Lipoly Moly and Droplet Size in obese Individuals

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1 See Commentary by Hesselink, Mensink, and Schrauwen on pages Effects of Weight Loss and Physical Activity on Muscle Lipid Content and Droplet Size Jing He, Bret H. Goodpaster, and David E. Kelley Abstract HE, JING, BRET H. GOODPASTER, AND DAVID E. KELLEY. Effects of weight loss and physical activity on muscle lipid content and droplet size. Obes Res. 2004;12: Objectives: To address the potential effects of weight loss and physical activity (WL Ex) on intramyocellular lipid (IMCL) and lipid droplet size in overweight and obese previously sedentary individuals. Research Methods and Procedures: IMCL and lipid droplet size was determined in vastus lateralis, obtained by percutaneous biopsy, from 21 obese volunteers (9 men/12 women), using Oil Red O staining, along with succinate dehydrogenase histochemistry and mitochondrial immunohistochemistry as measures of skeletal muscle oxidative capacity. Insulin sensitivity (IS) was determined by glucose clamp. Results: A 4-month WL Ex intervention resulted in 10% WL and 15% increase in maximal oxygen uptake, leading to a 46% increase in IS (all p 0.01). IMCL did not significantly change (p 0.36). However, the size of lipid droplets decreased after WL Ex (p 0.01), and this decrease in lipid droplet size correlated with increased IS (p 0.01) and the amount of physical activity (p 0.05). Succinate dehydrogenase activity and mitochondrial labeling increased significantly (p 0.01), without a significant shift in fiber type distribution. Discussion: In summary, IMCL does not decrease in response to WL Ex in obese, previously sedentary individuals, yet the lipid within muscle is dispersed into smaller droplets. This change in the size of lipid droplets, likely Received for review June 30, Accepted in final form March 18, The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Address correspondence to David E. Kelley, 807N Montefiore-University Hospital, 3459 Fifth Avenue, Pittsburgh, PA kelley@msx.dept-med.pitt.edu Copyright 2004 NAASO coupled with a concomitant increase in oxidative enzyme capacity, is correlated to improved IS. Key words: insulin resistance, skeletal muscle, diabetes, intramyocellular lipid, mitochondria Introduction Lipid content in skeletal muscle, both extracellular and intramyocellular lipid (IMCL), 1 is increased in obesity and type 2 diabetes (1 4). A number of investigations have found an association between increased IMCL and insulin resistance (IR) (5 9). The mechanisms that account for these associations are unclear. One intriguing line of investigation postulates that an increase in IMCL induces IR by activation of inflammatory pathways and serine phosphorylation of insulin receptor substrate-1 (10). In support of this, experimental elevation of IMCL in healthy humans by infusion of lipid emulsion caused IR (11) and activation of protein kinase C (12). However, elevated IMCL is not always associated with skeletal muscle IR. IMCL is increased in lean, highly trained individuals (13,14), a group characterized by heightened insulin sensitivity (IS). IMCL is an important energy source during activity, especially in physically trained individuals (15,16). However, unlike in trained individuals, skeletal muscle in obesity and type 2 diabetes characteristically has a reduced oxidative enzyme activity (17,18) and reduced reliance on lipid oxidation during fasting conditions (19 21). He et al. (3) observed that skeletal muscle in obesity and type 2 diabetes has both increased IMCL and diminished oxidative enzyme activity regardless of fiber type, suggesting that proportionality between IMCL and oxidative enzyme capacity shapes the association between IMCL and IR. Ultrastructural investigations of human skeletal muscle have shown that lipid droplets normally account for 1% of cell volume within muscle cells in lean, healthy individuals (22). The volume of lipid droplets in skeletal muscle is 1 Nonstandard abbreviations: IMCL, intramyocellular lipid; IR, insulin resistance; IS, insulin sensitivity; WL, weight loss; Ex, exercise; SDH, succinate dehydrogenase; FM, fat mass; HR, heart rate; FFM, fat-free mass; VO 2max, maximal oxygen uptake. OBESITY RESEARCH Vol. 12 No. 5 May

2 increased several fold in obesity and type 2 diabetes (1). Weight loss (WL) and increased physical exercise (Ex) ameliorate IR in obesity (23 28). Reductions in fat mass (FM) and central adiposity are related to the improvement in IR induced by WL (24,27), and it is plausible that reductions in IMCL may be part of this reduction in adiposity. In support of this concept, Goodpaster et al. observed that a 15% WL was associated with decreased IMCL in obesity (1), and Greco et al. (29), found that substantial WL after bariatric surgery caused a marked decrease in IMCL concomitant with improvement in IR. On the other hand, physical activity interventions, even among overweight individuals, has been observed to either increase IMCL or lead to unchanged IMCL and yet to decrease IR (30 32). Combined WL Ex is the intervention recommended for IR and prevention of type 2 diabetes in high-risk individuals (33,34); thus, it is of physiological interest to examine the effect on IMCL. The current study was undertaken to address this issue and of the effect of WL Ex intervention on muscle oxidative enzyme capacity in relation to IMCL. Research Methods and Procedures Subjects The research volunteers for this investigation were overweight or obese men (n 9, age years) and women (n 12, age years); metabolic data concerning this intervention have been previously published (35). Before participation, all potential volunteers had a medical examination. For inclusion, volunteers were required to have maintained a stable weight ( 2 kg) for at least 6 months before the study. None of the volunteers had type 2 diabetes, nor were they participating in any regular exercise before the study. The protocol was approved by the University of Pittsburgh Institutional Review Board, and all volunteers gave written informed consent. Muscle Biopsy and Tissue Microscopy A percutaneous muscle biopsy procedure was performed as previously described (21) to obtain samples of vastus lateralis skeletal muscle. This was done after an overnight fast and approximately 1 hour before starting an insulin infusion, as described below. At biopsy, to prepare samples for light microscopy, samples were dissected free of adipose and connective tissue and mounted in Cryomatrix (Shandon, Pittsburgh, PA), then frozen directly in isopentane cooled in liquid nitrogen. Biopsy samples were maintained within sealed containers at 80 C until microscopy studies. Tissue blocks from pre- and postintervention were mounted and prepared in parallel, as previously described (3). From each tissue block, serial transverse sections (8 m) were cut using a cryostat at 20 C and then mounted on Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA). Adjacent sections were incubated in different media for succinate dehydrogenase (SDH) and Oil Red O staining solution (36) to assess oxidative enzyme activity and lipid content, respectively. As negative control for the SDH reaction, to assess background staining, sections were incubated in media without the substrate succinate. For a control slide of Oil Red O staining, slides were incubated in 100% acetone for 10 minutes before immersing into Oil Red O staining solution. Image analysis was performed using a computer-based system, with images captured by an optical microscope (Olympus, Tokyo, Japan) connected to a charged coupled device video camera (Sony, Tokyo, Japan). Digitally captured images were processed and analyzed using Northern Eclipse 6.0 software (Empix Imaging, Inc., North Tonawanda, NY). Muscle fibers were classified as fiber types I and II based on myosin monoclonal antibody immunostaining. In serial sections, individual fibers identified on the slides prepared for fiber type determination were then re-identified on the sections stained for SDH and Oil Red O and assessed for oxidative enzyme activity and lipid content. Confocal laser scanning microscopy was used to obtain higher resolution of images and permit more detailed assessment of IMCL droplets and mitochondria in 8- m cryosections. The size of lipid droplets was assessed based on fluorescence signals by using a Texas red excitation filter (540 to 580 nm) to excite the autofluorescence of lipid droplet on Oil Red O staining and assessed with a quantitative image analysis program (Northern Eclipse 6.0). To more precisely estimate the size of lipid droplets, calibration factors were determined using fluorescent microspheres of known size (4- m diameter; Molecular Probes, Eugene, OR). These spheres were imaged using variations in each of the following parameters (photomultiplier tube settings, gain, and offset). For each of the factors, examined separately, a linear relation was evident (p 0.01) with respect to measured sphere area. Based on this observation, the spheres were imaged using the exact parameters used during the imaging of muscle cryosections, a calibration factor was determined to relate measured sphere area to its known size, and this calibration factor was applied to the measured size of lipid droplets and mitochondria size. To assess whether there were changes in the size of mitochondria, the size of the labeling signal was obtained using a fluorescein isothiocyanate excitation filter (465 to 495 nm) and measured with computerized image analysis. Mouse antihuman mitochondrial 65-kilodalton protein antibody (antibody was raised from the RAJI BURKITTS cell line, dilution 1:100; Chemicon International, Inc., Temecula, CA), and mouse antimyosin heavy chain monoclonal antibodies (dilution 1:200, Bio- Genex, San Ramon, CA) were used as primary antibodies for detecting the signal size of mitochondria and muscle fiber type, respectively. Alexa Fluor@ 488 goat antimouse IgG (H L) conjugated secondary antibody was used to 762 OBESITY RESEARCH Vol. 12 No. 5 May 2004

3 produce the fluorescent signals (dilution 1:500; Molecular Probe Inc.). A laser scanning confocal microscope (Olympus BX60) with a connected charged coupled device video camera was used to capture images. IS, Body Composition, and Fitness At baseline and at completion of the WL Ex intervention, IS was determined using the glucose clamp method (37), as previously described for our laboratory (5). IS was calculated as the mean rate of glucose disposal, expressed per fat-free mass (FFM), during the final 30 minutes of a 4-hour, euglycemic, insulin infusion (40 mu/m 2 per minute), performed after an overnight fast. Catheters were placed in a forearm vein for insulin (Humulin; Eli Lilly, Indianapolis, IN) and 20% dextrose infusion, and a second catheter was inserted into a radial artery for blood sampling. Blood samples were collected for determination of serum insulin. After intervention, IS was measured 36 to 48 hours after the last exercise bout to avoid any confounding effects of acute exercise on IS. Plasma glucose during the oral glucose tolerance test and glucose clamp was measured using an automated glucose oxidase reaction (YSI 2300 Glucose Analyzer; YSI Inc., Yellow Springs, OH). Serum insulin was determined using commercially available radioimmunoassay kits (Pharmacia, Uppsala, Sweden). FM and FFM were assessed by DXA (model DPX-L; Lunar, Madison, WI) using software version 1.3Z. Maximal oxygen uptake (VO 2max ) was measured using an incremental protocol on an electronically braked cycle ergometer (Sensormedics, Yorba Linda, CA), as previously described (21). HR, blood pressure, and electrocardiogram were recorded before, during, and immediately after this test. Systemic gas exchange measurements were performed with a metabolic cart (Sensormedics 2900) for analysis of CO 2 and O 2 fractions to calculate oxygen consumption and the HR-oxygen consumption relationship was plotted for each person to provide individualized exercise intensity prescriptions and estimate energy expenditure during exercise sessions. Exercise Training and WL Intervention After completion of the baseline assessments, research volunteers participated in a 16-week program of WL Ex, as previously described (35). In brief, volunteers were asked to participate in a minimum of four and a maximum of six exercise sessions weekly, with at least one supervised session per week. A wireless heart rate (HR) monitor (Polar, Kempele, Finland) was used to record exercise duration and mean HR for estimation of weekly caloric expenditure, and volunteers maintained logs of their exercise sessions. The target exercise intensity for each participant was 65% to 75% of their measured maximal HR. The goal of the WL intervention was to produce a WL of 10% body weight; to achieve this, a 500- to 1000-kcal/d reduction in calories was prescribed on the basis of recent food records and to achieve a low-fat ( 30% of calories from fat) diet. The WL program was administered individually and a nutritionist met weekly with each subject. Statistical Analysis Data are presented as mean SEM, unless otherwise indicated. Changes in IS, physical fitness (VO 2max ), body composition, mitochondria and lipid droplet size, SDH activity, and lipid total content were compared using paired Student s t tests. Linear regression analysis was used to determine whether the changes in skeletal muscle lipid, mitochondria, or oxidative capacity were associated with improvements in IS. All statistics were performed using JMP version for the Macintosh (SAS, Inc., Cary, NC). Results Physiological Responses to Intervention The clinical and physiological characteristics of the research volunteers at baseline and at completion of the WL Ex intervention are shown in Table 1. There were significant decreases of weight and FM of 10% and 17%, respectively, whereas FFM did not significantly change. There was a significant increase in VO 2max of 16%, and there was a significant increase in IS of 49% as previously reported (35). Thus, the intervention achieved the goal of an 10% WL combined with an increased aerobic fitness and improvement of IR. Light Microscopy of IMCL and Oxidative Enzyme Capacity Data from light microscopy assessments of IMCL and oxidative enzyme capacity are shown in Table 2. The intervention did not result in a significant change in the percentage of type I muscle fibers ( % vs %; pre- vs. postintervention; p 0.2). There was, however, a highly significant change (p ) in oxidative enzyme capacity, assessed on the basis of the intensity of SDH staining. An increase in oxidative enzyme capacity occurred within both type I ( % increase) and type II muscle fibers ( %). There was not a significant change in IMCL in either fiber type, as reflected in quantitative assessments of Oil Red O. Confocal Microscopy of Muscle Fibers The mean size of lipid droplets within muscle fibers and the mean size of mitochondria labeling were assessed using confocal microscopy, and these data are shown in Table 3. The mean size of lipid droplets decreased significantly (p 0.001), by 9%. There was also a significant decrement (p 0.01) in the mean intensity of the autofluorescence of Oil Red O staining per lipid droplet, from 69 3to65 OBESITY RESEARCH Vol. 12 No. 5 May

4 Table 1. Effect of WL Ex on weight, VO 2max, and IS Baseline Postintervention p Weight (kg) BMI (kg/m 2 ) FM (kg) FFM (kg) NS VO 2max (ml/min per kilogram FFM) IS (mg/min per kilogram FFM) Fasting glucose (mg/dl) Fasting insulin (pm) All values are means SE. Differences determined using paired Student s t tests. NS, nonsignificant. 3 arbitrary units of staining density, on a white to gray digitized scale. The mean size of immunohistochemical labeling of mitochondria increased by 14% (p 0.01). A representative photograph of the staining for IMCL and mitochondrial labeling is shown in Figure 1. The reduction in the mean size of lipid droplets was highly correlated with improved IS (r 0.69; p 0.05) as shown in Figure 2, indicating that an increase in IS was associated with the change toward smaller lipid droplets. The decrease in size of lipid droplets was also significantly correlated with the increase in mitochondria protein signal size (r 0.46; p 0.05) and with improved VO 2max (r 0.46; p 0.05) but was not significantly correlated with WL or loss of FM. The increase in the mitochondria protein signal was also associated with improvement in IS (r 0.59; p 0.05) as shown in Figure 3. Muscle Lipid Droplet Size in Type 2 Diabetes The current findings on decreases in muscle lipid droplet size after WL Ex intervention and correlation to changes in IS are novel observations. Therefore, we undertook additional assessments of lipid droplet size using muscle biopsy samples previously obtained from lean, obese, and type 2 diabetes groups. Data from a cross-sectional comparison for IMCL, fiber type, and muscle oxidative enzyme activity has previously been published using these biopsy samples (3). Using newly prepared sections from these tissue blocks, lipid droplet size was measured, and the results are shown in Figure 4. The size of lipid droplets was larger in type 2 diabetes than in muscle from lean, sedentary, nondiabetic volunteers. The findings in obese, sedentary, nondiabetic volunteers resemble type 2 diabetes. These data further suggest that in addition to IMCL, lipid droplet size is a morphological characteristic pertinent to IR. Discussion During the past decade, the concept of lipotoxicity has emerged as a potential mechanism for the association Table 2. Fluorescence microscopy of Vastus Lateralis for muscle lipid and oxidative enzyme activity, before and after weight loss and physical activity intervention Baseline Postintervention p IMCL type I fibers NS (0.65) IMCL type II fibers NS (0.11) SDH All values are means SE. All values are arbitrary units of staining density. Differences determined using paired Student s t tests. NS, nonsignificant. Table 3. Confocal microscopy of muscle lipid droplets and mitochondria Baseline Postintervention p Lipid droplet size ( m 2 ) Lipid droplet intensity (AU) Mitochondria label size ( m 2 ) All values are means SE. AU, arbitrary units of staining density. Differences determined using paired Student s t tests. 764 OBESITY RESEARCH Vol. 12 No. 5 May 2004

5 Figure 1: A representative photograph at of confocal microscopy of immunohistochemical labeling of mitochondria (40 magnification) before (pre-) and after (post-) intervention (upper panels) and lipid droplets stained with Oil Red O (60 magnification) from biopsy samples obtained before and after WL Ex intervention (lower panels). Inset for each panel represents a 100 magnification. of IR with adiposity (38). One of the key aspects of lipotoxicity concerns increased IMCL because muscle is a principal site of insulin-stimulated glucose disposal. A number of cross-sectional studies have observed an association between IMCL and IR (6 9,11,39). The current study addressed the relationship between IMCL and IR from the perspective of a combined WL and Ex intervention, the type of intervention strongly recommended for treatment of IR and prevention of type 2 diabetes (33,34). We found that improvement in IS achieved by WL Ex is not associated with decreased IMCL; in fact, there was a nonsignificant trend toward increased IMCL. This finding, which is consistent with that of several other recent studies that examined effects of Ex intervention on IMCL (30 32), might infer that IMCL is unrelated to IR or at least that improvement in IR is not dependent on concomitant change in IMCL. Yet, such a conclusion would overlook a key novel observation of the current study, which is that the size of lipid droplets is significantly smaller after WL Ex and that the improvement in IR is correlated with this change. The decrease in size of lipid droplets was significantly correlated with improved aerobic fitness, although not with WL per se. This observation of smaller lipid droplets, taken together with the finding of an absence of change in IMCL, denotes a change in the storage of triglyceride within muscle fibers, with dispersion into smaller and more numerous lipid droplets. Physically, this is a change that increases surface area in relation to volume, suggesting greater accessibility for use and, perhaps, higher rates of triglyceride turnover, as will be discussed below. Most of the studies that have examined the association between IMCL and IR have been cross-sectional, although one valuable exception was the recent study by Greco et al. (29). In that study, after marked WL achieved by bariatric surgery, there was a substantial decrease in IMCL that was related to the improvement in IR (29). A previous report from our laboratory indicated that IMCL in obesity and type 2 diabetes tends to decrease after a more moderate WL (1). These WL interventions did not specifically involve in- OBESITY RESEARCH Vol. 12 No. 5 May

6 Figure 2: Association between the change in the size of lipid droplets and improved IS. Figure 3: Association between the change in the size of mitochondria protein signal and improved IS. creased Ex. In a prior study of WL intervention (1), we did not observe a change in fitness, and activity of oxidative enzymes did not change (18), yet there was a decline in IMCL associated with improved IR, suggesting that lessening lipotoxicity improves IR in obesity and type 2 diabetes. In the current study, which entailed WL and Ex, there was an increase in muscle oxidative enzyme activity, and this change was related to improvement in IR. We postulate that improvements in muscle oxidative enzyme activity leverage a shift in the relationship between IMCL and IR. One can infer this from the findings of increased IMCL in highly trained athletes (14), in which there is also heightened oxidative enzyme capacity and IS. In the current study, the change in oxidative enzyme activity was correlated with the decreased lipid droplet size, and we interpret this to reflect more active use of IMCL after increased physical activity. Induction of increased oxidative enzyme activity by aerobic exercise is one of the classic manifestations of metabolic malleability of skeletal muscle (40), and as observed in the current study, this can occur without changes in fiber type distribution (22). In the current study, the increase in oxidative enzyme activity was manifest both by histochemical staining for SDH activity and by immunohistochemical labeling of mitochondria, assessed by confocal microscopy, and these changes were related to the amounts of physical activity. Taken together, smaller, more numerous lipid droplets and increased mitochondria denote more active catabolism of stored lipid, and this is supported by concomitant physiological observations in these volunteers. Recently, we reported an increased reliance on lipid oxidation during fasting conditions after this intervention and that enhanced fat oxidation during fasted conditions was a strong correlate of improved IS, more robust than either WL or change in VO 2max (35). Skeletal muscle is a major site for lipid oxidation during fasting conditions (41). Our laboratory has advanced an hypothesis of metabolic inflexibility in lipid oxidation by muscle as one of the components of muscle IR in obesity and type 2 diabetes (42). The volunteers in the current study showed a restitution of metabolic flexibility in lipid oxidation after the WL Ex intervention (35), and the microscopy findings reported herein begin to delineate some of the cellular mechanisms or at least morphological changes underlying the improvements in nutrient partitioning and IR in muscle. Although the mean value for IMCL did not change with WL Ex intervention, there was considerable variability across the participants in the current study. The findings in one individual are of particular interest. This participant expended an average of 3623 kcal/wk during structured physical activity, or 3-fold higher than the mean of the group, which was kcal/wk, and he also had a 60% increase in IMCL and a 60% increase in IS. These data, together with the reported high degree of variability in the exercise training-induced changes in IMCL (43), suggest that exercise tends to augment IMCL yet concomitantly increases IS. This postulate is supported by the findings of 766 OBESITY RESEARCH Vol. 12 No. 5 May 2004

7 Figure 4: Size of lipid droplets and size of mitochondria protein signal in obese subjects with type 2 diabetes, obese subjects without type 2 diabetes (obese), and lean subjects without type 2 diabetes (lean). An asterisk denotes significant difference between lean vs. obese and type 2 diabetes. several recent Ex intervention studies (30 32). More precise characteristics of IMCL, notably lipid droplet size and relation to oxidative capacity, might be more germane to the relation to IR than IMCL per se. Using confocal microscopy, it was technically possible to assess lipid droplet size and, thereby, detect a decrease in the average size of lipid droplets, an approach we corroborated with the use of microspheres of known size to calibrate these measurements. Another, related technical modification used in the current study was based on the recently reported method of exploiting autofluorescence of Oil Red O staining in tissue section (44). To our knowledge, the current study is the first to explore whether there is an association of lipid droplet size within the putative association between IMCL and IR. After intervention, as well as the decrease in size of lipid droplets, there was a significant decrease in the imaging intensity of the Oil Red O staining of the lipid droplets. This biophysical property is likely related to the proportionality between polar phospholipids (that have less binding of Oil Red O) and triglyceride (that have avid binding of Oil Red O) and, thus, is corroborative of a smaller size of lipid droplets because this would reflect an increase in the ratio of surface area (phospholipids) to lipid volume (triglyceride content) arising from a predominance of smaller lipid droplets. Intrigued by this new observation concerning the size of lipid droplets, new examinations were conducted using tissue blocks from a prior cross-sectional study involving lean, obese, and type 2 diabetes research volunteers (3). In the study by He et al. (3), it was observed that increased IMCL in obesity and type 2 diabetes was associated with decreased oxidative enzyme activity. A reexamination of these biopsy samples using this newly developed confocal microscopy technique revealed group differences in lipid droplet size and in a manner that follows the group differences in IR, oxidative capacity, and IMCL. Thus, both in the context of interventioninduced changes and in cross-sectional group comparisons, increased lipid droplet size is a characteristic of importance in the relation between IMCL and IR. In summary, the current study provides novel information on the impact of WL Ex on IMCL and the relationship to IR. The combination of WL Ex augmented mitochondria, decreased IR, and reduced the size of lipid droplets, yet did not change overall IMCL. That improvement in IR was associated with a dispersion of stored lipid into smaller droplets and with increased oxidative enzyme activity suggests that it is not simply the amount of IMCL that determines lipotoxicity and IR but rather a dependence of the mass of the stored lipid on a metabolic capacity for its catabolism that underlies lipotoxic IR in skeletal muscle in obesity and type 2 diabetes. Acknowledgments This work was funded by National Institues of Health, National Institute of Diabetes & Digestive & Kidney Diseases R01DK , K24 DK02782, and National Institutes of Health-National Institute on Aging K01-AG and also by the Obesity Nutrition Research, National Institutes of Health, National Institute of Diabetes & Digestive & Kidney Diseases (1P30DK46204) and General Clinical Research (5M01RR00056) Centers. References 1. Goodpaster BH, Theriault R, Watkins SC, Kelley DE. Intramuscular lipid content is increased in obesity and decreased by weight loss. Metabolism. 2000;49: Goodpaster BH, Thaete FL, Kelley DE. Thigh adipose tissue distribution is associated with insulin resistance and obesity in type 2 diabetes mellitus. Am J Clin Nutr. 2000;71: 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. 2001;50: Thamer C, Machann J, Bachmann O, et al. Intramyocellular lipids: anthropometric determinants and relationships with maximal aerobic capacity and insulin sensitivity. J Clin Endocrinol Metab. 2003;88: Goodpaster BH, Thaete FL, Simoneau JA, Kelley DE. Subcutaneous abdominal fat and thigh muscle composition predict insulin sensitivity independently of visceral fat. Diabetes. 1997;46: Jacob S, Machann J, Rett K, et al. Association of increased OBESITY RESEARCH Vol. 12 No. 5 May

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