diminished insulin action in men, but not women, in the progression toward diabetes. Obese men with PD had higher IMTG concentration and a lower FSR commensurate with lower insulin sensitivity compared to a matched control group with NGT. No such differences were seen comparing obese women with PD vs. NGT. The latter finding is in marked contrast to our observation when the groups are pooled. Specifically, we found that inflexibility in IMTG FSR distinguishes PD from simple obesity in humans (32). Together, these data suggest that the transition from obesity to PD represents a more severe metabolic derangement in men than women, especially with respect to processes in skeletal muscle.

Over the past two decades, considerable attention has been paid to the frequent association between high IMTG concentration and insulin resistance in both men and women (912). Nevertheless, an increasing body of evidence shows that this relationship can be dissociated (33,34) leading to speculation about the role of IMTG synthesis, rather than total concentration, as a link to insulin resistance (35). Our data are consistent with recent studies in both animals (34) and humans (36,37) that insulin action is particularly impaired in an IMTG pool with a low FSR. However, this was only seen in men and not women. Our PD men had a lower MCR (and trend for lower Rd) during the high-dose insulin infusion concurrent with a higher IMTG concentration and lower FSR than men with NGT. The seeming paradox of higher IMTG concentration amidst lower FSR can be reconciled when considering the higher circulating FFA and glycerol concentrations during the high-dose clamp (implying higher postprandial substrate delivery) in the face of a lower oxidative capacity. Whether the lower oxidative capacity was the cause or result of higher IMTG, and how much this contributed to the lesser insulin action vs. the lower FSR could not be discerned. In any fashion, we believe that our data are the first to demonstrate that changes in intramuscular lipid metabolism may be more relevant in men than women in the early progression to diabetes.

In contrast to the PD vs. NGT men, obese women with PD were of similar insulin sensitivity, and had comparable IMTG concentration and FSR as those with NGT. One of the only differences seen comparing PD and NGT women was a lower palmitate Rd in PD women, with no group differences noted in palmitate incorporation into IMTG or whole-body palmitate oxidation. This observation would suggest that PD women had less nonoxidative disposal to other (nonmuscle) tissues compared to NGT women. Further support for altered intramuscular lipid metabolism being less related to insulin action in women vs. men is seen comparing our men and women with NGT. Ours is the second study (12) to show that women with NGT have paradoxically higher IMTG concentration than men with NGT, and the first to show low IMTG FSR in NGT women vs. men, and that these occur without a negative impact on insulin action. This is not to contend that interventions aimed at enhancing IMTG flux do not positively influence insulin action in women (36,37), but simply that it may be less clinically relevant than in men.

Classification as having “PD” may denote greater short-term risk of diabetes in men than women. Our PD men had significantly greater hepatic and skeletal muscle insulin resistance, despite the fact that PD women had significantly more body fat and less FFM. Although no difference in IMTG concentration or FSR was observed in PD men vs. women, PD men had a lower oxidative capacity and DAG FSR. As with IMTG, the turnover of the DAG pool may be more important than the total concentration. DAG, in particular, appears central in modifying insulin action in muscle through activation of PKCε, PKCθ, and PKCδ, with subsequent inhibition of insulin signaling (38). Although we found no difference in PKCε IRS-1ser636, or ratio of IRS-1ser636/IRS-1 total between groups, these measures were made in the basal (noninsulin stimulated) state, and we did not determine sarcolemmal vs. cytosolic localization of PKCε; thus, we cannot refute its importance in the pathogenesis of insulin resistance in muscle. In addition, no group differences were observed in markers of lipid tracking (PPAR-α) or uptake (CPT-1), or in systemic processes related to lipid peroxidation (4-HNE) or inflammation (MAP4K4). Altogether, the greater capacity for whole-body fat storage may be vital for maintaining insulin sensitivity in women, whereas the ability to synthesize (and degrade) tissue lipids may be more important in men.

Tissue lipid uptake and use are regulated, in part, by the sex steroids and cytokines. Thus, analyses were undertaken to examine these as possible explanations for our results. Although testosterone was higher in men than women, no difference was seen comparing PD and NGT men, and therefore is unlikely to explain the marked metabolic differences between these groups. Leptin and adiponectin have each been shown to increase fatty-acid oxidation and decrease IMTG concentration (39,40), but their actions did not appear to predominate in our cohort. For example, adiponectin, leptin, and IMTG were all higher in NGT women vs. men. Furthermore, adiponectin was not different between PD men and women, despite the difference in insulin sensitivity. In sum, neither circulating sex steroids nor the measured cytokines could explain the observed sex differences.

There are several limitations of the current study worth noting. In order to focus on metabolic differences between simple obesity and PD, no lean control group was studied. Furthermore, all types of PD were grouped together. Although this likely diminished finding differences between PD and “normal,” the design was vital in differentiating high- vs. low-risk individuals. Second, DAG FSR may have been underestimated because only the intramuscular FFA precursor pool, and not phospholipid pool, was labeled using the palmitate stable isotope. Additionally, measures of IMTG and DAG FSR were made in the fasted state and are likely more relevant to the pathogenesis of diabetes when made in the postprandial or postexercise state. These limitations should be taken into consideration when interpreting our conclusions. Last, true progression to diabetes was not assessed in this cross-sectional study; thus, longitudinal studies should be conducted to confirm our findings.

In conclusion, the transition from simple obesity to PD likely represents a more dramatic metabolic change in men than women. Men develop features of diminished skeletal muscle glucose metabolism that relate to lower IMTG oxidation and turnover with subsequent accumulation of IMTG. Once in the prediabetic state, men maintain lower insulin sensitivity than their female counterparts possibly because of lower oxidative capacity and/or synthesis of the DAG pool. No such differences in insulin action or intramuscular lipid metabolism were seen in women with simple obesity vs. PD. Together, this implies a more significant role of altered intramuscular metabolism for men at this disease juncture. The role of altered intramuscular lipid metabolism in the development of diabetes in women likely occurs later in disease development.

Acknowledgments

We owe the success of this work to the research subjects who volunteered their time to participate, the committed staff of the General Clinical Research Center, as well as to the National Institutes of Health, who funded this work (grant no. NIH DK-064811, DK-059739, and RR-0036).

Footnotes

DISCLOSURE

The authors declared no conflict of interest.

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