Higher intramuscular triacylglycerol in women does not impair insulin sensitivity and proximal insulin signaling

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1 J Appl Physiol 107: , First published July 2, 2009; doi: /japplphysiol Higher intramuscular triacylglycerol in women does not impair insulin sensitivity and proximal insulin signaling Louise Høeg,* Carsten Roepstorff,* Maja Thiele, Erik A. Richter, Jørgen F. P. Wojtaszewski, and Bente Kiens The Copenhagen Muscle Research Centre, Molecular Physiology Group, Section of Human Physiology, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark Submitted 18 October 2008; accepted in final form 29 June 2009 Høeg L, Roepstorff C, Thiele M, Richter EA, Wojtaszewski JF, Kiens B. Higher intramuscular triacylglycerol in women does not impair insulin sensitivity and proximal insulin signaling. J Appl Physiol 107: , First published July 2, 2009; doi: /japplphysiol Women have been shown to have higher muscle triacylglycerol (IMTG) levels than men and could therefore be expected to have lower insulin sensitivity than men, since previous studies have linked high IMTG to decreased insulin sensitivity. Therefore, insulin sensitivity of whole body and leg glucose uptake was studied in 9 women in the follicular phase and 8 men on a controlled diet and matched for maximal oxygen uptake per kilogram of lean body mass and habitual activity level. A 47% higher (P 0.05) IMTG level was found in women than in men, and, at the same time, women also displayed 22% higher whole body insulin sensitivity (P 0.05) and 29% higher insulin-stimulated leg glucose uptake (P 0.05) during an euglycemic-hyperinsulinemic ( 70 U/ml) clamp compared with matched male subjects. The higher insulin sensitivity in women could not be explained by higher expression of muscle glucose transporter GLUT4, insulin receptor, or Akt expression or by the ability of insulin to stimulate Akt Thr 308 or Akt Ser 473 phosphorylation. However, a 30% higher (P 0.05) capillary density and 31% more type 1 muscle fiber expressed per area in the vastus lateralis muscle were noted in women than in matched men. It is concluded that despite 47% higher IMTG levels in women in the follicular phase, whole body as well as leg insulin sensitivity are higher than in matched men. This was not explained by sex differences in proximal insulin signaling in women. In women, it seems that a high capillary density and type 1 muscle fiber expression may be important for insulin action. muscle triacylglycerol; sex paradox; insulin action CONTROVERSY EXISTS whether whole body insulin sensitivity is different between men and women. When the euglycemichyperinsulinemic clamp technique was applied in healthy women and men matched with respect to body mass index (BMI), a difference in whole body insulin sensitivity was not found in some studies (3, 36), whereas in other studies a higher whole body insulin sensitivity was found in women (4, 14, 30). Recently, a higher whole body insulin sensitivity also was found in obese women compared with obese men when matched with respect to maximal oxygen uptake per kilogram of lean body mass (LBM) (51). Skeletal muscle is an important determinant of whole body insulin sensitivity, since earlier studies in men have indicated * L. Høeg and C. Roepstorff contributed equally to this work. Address for reprint requests and other correspondence: B. Kiens, The Copenhagen Muscle Research Centre, Molecular Physiology Group, Dept. of Human Physiology, Institute of Exercise and Sport Sciences, Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark ( ku.dk). that skeletal muscle is responsible for 50 70% of insulinstimulated whole body glucose uptake (10). Whether insulinstimulated glucose uptake specifically in muscle is different in women from that in men is not known. Muscle insulin sensitivity could be ascribed to sensitivity of the insulin signaling molecules. When insulin binds to its receptor, the insulin receptor (IR) phosphorylates insulin receptor substrate-1 (IRS-1) at the tyrosine residues, which then binds phosphatidylinositol 3-kinase (PI3-kinase), leading to its activation (47). When PI3-kinase is activated, its product, phosphatidylinositol 3,4,5-trisphosphate, activates pyruvate dehydrogenase kinase, in turn phosphorylating and activating the kinase Akt. Akt has been implicated in translocation of the glucose transporter GLUT4 to the plasma membrane, a critical event in insulinstimulated glucose uptake (47). In human skeletal muscle, possible sex differences in insulin signaling have never been investigated. Previously, whole body insulin sensitivity has in many instances been related to the amount of intramuscular triacylglycerol (IMTG) (32, 34), and it is still mentioned in recent literature that an inverse relationship between IMTG content and whole body insulin sensitivity exists (5, 22). This inverse relationship between IMTG content and whole body insulin sensitivity is, however, not always observed (17). Recently, it was demonstrated that IMTG content is higher in skeletal muscle in women than in matched men (38, 39, 43, 44). The aims of this study were therefore to examine 1) whether insulin sensitivity on a whole body and/or muscular level is similar to or higher in healthy women despite the higher IMTG content compared with that in matched men and 2) whether the central molecular mechanisms involved in insulin signaling to stimulate glucose uptake in skeletal muscle are expressed or activated differently between women and men. To fulfill these aims, women and men, matched according to strict criteria, underwent an experiment in which whole body and leg muscle insulin sensitivity were measured using the euglycemic-hyperinsulinemic clamp procedure combined with the leg balance technique and muscle biopsies obtained from the vastus lateralis muscle. MATERIALS AND METHODS Subjects. Nine women and eight men were recruited to participate in the study (Table 1). Before volunteering, all subjects were given full verbal and written information about the course of the study and possible risks associated with participation. Written consent was obtained from each subject. The study was approved by The Copenhagen Ethics Committee (KF /02) and conformed to the code of ethics of the World Medical Association (Declaration of Helsinki II). All subjects were young and healthy with normal body weight and were nonsmokers with no family history of diabetes. All subjects were /09 $8.00 Copyright 2009 the American Physiological Society

2 Table 1. Subject characteristics Women n 8 8 Age, yr Height, m * BM, kg * Body fat, % * LBM, kg V O2peak * l/min * ml kg BM 1 min * ml kg LBM 1 min Maximal citrate synthase activity, mol g dry wt 1 min Training history Frequency, workouts/wk Duration, h/wk Data are means SE; n no. of subjects. BM, body mass; LBM, lean body mass; V O2peak, peak oxygen uptake. *P 0.001, sex difference. Men moderately trained and participated on a regular basis (3 4 h/wk) in leisure time activities such as running, cycling, strength training, and different game sports. The women were eumenorrheic with a menstrual cycle length between 28 and 35 days and did not take any oral contraception. All preexperimental testing and the main experiments in women were carried out during the midfollicular phase of their menstrual cycle (between days 7 and 10 counted from the first day of menstruation day). Preexperimental testing. For determination of whole body peak oxygen uptake (V O2peak), all subjects performed an incremental exercise test on a Monark Ergomedic 839E bicycle ergometer (Monark, Varberg, Sweden). In addition, they filled out a questionnaire regarding habitual physical activity and exercise training. Whole body fat percentage was determined by hydrostatic weighing (42) with a correction for residual lung volume measured using the oxygen dilution method (29). Leg composition was determined by dual-energy X-ray absorptiometry (DEXA; DPX-IQ version 4.7; Lunar, Madison, WI) from a whole body scan by a pelvis cut angled through the femoral necks according to the manufacturer s directions. Both determinations of body composition were carried out after a 4-h fasting period when subjects refrained from all food and liquids. Female and male groups were matched according to V O2peak/kg LBM, habitual physical activity level, and exercise training history. Diet. For 8 days preceding the main trial, all subjects consumed an isoenergetic diet containing 65 energy percent (E%) carbohydrate, 20 E% fat, and 15 E% protein. The constituents of the diet were weighed out at the laboratory and delivered to the subjects. The amount of energy to be consumed was individually determined from body weight, sex, and habitual physical activity level based on guidelines from World Health Organization (54). To ensure that subjects kept their body mass steady during the 8-day dietary regimen, we adjusted energy consumption during the diet period after 4 days based on daily monitoring of body mass. Experimental protocol. Subjects arrived at the laboratory in the morning after an overnight fast by bus, train, or car. The subjects had abstained from exercise training during the 2 days preceding the experimental day. After subjects had been at rest for 30 min in the supine position, Teflon catheters were inserted under local anesthesia into the femoral artery and femoral vein (same leg) below the inguinal ligament. A thermistor (Edslab probe F; Baxter, Allerød, Denmark) was inserted through the femoral venous catheter and advanced 8 cm proximal to the catheter tip for determination of femoral venous blood temperature. Two venous catheters were inserted into antecubital veins for infusion of insulin and glucose. After subjects had been at rest for an hour in a supine position, expired air SEX AND INSULIN SENSITIVITY 825 was sampled in a Douglas bag for determination of pulmonary oxygen uptake and carbon dioxide excretion. Hereafter, blood was sampled from the arterial and femoral venous catheters, femoral venous blood flow was determined, and a muscle biopsy was obtained from the vastus lateralis muscle under local anesthesia of the skin and fascia. Subjects then underwent a 120-min euglycemic-hyperinsulinemic clamp [1.1 mu min 1 kg body mass (BM) 1 ] that was initiated with a bolus injection of insulin (6 mu/kg BM 1 Actrapid; Novo Nordisk, Bagsværd, Denmark). During the clamp, blood samples were drawn simultaneously from the femoral catheters after 20, 40, 60, 75, 90, 105, and 120 min of insulin infusion. After each blood sampling, femoral venous blood flow was measured. At 90 and 120 min of the clamp, expired air was sampled in a Douglas bag. During the clamp, arterial blood samples were frequently taken and rapidly analyzed for glucose to adjust the glucose infusion rate. After 120 min of the clamp, another muscle biopsy was obtained from the vastus lateralis muscle, this time in the contralateral leg. Breath samples. Expired volumes of air in the Douglas bags were measured with a chain-suspended Collins spirometer, and mixed expiratory air was analyzed for O 2 (Servomex S-3A) and CO 2 (Beckman LB2). Respiratory exchange ratio (RER) was calculated as the ratio between pulmonary CO 2 excretion and O 2 uptake. Blood samples. Blood O 2 and CO 2 concentrations were determined, and leg respiratory quotient (RQ) was calculated as described previously (39). Blood glucose concentration was measured on an ABL510 (Radiometer Medical, Copenhagen, Denmark). Plasma fatty acid (FA) concentration was measured using a colorimetric commercial assay kit (Wako Chemicals, Richmond, VA) using a COBAS FARA 2 autoanalyzer (Roche Diagnostic, Rotkreuz, Switzerland). Concentrations of insulin (Pharmacia insulin radioimmunoassay 100; Pharmacia & Upjohn Diagnostics, Uppsala, Sweden), epinephrine, and norepinephrine (KatCombi radioimmunoassay; Immuno-Biological Laboratories, Hamburg, Germany) in plasma were determined by radioimmunoassay. Blood flow. Venous blood flow was measured using the thermodilution technique by constant infusion of ice-cold saline (1, 37). Insulin sensitivity. Insulin sensitivity on a whole body level was expressed as area under the curve of the glucose infusion rate during the last 30 min of the clamp. Insulin sensitivity across the leg was expressed as area under the curve of the glucose infiltration rate minus the individual basal level. Muscle biopsies. The biopsies were divided in two. One part was immediately frozen in liquid nitrogen and stored at 80 C for subsequent analysis. The other part was mounted in embedding medium, frozen in precooled isopentane, and stored at 80 C for subsequent histochemical analysis. Muscle tissue ( 80 mg wet wt) was freeze-dried and dissected free of all visible adipose tissue, connective tissue, and blood under a microscope. The dissected muscle fibers were pooled and then divided into subpools for the respective analyses. IMTG. The concentration of IMTG was determined in freeze-dried and dissected muscle tissue as described previously (21, 43). In short, the freeze-dried, dissected tissue was incubated overnight in tetraethylammonium hydroxide. Glycerol was then analyzed fluorometrically. Muscle glycogen. The muscle glycogen concentration was determined as glycosyl units after acid hydrolysis of freeze-dried and dissected muscle tissue using a fluorometric method (28). Maximal citrate synthase activity. Maximal activity of citrate synthase was measured fluorometrically (28). Muscle lysates. Freeze-dried and dissected muscle tissue was homogenized [1:80 (wt/vol)] in ice-cold buffer containing 50 mm HEPES (ph 7.5), 150 mm NaCl, 20 mm sodium pyrophosphate, 20 mm -glycerophosphate, 10 mm NaF, 2 mm sodium orthovanadate, 2 mm EDTA, 1% Nonidet P-40, 10% glycerol, 2 mm PMSF, 1 mm MgCl 2, 1 mm CaCl 2,10 g/ml leupeptin, 10 g/ml aprotinin, and 3 mm benzamidine. Homogenates were rotated end over end for 1 h at 4 C and then cleared by centrifugation at 17,500 g at 4 C for 30 min.

3 826 SEX AND INSULIN SENSITIVITY Protein content in the supernatant was measured using the bicinchoninic acid method (Pierce Chemical, Rockford, IL). The lysates were boiled in Laemmli buffer before being subjected to SDS-PAGE and immunoblotting. Western blotting. Expression of IR, GLUT4, and Akt and phosphorylation of Akt Thr 308 and Akt Ser 473 were determined by Western blotting on muscle lysates. Primary monoclonal antibody was mouse anti-ir (raised against the COOH-terminal end of the IR -subunit; a gift from Dr. Ken Siddle, Cambridge University, UK), and primary polyclonal antibodies were rabbit anti-glut4 (AB1346; Chemicon International, Temecula, CA), rabbit anti- Akt1 (Upstate Biotechnology, Lake Placid, NY), rabbit anti-phospho Akt1 Thr 308 (05-802; Upstate Biotechnology), and rabbit anti-phospho Akt Ser 473 (Cell Signaling Technology, Danvers, MA). Secondary antibodies were horseradish peroxidase-conjugated anti-mouse and anti-rabbit (P0161 and P0448; DAKO, Glostrup, Denmark). Antigen-antibody complexes were visualized using enhanced chemiluminescence (ECL ; Amersham Biosciences, Little Chalfont, UK) and quantified using a Kodak Image Station 2000MM (Ballerup, Denmark). ATPase and capillary staining. Serial cross sections(10 m) were cut and stained for myofibrillar ATPase to identify type 1, 2A, and 2X muscle fibers (6). On an additional cross section, capillaries were stained using a double staining method with primary antibodies against caveolin-1 and collagen type IV (35). Muscle fiber type composition and capillary density were analyzed in all subjects by the same blinded observer using the TEMA image analysis software (CheckVision, Støvring, Denmark). Statistics. Data are means SE. For variables independent of time, a t-test was performed to test for differences between women and men. For variables measured before and during the euglycemichyperinsulinemic clamp, a two-way analysis of variance, with repeated measures for time and sex, was performed to test for sex differences or changes during the clamp. When significant main effects were found or when significant interaction between effects of sex or time was found, pairwise differences were tested using Tukey s post hoc test. In all cases, a probability of 0.05 was used as the level of significance. RESULTS Subject characteristics. V O2peak was and ml O 2 min 1 kg BM 1 in women and men, respectively (Table 1). When V O2peak was expressed relative to LBM, differences were not present between sexes (Table 1). RER and leg RQ. Whole body RER at basal level was higher (P 0.05) in men than in women at basal level, indicating a higher rate of lipid oxidation in women (Table 2). RER increased (P 0.001) during the euglycemic-hyperinsulinemic clamp in both women and men and was similar between sexes at the end of the clamp [no significant difference (NS)]. Although basal leg RQ tended to be higher in men, the difference was not significant. Leg RQ increased (P 0.001) during the clamp to the same level in women and men (NS) (Table 2). Femoral venous blood flow. Basal femoral venous blood flow did not differ significantly between women and men (Table 2). During the euglycemic-hyperinsulinemic clamp, femoral venous blood flow remained unchanged in women (NS) but was increased in men (P 0.01). During the last 30 min of the clamp, femoral venous blood flow was 37% higher (P 0.05) in men than in women. Blood glucose concentration. The arterial blood glucose concentration was similar in women and men at basal level (NS) and it was clamped at the basal level in both sexes (Table 2). Insulin sensitivity. During the last 30 min of the clamp, the glucose infiltration rate per kilogram of BM was not different in women and men (data not shown). When the glucose infiltration rate was expressed per kilogram of LBM, a 22% higher (P 0.05) whole body insulin sensitivity was observed in women than in men (Fig. 1A). Leg glucose uptake per kilogram of lean leg mass (LLM) increased (P 0.001) continuously from basal level to 120 min of the euglycemic-hyperinsulinemic clamp in both sexes (Fig. 1B). Leg glucose uptake per kilogram of LLM did not differ significantly between women and men at the basal state but was 29% higher (P 0.05) in women compared with men the last 30 min of the clamp (Fig. 1C). Plasma FA. The basal arterial plasma FA concentration did not differ significantly between women and men, but a decrease (P 0.001) was observed in response to insulin in both sexes (Table 2). Hormones. The basal arterial plasma insulin concentration was similar in women and men (NS), and in response to insulin infusion, it increased (P 0.001) to similar levels in the two trials (NS; Table 2). At basal level, the arterial concentrations of epinephrine and norepinephrine were both similar between sexes (NS; Table 2). In both sexes, the arterial norepinephrine concentration increased (P 0.01) during the clamp, whereas arterial epinephrine concentration remained unchanged (NS). Table 2. RER, leg RQ, and arterial plasma substrate and hormone concentrations before and during a euglycemichyperinsulinemic clamp in women and men Basal State Insulin Women Men Women Men RER * Leg RQ Femoral venous blood flow, ml/min * Arterial blood glucose concentration, mm Plasma FA concentration, M Plasma insulin concentration, U/ml Plasma epinephrine concentration, nm Plasma norepinephrine concentration, nm Data are means SE. RER, respiratory exchange ratio; RQ, respiratory quotient; FA, fatty acids. Values during the clamp are averaged from 90 to 120 min. *P 0.05, different from women at same time point. P 0.01; P 0.001, main effect: insulin vs. basal state.

4 SEX AND INSULIN SENSITIVITY Table 3. Fiber type composition and capillary density in women and men Women Men 827 Fiber type composition, % Type Type 2A Type 2X Fiber type composition, area% Type * 49 3 Type 2A 25 4* 35 2 Type 2X Mean area per fiber, m 2 Type 1 3, , Type 2A 3, * 5, Type 2X 3, * 5, Capillary density Capillary per fiber Capillary per mm * Data are means SE. *P 0.05, sex difference. IMTG. The IMTG concentration in the vastus lateralis muscle was 47% higher (P 0.05) in women than in men (Fig. 2A). The IMTG concentration did not change during the clamp (NS). Fig. 1. Glucose infusion rate and insulin-stimulated leg glucose uptake in women and men. A: glucose infusion rate during the last 30 min of the clamp, expressed per minute. LBM, lean body mass. *P 0.05, sex difference. B: insulin-stimulated glucose uptake before and during the 120-min clamp. *P 0.05; #P 0.07, sex difference. C: insulin-stimulated glucose uptake during the last 30 min of the clamp, expressed per minute. LLM, lean leg mass. *P 0.05 sex difference. Fiber type composition, fiber area, and capillary density. The percentage of type 1 fiber expressed relative to area (area%) was 31% higher (P 0.05) in women than in men, and the area% of type 2A was 40% higher (P 0.05) in men than in women (Table 3). The average area of type 2A and 2X fibers was smaller (P 0.05) in women compared with men. The capillary density (capillaries per mm 2 ) was 30% higher (P 0.05) in women than in men (Table 3). Fig. 2. Intramuscular triacylglycerol (IMTG) and glycogen concentrations in the vastus lateralis muscle before and after a 120-min euglycemic-hyperinsulinemic clamp in women and men. A: IMTG concentration (conc). *P 0.05, main effect: men vs. women. B: muscle glycogen content. *P 0.05, main effect: insulin vs. basal state.

5 828 SEX AND INSULIN SENSITIVITY Muscle glycogen. The glycogen concentration in the vastus lateralis muscle did not differ between women and men (NS; Fig. 2B). Muscle glycogen increased 8% (P 0.05) during the euglycemic-hyperinsulinemic clamp. IR and GLUT4 protein expression. Because IR and GLUT4 protein expression are unlikely to change during the 2-h clamp, these variables were only measured in the preclamp biopsy. There was no sex difference in basal IR and GLUT4 protein expression in the vastus lateralis muscle (Fig. 3). Akt1/2 protein expression. Akt1/2 protein expression did not differ between women and men (NS) at basal level and did not change during the euglycemic-hyperinsulinemic clamp (Fig. 4). Akt Thr 308 and Ser 473 phosphorylation were not detectable in the basal state. Therefore, quantitative analysis was only performed on insulin stimulated samples (Fig. 4). Both Akt Thr 308 and Akt Ser 473 phosphorylation were increased by insulin but were not different between Fig. 4. Akt protein expression and Akt Thr 308 and Ser 473 phosphorylation in the vastus lateralis muscle before and after a 120-min euglycemic-hyperinsulinemic clamp in women and men. A: Akt protein expression. B: Akt Thr 308 phosphorylation. C: Akt Ser 473 phosphorylation. ND, not detectable. women and men after the euglycemic-hyperinsulinemic clamp. DISCUSSION Fig. 3. Insulin receptor and glucose transporter GLUT4 expression in the vastus lateralis muscle before the 120-min euglycemic-hyperinsulinemic clamp in women and men. A: insulin receptor expression. B: GLUT4 expression. In the present study it was shown that insulin-stimulated glucose uptake in skeletal muscle was higher in women than in men carefully matched with respect to BMI, age, fitness level, and regular level of physical activity and investigated on a controlled diet in the midfollicular phase of the menstrual

6 cycle. In accordance with the different insulin sensitivity at the muscular level, a higher whole body insulin sensitivity per kilogram of LBM was demonstrated in women compared with men. Because skeletal muscle is responsible for 50 70% of insulin-stimulated whole body glucose uptake (10), the higher whole body insulin sensitivity in women can to a large extent be explained by a higher insulin sensitivity in skeletal muscle. Interestingly, the higher insulin-stimulated glucose uptake in skeletal muscle in women was found despite a higher IMTG concentration in women than in men. The higher insulin sensitivity at the muscular level in women could not be explained by higher protein expression of the IR, Akt, or GLUT 4 or a higher ability of insulin to stimulate signaling through Akt in women, but it may be related to a higher capillary density and a higher expression of type 1 muscle fibers in women than in men. In the literature there has been some controversy whether insulin sensitivity in premenopausal women and men is different. Difference in body fat, body fat distribution, and physical fitness level between sexes may be part of this controversy. When whole body insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp technique in young, healthy, normal weight subjects in the follicular phase of the menstrual cycle, sex differences were not obtained when insulin sensitivity was expressed as glucose infusion per kilogram of body weight in some studies (3, 36), whereas in other studies, insulin sensitivity expressed as glucose infusion rate either per kilogram of body weight (4, 30) or per kilogram of LBM (14) was higher in women than in men. However, in these studies, subjects were only matched with respect to BMI, and the physical fitness level of the subjects was not taken into considerations. It has been shown that exercise training increases insulin sensitivity in skeletal muscle in young healthy men (12) and type 2 diabetic individuals (11). Accordingly, it has been suggested that women and men with similar V O2peak per kilogram of LBM represent comparable aerobic physical fitness levels (45). When obese women and men were matched with respect to maximal oxygen uptake per kilogram of LBM, whole body insulin sensitivity was higher in women than in men when expressed as glucose infusion rate per kilogram of LBM (51). Moreover, in a study by Nuutila et al. (31) in which young men and women were matched with respect to maximal aerobic capacity (V O2 max ) and BMI, whole body insulin sensitivity measured by position emission tomography (PET) under hyperinsulinemic, normoglycemic conditions was 41% greater in women than in men, and this difference was explained by a 47% greater rate of glucose uptake by femoral muscles in women than in men (31). These findings are in agreement with the present findings, where the 22% higher glucose infusion rate in women than in men can be explained by the 29% higher glucose uptake in leg skeletal muscle in women than in men. Another important factor to consider when studying sex difference is sex hormones and the phase of the menstrual cycle in which the study is performed. In the present study, we cannot be ruled out the possibility that sex hormones and/or the phase of the menstrual cycle may play a role in the higher insulin sensitivity in women than in men. Studies in postmenopausal women have shown that increased androgenicity induces a decrease in whole body insulin sensitivity and that hormonal treatment with low doses of estradiol at levels similar SEX AND INSULIN SENSITIVITY 829 to those seen in the early follicular phase in premenopausal women reversed the androgen-induced decrease in whole body insulin sensitivity (8, 24). This indicates that serum estradiol may play a role in insulin sensitivity and could be of importance when studying metabolic processes. However, the relationship between female sex hormones and insulin sensitivity is not simple. The luteal phase is characterized by increased levels of serum estradiol and progesterone compared with the follicular phase. Insulin sensitivity previously has been shown to decrease significantly in a stepwise fashion from the follicular to the luteal phase of the menstrual cycle in some studies (15, 48), whereas in one study (2), no difference in insulin sensitivity between the luteal phase and the follicular phase was obtained in 13 healthy premenopausal women. If serum estradiol plays a role in insulin sensitivity, the most comparable period between sexes therefore seems to be the follicular phase, where the female sex hormone concentration in plasma is lowest in women and only a little higher than in men. In the present study, where a higher insulin sensitivity was obtained in women than in men, all women were studied in the midfollicular phase of the menstrual cycle (between days 7 and 10 counted from the first day of menstruation). The possibility cannot be ruled out that insulin sensitivity might have been lower if the women had been studied in the luteal phase, although, as explained above, there is no clear consensus about the effect of menstrual cycle on insulin sensitivity. In the present study, a 47% higher (P 0.05) IMTG content in the vastus lateralis muscle was observed in women compared with men (Fig. 4A). The higher IMTG content in women than in men previously has been found in studies from our laboratory (38, 39, 43) as well as in other studies (13, 44). In the present study, the higher IMTG content in women was observed concomitantly with an enhanced whole body insulin sensitivity and also an enhanced insulin-stimulated glucose uptake in skeletal muscle of women compared with men (Fig. 1, A and B). An inverse relationship between muscle content of IMTG and whole body insulin sensitivity has been demonstrated in obese or severely obese subjects (17, 32, 41), in normal weight insulin resistant individuals (20), or in type 2 diabetics (18, 25). Still, in normal weight subjects, an association between IMTG content and impaired whole body insulin sensitivity also has been observed when IMTG was measured using 1 H MRS in the soleus muscle (23, 46, 52), the tibialis anterior muscle (46), or the vastus lateralis muscle (50). From these studies, a link between high IMTG content and insulin resistance has been indicated. However, in contrast, an inverse relationship between IMTG content and insulin sensitivity on a whole body level was not supported in other studies. Thus, in the study by Kiens and Richter (21), a higher whole body insulin sensitivity was seen in healthy young men concomitantly with an increase in IMTG content in the vastus lateralis muscle when a diet consisting of primarily high glycemic index (GI) food items was ingested for 4 wk. Also, when IMTG was normalized in type 2 diabetic individuals after 8 wk of exercise training, whole body insulin sensitivity did not change (7), and moreover, 4 wk of endurance training of healthy males subjects was not followed by changes in IMTG content, whereas an increased insulin-stimulated leg glucose uptake was observed (19). Studies using the oil-red O staining technique (17) or 1 H MRS (9, 41) to measure IMTG content support the notion that

7 830 SEX AND INSULIN SENSITIVITY a relationship between IMTG content and whole body insulin sensitivity does not exist in normal weight, healthy, insulinsensitive subjects (41) and in endurance-trained subjects (9, 17). It is apparent from these findings, together with the data from the present study, that a clear association does not exist between triacylglycerol content in skeletal muscle and whole body insulin sensitivity or insulin-stimulated muscle glucose uptake in healthy, normal weight women and men, suggesting that there is no causal relationship between IMTG content and insulin sensitivity. Sex differences in insulin sensitivity could be due to sex differences in the insulin signaling cascade. However, no sex differences were observed in protein expression of IR (Fig. 3A), Akt, or insulin-stimulated site-specific Akt phosphorylation on Akt Ser 473 or Akt Thr 308 (Fig. 4). Moreover, sex differences were not seen in GLUT4 protein expression (Fig. 3B). Still, differences in insulin sensitivity are not always accompanied by differences in insulin signaling. For example, increased insulin sensitivity of glucose uptake after exercise is not accompanied by increased activation of Akt (53), and in cell culture studies, malonyl coenzyme A inhibits insulin action without detectable changes in insulin signaling (33). An alternative explanation for the higher insulin-stimulated leg glucose uptake in women compared with men could possibly be linked to muscle morphology. In the present study, the percentage area of oxidative type 1 fibers as well as the capillary density in the vastus lateralis muscle were higher in women than in men (Table 3), in accordance with previous findings from our laboratory (40, 43). A higher percentage of type 1 fibers and a higher capillary density in skeletal muscle have been shown to be closely associated with increased insulin sensitivity (26, 27). It is therefore reasonable to believe that the higher capillary density and proportion of type 1 fibers in women compared with men can partly explain the higher muscle insulin sensitivity in women. Along these lines, a high capillary density also provides the opportunity for a higher capillary recruitment, also termed nutritive flow, during insulin stimulation (49), and the possibility exists that a higher nutritive flow in women than in men, and thereby an improved delivery of blood borne substrates to the muscle fibers, can be another explanation for why women had a higher insulinstimulated leg glucose uptake compared with men. In conclusion, the present data revealed a 22 and 29% higher insulin sensitivity on a whole body and a muscular level, respectively, in women compared with men despite a higher IMTG content in women. These data indicate that impaired insulin sensitivity is not directly associated with high IMTG content. The higher insulin sensitivity in skeletal muscle in women compared with men, measured in the midfollicular phase of the menstrual cycle, could not be explained by improved proximal insulin signaling in women but may be related to sex differences in muscle morphology, particularly a higher proportion of type 1 muscle fibers and a higher capillary density in women compared with men. ACKNOWLEDGMENTS We acknowledge the skilled technical assistance of Irene Bech Nielsen, Nicoline Resen Andersen, and Winnie Taagerup. We are grateful to Dr. Ken Siddle, Cambridge University, Cambridge, UK, for kindly donating the insulin receptor antibody. GRANTS This study was supported by the Danish Agency for Science, Technology and Innovation, the Ministry of Food, Agriculture and Fisheries, the Danish Medical and Natural Science Research Councils, the Novo Nordisk Foundation, the Danish Diabetes Association, Michaelsen Fonden, and Integrated Project Grant LSHM-CT funded by the European Commission. J. F. P. Wojtaszewski was the recipient of a Hallas Møller fellowship from the Novo Nordisk Foundation. REFERENCES 1. Andersen P, Saltin B. Maximal perfusion of skeletal muscle in man. J Physiol 366: , Bingley CA, Gitau R, Lovegrove JA. Impact of menstrual cycle phase on insulin sensitivity measures and fasting lipids. Horm Metab Res 40: , Binnert C, Ruchat S, Nicod N, Tappy L. Dexamethasone-induced insulin resistance shows no gender difference in healthy humans. Diabetes Metab 30: , Borissova AM, Tankova T, Kirilov G, Koev D. Gender-dependent effect of ageing on peripheral insulin action. Int J Clin Pract 59: , Brehm A, Krssak M, Schmid AI, Nowotny P, Waldhausl W, Roden M. Increased lipid availability impairs insulin-stimulated ATP synthesis in human skeletal muscle. Diabetes 55: , Brooke MH, Kaiser KK. Three myosin adenosine triphosphatase systems: the nature of their ph lability and sulfhydryl dependence. J Histochem Cytochem 18: , Bruce CR, Kriketos AD, Cooney GJ, Hawley JA. Disassociation of muscle triglyceride content and insulin sensitivity after exercise training in patients with type 2 diabetes. Diabetologia 47: 23 30, Cagnacci A, Soldani R, Carriero PL, Paoletti AM, Fioretti P, Melis GB. Effects of low doses of transdermal 17 beta-estradiol on carbohydrate metabolism in postmenopausal women. J Clin Endocrinol Metab 74: , Decombaz J, Schmitt B, Ith M, Decarli B, Diem P, Kreis R, Hoppeler H, Boesch C. Postexercise fat intake repletes intramyocellular lipids but no faster in trained than in sedentary subjects. Am J Physiol Regul Integr Comp Physiol 281: R760 R769, DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30: , Dela F, Larsen JJ, Mikines KJ, Galbo H. Normal effect of insulin to stimulate leg blood flow in NIDDM. Diabetes 44: , Dela F, Mikines KJ, Von LM, Secher NH, Galbo H. Effect of training on insulin-mediated glucose uptake in human muscle. Am J Physiol Endocrinol Metab 263: E1134 E1143, Devries MC, Lowther SA, Glover AW, Hamadeh MJ, Tarnopolsky MA. IMCL area density, but not IMCL utilization, is higher in women during moderate-intensity endurance exercise, compared with men. Am J Physiol Regul Integr Comp Physiol 293: R2336 R2342, Donahue RP, Prineas RJ, DeCarlo DR, Bean JA, Skyler JS. The female insulin advantage in a biracial cohort: results from the Miami Community Health Study. Int J Obes Relat Metab Disord 20: 76 82, Escalante Pulido JM, Alpizar SM. Changes in insulin sensitivity, secretion and glucose effectiveness during menstrual cycle. Arch Med Res 30: 19 22, Goodpaster BH, He J, Watkins S, Kelley DE. Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes. J Clin Endocrinol Metab 86: , He J, Watkins S, Kelley DE. Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity. Diabetes 50: , Helge JW, Dela F. Effect of training on muscle triacylglycerol and structural lipids: a relation to insulin sensitivity? Diabetes 52: , Jacob S, Machann J, Rett K, Brechtel K, Volk A, Renn W, Maerker E, Matthaei S, Schick F, Claussen CD, Haring HU. Association of increased intramyocellular lipid content with insulin resistance in lean nondiabetic offspring of type 2 diabetic subjects. Diabetes 48: , 1999.

8 SEX AND INSULIN SENSITIVITY Kiens B, Richter EA. Types of carbohydrate in an ordinary diet affect insulin action and muscle substrates in humans. Am J Clin Nutr 63: 47 53, Kraegen EW, Cooney GJ. Free fatty acids and skeletal muscle insulin resistance. Curr Opin Lipidol 19: , Krssak M, Falk PK, Dresner A, DiPietro L, Vogel SM, Rothman DL, Roden M, Shulman GI. Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study. Diabetologia 42: , Lee CC, Kasa-Vubu JZ, Supiano MA. Androgenicity and obesity are independently associated with insulin sensitivity in postmenopausal women. Metabolism 53: , Levin K, Daa SH, Alford FP, Beck-Nielsen H. Morphometric documentation of abnormal intramyocellular fat storage and reduced glycogen in obese patients with type II diabetes. Diabetologia 44: , Lillioja S, Young AA, Culter CL, Ivy JL, Abbott WG, Zawadzki JK, Yki-Jarvinen H, Christin L, Secomb TW, Bogardus C. Skeletal muscle capillary density and fiber type are possible determinants of in vivo insulin resistance in man. J Clin Invest 80: , Lithell H, Lindgarde F, Nygaard E, Saltin B. Capillary supply and lipoprotein-lipase activity in skeletal muscle in man. Acta Physiol Scand 111: , Lowry OH, Passonneau JV. A Flexible System of Enzymatic Analysis. New York: Academic, Lundsgaard C, van Slyke DD. Studies of lung volume I: Relation between thorax size and lung volume in normal adults. J Exp Med 27: 65 85, Nilsson PM, Lind L, Pollare T, Berne C, Lithell H. Differences in insulin sensitivity and risk markers due to gender and age in hypertensives. J Hum Hypertens 14: 51 56, Nuutila P, Knuuti MJ, Maki M, Laine H, Ruotsalainen U, Teras M, Haaparanta M, Solin O, Yki-Jarvinen H. Gender and insulin sensitivity in the heart and in skeletal muscles Studies using positron emission tomography. Diabetes 44: 31 36, Pan DA, Lillioja S, Kriketos AD, Milner MR, Baur LA, Bogardus C, Jenkins AB, Storlien LH. Skeletal muscle triglyceride levels are inversely related to insulin action. Diabetes 46: , Patil PB, Minteer SD, Mielke AA, Lewis LR, Casmaer CA, Barrientos EJ, Ju JS, Smith JL, Fisher JS. Malonyl coenzyme A affects insulinstimulated glucose transport in myotubes. Arch Physiol Biochem 113: 13 24, Perseghin G, Scifo P, Pagliato E, Battezzati A, Benedini S, Soldini L, Testolin G, Del MA, Luzi L. Gender factors affect fatty acids-induced insulin resistance in nonobese humans: effects of oral steroidal contraception. J Clin Endocrinol Metab 86: , Qu Z, Andersen JL, Zhou S. Visualisation of capillaries in human skeletal muscle. Histochem Cell Biol 107: , Rattarasarn C, Leelawattana R, Soonthornpun S, Setasuban W, Thamprasit A. Gender differences of regional abdominal fat distribution and their relationships with insulin sensitivity in healthy and glucoseintolerant Thais. J Clin Endocrinol Metab 89: , Richter EA, Mikines KJ, Galbo H, Kiens B. Effect of exercise on insulin action in human skeletal muscle. J Appl Physiol 66: , Roepstorff C, Donsmark M, Thiele M, Vistisen B, Stewart G, Vissing K, Schjerling P, Hardie DG, Galbo H, Kiens B. Sex differences in hormone-sensitive lipase expression, activity, and phosphorylation in skeletal muscle at rest and during exercise. Am J Physiol Endocrinol Metab 291: E1106 E1114, Roepstorff C, Steffensen CH, Madsen M, Stallknecht B, Kanstrup IL, Richter EA, Kiens B. Gender differences in substrate utilization during submaximal exercise in endurance-trained subjects. Am J Physiol Endocrinol Metab 282: E435 E447, Roepstorff C, Thiele M, Hillig T, Pilegaard H, Richter EA, Wojtaszewski JF, Kiens B. Higher skeletal muscle alpha2ampk activation and lower energy charge and fat oxidation in men than in women during submaximal exercise. J Physiol 574: , Sinha R, Dufour S, Petersen KF, LeBon V, Enoksson S, Ma YZ, Savoye M, Rothman DL, Shulman GI, Caprio S. Assessment of skeletal muscle triglyceride content by 1 H nuclear magnetic resonance spectroscopy in lean and obese adolescents: relationships to insulin sensitivity, total body fat, and central adiposity. Diabetes 51: , Siri WE. The gross composition of the body. Adv Biol Med Phys 4: , Steffensen CH, Roepstorff C, Madsen M, Kiens B. Myocellular triacylglycerol breakdown in females but not in males during exercise. Am J Physiol Endocrinol Metab 282: E634 E642, Tarnopolsky MA, Rennie CD, Robertshaw HA, Fedak-Tarnopolsky SN, Devries MC, Hamadeh MJ. Influence of endurance exercise training and sex on intramyocellular lipid and mitochondrial ultrastructure, substrate use, and mitochondrial enzyme activity. Am J Physiol Regul Integr Comp Physiol 292: R1271 R1278, Tarnopolsky MA, Ruby BC. Sex differences in carbohydrate metabolism. Curr Opin Clin Nutr Metab Care 4: , Thamer C, Machann J, Bachmann O, Haap M, Dahl D, Wietek B, Tschritter O, Niess A, Brechtel K, Fritsche A, Claussen C, Jacob S, Schick F, Haring HU, Stumvoll M. Intramyocellular lipids: anthropometric determinants and relationships with maximal aerobic capacity and insulin sensitivity. J Clin Endocrinol Metab 88: , Thong FS, Dugani CB, Klip A. Turning signals on and off: GLUT4 traffic in the insulin-signaling highway. Physiology 20: , Valdes CT, Elkind-Hirsch KE. Intravenous glucose tolerance test-derived insulin sensitivity changes during the menstrual cycle. J Clin Endocrinol Metab 72: , Vincent MA, Clerk LH, Rattigan S, Clark MG, Barrett EJ. Active role for the vasculature in the delivery of insulin to skeletal muscle. Clin Exp Pharmacol Physiol 32: , Virkamaki A, Korsheninnikova E, Seppala-Lindroos A, Vehkavaara S, Goto T, Halavaara J, Hakkinen AM, Yki-Jarvinen H. Intramyocellular lipid is associated with resistance to in vivo insulin actions on glucose uptake, antilipolysis, and early insulin signaling pathways in human skeletal muscle. Diabetes 50: , Vistisen B, Hellgren LI, Vadset T, Scheede-Bergdahl C, Helge JW, Dela F, Stallknecht B. Effect of gender on lipid-induced insulin resistance in obese subjects. Eur J Endocrinol 158: 61 68, White LJ, Ferguson MA, McCoy SC, Kim HW, Castellano V. Cardiovascular/non-insulin-dependent diabetes mellitus risk factors and intramyocellular lipid in healthy subjects: a sex comparison. Metabolism 55: , Wojtaszewski JF, Hansen BF, Gade Kiens B, Markuns JF, Goodyear LJ, Richter EA. Insulin signaling and insulin sensitivity after exercise in human skeletal muscle. Diabetes 49: , World Health Organization. Energy and Protein Requirements: Report of a Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series No Geneva: WHO, 1985.