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Skeletal Muscle Fat Oxidation Is Increased in African-American and White Women after 10 days of Endurance Exercise Training Ronald N. Cortright,*† Kimberly M. Sandhoff,* Jessica L. Basilio,* Jason R. Berggren,* Robert C. Hickner,*† Matthew W. Hulver,‡ G. Lynis Dohm,† and Joseph A. Houmard*

Abstract CORTRIGHT, RONALD N., KIMBERLY M. SANDHOFF, JESSICA L. BASILIO, JASON R. BERGGREN, ROBERT C. HICKNER, MATTHEW W. HULVER, G. LYNIS DOHM, AND JOSEPH A. HOUMARD. Skeletal muscle fat oxidation is increased in African-American and white women after 10 days of endurance exercise training. Obesity. 2006;14:1201–1210. Objective: Obesity is associated with lower rates of skeletal muscle fatty acid oxidation (FAO), which is linked to insulin resistance. FAO is reduced further in obese AfricanAmerican (AAW) vs. white women (CW) and may also be lower in lean AAW vs. CW. In lean CW, endurance exercise training (EET) elevates the oxidative capacity of skeletal muscle. Therefore, we determined whether EET would elevate skeletal muscle FAO similarly in AAW and CW with a lower lipid oxidative capacity. Research Methods and Procedures: In vitro rates of FAO were assessed in rectus abdominus muscle strips using [1-14C] palmitate (Pal) from lean AAW [BMI ⫽ 24.2 ⫾ 0.9 (standard error) kg/m2] and CW (23.6 ⫾ 0.8 kg/m2) undergoing voluntary abdominal surgery. Lean AAW (22 ⫾ 0.9 kg/m2) and CW (24 ⫾ 0.8 kg/m2) and obese AAW (36 ⫾ 1.2 kg/m2) and CW (40 ⫾ 1.3 kg/m2) underwent 10 consecutive days of EET on a cycle ergometer (60 min/d, 75% peak oxygen uptake). FAO was measured in vastus lateralis homogenates as captured 14CO2 using [1-14C]

Received for review August 19, 2005. Accepted in final form May 10, 2006. 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. *The Human Performance Laboratory, Department of Exercise and Sport Science, College of Health and Human Performance, and †Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina; and ‡Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana. Address correspondence to Ronald N. Cortright, 371 Ward Sports Medicine Building, The Human Performance Laboratory, East Carolina University, Greenville, NC 27858. E-mail: [email protected] Copyright © 2006 NAASO

Pal, palmitoyl-CoA (Pal-CoA), and palmityl-carnitine (PalCar). Results: Muscle strip experiments showed suppressed rates of FAO (p ⫽ 0.03) in lean AAW vs. CW. EET increased the rates of skeletal muscle Pal oxidation (p ⫽ 0.05) in both lean AAW and CW. In obese subjects, Pre-EET Pal (but not Pal-CoA or Pal-Car) oxidation was lower (p ⫽ 0.05) in AAW vs. CW. EET increased Pal oxidation 100% in obese AAW (p ⬍ 0.05) and 59% (p ⬍ 0.05) in obese CW. Similar increases (p ⬍ 0.05) in post-EET FAO were observed for Pal-CoA and Pal-Car in both groups. Discussion: Both lean and obese AAW possess a lower capacity for skeletal muscle FAO, but EET increases FAO similarly in both AAW and CW. These data suggest the use of EET for treatment against obesity and diabetes for both AAW and CW. Key words: fatty acid oxidation, race, diabetes, acylCoA synthetase, acyl-CoA synthetase long chain

Introduction A number of epidemiological studies have identified differences in the development and maintenance of obesity in African-American (AAW)1 compared with white women (CW) (1–3). For example, Burke et al. (4) found that AAW have a propensity to gain more weight at an earlier age than CW. In addition, Foryet (5) and Kumanyika (6) observed that obese AAW show a slower rate of weight loss than CW of similar weight, possibly contributing to their higher incidence of obesity. Suggested explanations for these findings have included differences in dietary habits and lower rates of physical activity energy expenditure (7,8). More recently, however, mechanism-based studies have suggested metabolic differences in obese AAW compared with

1 Nonstandard abbreviations: AAW, African-American women; CW, white women; FAO, fatty acid oxidation; VO2peak, peak oxygen uptake; CPT-1, carnitine palmitoyl transferase-1; PKC, protein kinase C.

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Skeletal Muscle Fatty Acid Oxidation in African-Americans, Cortright et al.

obese CW (9,10). Therefore, although differences in income, socioeconomic status, or education may contribute to the higher incidence of obesity in AAW, inherent biochemical mechanisms also seem to play a role (6,10 –13). Rates of skeletal muscle oxidation of fatty acids have been shown to be lower in obese compared with lean individuals. To date, however, few studies have compared the capacity for skeletal muscle to oxidize fat between AAW and CW. However, one study by Privette et al. (10) did compare the in vitro rates of long-chain fatty acid oxidation (FAO) between the races and showed that obese AAW have a significantly reduced capacity to oxidize palmitic acid (10). Because fatty acids provide ⬃60% of the energy needs of skeletal muscle during rest in healthy adults, it was suggested that suppressed rates of lipid oxidation by muscle could result in shunting of this fuel to adipose tissue for storage. Similarly, reduced rates of skeletal muscle FAO could also contribute toward an intramyofibrilar lipotoxic environment characteristic of the insulinresistant state (14). It is well established that acute aerobic exercise is associated with increased lipid mobilization and oxidation by skeletal muscle if the exercise intensity is light to moderate [e.g., 40% to 65% peak oxygen uptake (VO2peak)]. It is equally well understood that aerobically trained individuals are characterized by an expansion in mitochondrial oxidative capacity (15), and recent data have shown a greater in vitro oxidation of fatty acids by skeletal muscle in trained vs. sedentary individuals (16). The shift in efficiency for metabolizing lipids by skeletal muscle in lean subjects occurs early with exercise training, as only 7 to 10 days of regular aerobic exercise increased post-absorptive fat oxidation without changing body composition (17). These data support the use of exercise training as a component in the treatment of obesity (18). However, there are few studies comparing the effects of aerobic exercise training on skeletal muscle FAO between obese and lean individuals, and those that do exist are currently equivocal (16,19,20). More so, even fewer studies exist that have considered the hypothesis that physiological differences may exist in the metabolic response to exercise between AAW and CW. One recent study, however, has shown preferential use of carbohydrates over fats during submaximal exercise in AAW compared with CW (20). A surprising outcome of this study was that, like obese subjects, lean AAW remained metabolically inflexible during an acute bout of endurance exercise. That is, like obese individuals, lean AAW did not shift toward the use of fat during aerobic exercise in the untrained state. This startling observation now raises the question as to whether non-obese AAW possess a pre-existing metabolic reduction in the capacity to oxidize fatty acids compared with lean CW, thus predisposing them to obesity and related diseases. To date, only two studies have addressed this issue, but both were performed during exercise 1202


and at the whole body level (9,20). Thus, direct evidence to support reductions in skeletal muscle lipid oxidative capacity in non-obese African Americans is lacking. Given the greater prevalence and severity of obesity in AAW, the purpose of these studies was to determine whether there are differences in skeletal muscle FAO rates between AAW and CW. We hypothesized that the observed lower rates of lipid oxidation in skeletal muscle from obese individuals is also present in lean AAW. Furthermore, because of the relative absence of data comparing skeletal muscle FAO after aerobic training in obese individuals as well as between AAW and CW, we determined the in vitro rates of FAO in non-obese and obese AAW and CW before and after 10 days of aerobic exercise training.

Research Methods and Procedures Materials [1-14C] palmitic acid, [1-14C] palmitoyl-CoA, and [1-14C] palmityl-carnitine were purchased from Perkin-Elmer (Boston, MA). Protein was determined by the bicinchoninic acid method (Pierce Laboratories, Rockford, IL). All other reagents were purchased from Sigma Chemicals (St. Louis, MO). Human Subjects FAO Studies in Rectus Abdominus Muscle Strips. To test whether reduced rates of skeletal muscle FAO exist in lean AAW, we determined the ability of muscle strips from the rectus abdominus to oxidize the long-chain fatty acid palmitate. Subjects were recruited from patients undergoing elective abdominal surgery including total hysterectomy. Lean subjects were defined as having a BMI ⬍27 kg/m2. None of the subjects had any diseases or were taking medications known to alter carbohydrate or lipid metabolism. All subjects had maintained a constant body mass during the year preceding surgery. After an overnight fast (10 hours), general anesthesia was initiated with a short-acting barbiturate and maintained with fentanyl and NO-oxygen mixture. For FAO, 12 non-obese (lean) women (N ⫽ 5, AAW; N ⫽ 7, CW) were newly recruited for this aspect of the study. Studies Investigating FAO in Vastus Lateralis. FAO was evaluated in whole muscle homogenates from subjects before and after endurance exercise training. For this aspect of the study, 29 participants (N ⫽ 6, lean AAW; N ⫽ 7, lean CW; N ⫽ 9, obese AAW; N ⫽ 7, obese CW) were newly recruited. All subjects were premenopausal, and AAW were at least second generation. Study protocols, including participant recruitment, were reviewed and approved by the institutional review board at East Carolina University, and conformed to the University, State of North Carolina, and Federal mandates for standard operating procedures. Whole Body Procedures on Exercise Trained Subjects Hydrostatic weighing methodology without head submersion was used to assess body composition. Height and


weight were measured to calculate the participant’s BMI. In addition, the participants performed a cycle VO2peak test to characterize their aerobic fitness. To minimize the potential effects of a variable energy intake and diet composition on substrate use, subjects were trained by a certified nutritionist on how to conduct a 24-hour diet recall, which included a detailed record of food intake (quantity and particular foods) preceding their initial muscle biopsy. Over the last 24 hours of the training protocol, subjects consumed the same diet (food items and quantities) as recorded previously. Subjects reported to the laboratory after an overnight fast (no food after 8:00 PM the night before). Each participant confirmed that caffeinated beverages were not consumed the morning of the biopsy procedure. In the fasted state, a muscle biopsy from the vastus lateralis was obtained from the study participants using a modification of the percutaneous biopsy procedure as previously reported (21). After 10 days of aerobic training, the second muscle biopsy was obtained 24 hours after the last exercise session in the fasted condition. Exercise Training Protocol The participants were endurance trained according to a modified protocol used previously in our laboratory (22). Subjects were matched for aerobic capacity and activity level before training. VO2peak was measured by an incremental exercise test using an electronically braked cycle ergometer (Lode, Diversified, and Brea, CA) in the upright position. Oxygen consumption was measured with open circuit spirometry with a metabolic cart (ParvoMedics, Sandy, UT). The VO2peak and maximum heart rate was used to set the workload for the participants over the exercise training period. The exercise regimen consisted of 10 consecutive days of exercise training for 60 minutes on a cycle ergometer at an exercise heart rate corresponding to 75% VO2peak. Previously, a similar regimen reported increased insulin action and oxidative capacity without altering body weight or composition in young and older subjects (17,22). During the training protocol, each participant was intermittently monitored for oxygen consumption, and workload adjustments were made to maintain the same relative percentage of VO2peak over the 10-day training protocol. Body weight was assessed on Day 0 and Day 10 of the protocol to show the absence of weight loss caused by the endurance exercise regimen. FAO Studies in Rectus Abdominus Muscle Strips FAO studies were conducted as previously described (23,24). In brief, muscle strips weighing ⬃25 mg were teased from the biopsy sample and clamped in lucite clips to maintain consistent resting muscle length and tension throughout the preparation. Contamination of muscle samples with extramyocellular lipids was minimized by carefully removing visible fat. Muscles were incubated in

warmed (30 °C) Krebs-Hesseleit buffer gassed with 95% O2–CO2 (pH 7.0) and containing 4% bovine serum albumin (fatty acid free), 5 mM glucose, and 1 mM palmitate. Palmitic acid was dissolved in ethanol, and a small volume (0.8% of total buffer volume) was added to the incubation buffer to achieve the final desired palmitate concentration. After a 30-minute pre-incubation period, muscle samples were incubated for 1 hour at 30 °C in the same incubation medium as above with the addition of [1-14C] palmitate. Total palmitate oxidation was determined by measuring and summing 14CO2 production and 14C-labeled watersoluble metabolites. The measurement of 14C-water soluble metabolites accounted for 14C label that did not result in 14 CO2 because of isotopic exchange in the tricarboxylic acid cycle. Gaseous 14CO2 produced from the oxidation of [1-14C] palmitate during the incubation was measured by transferring 1.0 mL of the incubation medium to a 20-mL glass scintillation vial containing 1.0 mL of H2SO4 and a 0.5-mL Fisher microcentrifuge tube containing benzethonium hydroxide. Liberated 14CO2 was trapped in benzethonium hydroxide for 60 minutes, and the microcentrifuge tube containing trapped 14CO2 was placed in a scintillation vial and counted. 14C-water-soluble metabolites were measured by sampling 0.5 mL of the aqueous phase after lipid extraction of incubated muscle, which was placed in a scintillation vial and counted. FAO Studies in Whole Muscle Homogenates Oxidation studies on muscle homogenates were determined by measuring production of 1-14CO2 (complete oxidation) as previously described (25,26). Briefly, vastus lateralis tissue samples (⬃70 mg) were placed in homogenization buffer (see below) and cleaned of any visual lipids and connective tissue before chemical analysis. Afterward, samples were blotted, weighed, and placed in 2 volumes of fresh ice-cold homogenization buffer (SET) containing (in mM) 250 sucrose, 10 Tris-HCl, 1 EDTA, and 2 ATP, pH 7.4. Samples were thoroughly minced with surgical scissors, and homogenization buffer was added to yield a 20-fold diluted (wt:vol) homogenate and transferred to a 3-mL Potter-Elvehjem glass homogenization vessel. Muscle suspensions were homogenized on ice with a Teflon pestle at 10 passes across 30 seconds at 1200 rpm. We earlier established that muscle homogenates prepared this way contain intact mitochondria (respiratory control ratio ⬎5.0 by polarography) in which palmitate oxidation is carnitine and CoA dependent (26). Subsequently, 40-␮L aliquots of the 20-fold diluted muscle homogenates were plated in quadruplet into a modified 48-well cell culture plate (Costar, Cambridge, MA). A small groove was engineered between adjacent wells so that CO2 could freely diffuse between the incubation and trap wells. Reactions were started with the addition of 160 ␮L of a reaction mixture (pH 7.4) yielding final concentrations of (in mM) OBESITY Vol. 14 No. 7 July 2006

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0.2 palmitate ([1-14C]palmitate at 0.5 ␮Ci/mL), 100 sucrose, 10 Tris-HCl, 5 potassium phosphate, 80 potassium chloride, 1 magnesium chloride, 0.1 malate, 2 ATP, 1 dithiothreitol, 0.2 EDTA, 1 l-carnitine, 0.05 coenzyme A, and 0.5% fatty acid free bovine serum albumin. Specific activity for all experiments was maintained between 8000 and 8500 dpm/nmol palmitate. Culture plates were sealed with parafilm and a siliconized rubber gasket and allowed to incubate in a shaking water bath at 37 °C. After 60 minutes, reactions were terminated in the sealed reaction plates by the addition of 100 ␮L 70% perchloric acid to the incubation wells. The incubation plate was transferred to an orbital shaker, and 14CO2 was trapped in the adjoining well in 200 ␮L of 1 N NaOH for 1 hour (earlier time-course experiments indicated optimal time for CO2 trapping). Radioactivity of CO2 was determined by liquid scintillation counting using 4 mL Uniscint BD (National Diagnostics, Atlanta, GA). Statistical Analyses GraphPad Prism Version 4.00 for Windows, (GraphPad Software, San Diego, CA) was used to analyze all of the data collected. A general linear model two-way (race ⫻ time/training status) repeated-measures ANOVA was used to determine significant differences in in vitro (vastus lateralis homogenates) skeletal muscle FAO between the races and pre- to post-exercise training. Mean differences between rates of palmitate oxidation from muscle strips of lean AAW and CW were made using a one-way ANOVA. Post hoc analyses were applied where appropriate if significant main or interaction effects were noted by the ANOVA. Significance was set a priori at p ⱕ 0.05.

Results Subjects Table 1A shows the characteristics for the lean subjects undergoing abdominal surgery. The BMI for both AAW and CW were ⬍25 kg/m2 and were not significantly different between groups. AAW and CW were also of similar age. For the exercise training study, participants completed an activity questionnaire to confirm they were sedentary (participated in 60 minutes or less per week of recreation or work requiring modest physical activity such as golf, table tennis, bowling, or yard work). In addition, subjects confirmed that they were premenopausal and AAW were of second generation or greater. There were no significant differences when comparing Day 1 and Day 10 body weights between the two groups. Additionally, we successfully monitored participant’s dietary choices before the first and last biopsy procedure (data not shown). We were successful in our attempt to have each person eat similarly 1204


Table 1. Subject characteristics for individuals participating in FAO experiments

(A) Lean subjects N Age (years) BMI (kg/m2) (B) Lean subjects N Age (years) BMI (kg/m2) Percent fat VO2peak(liters/min) mL/kg TBM/min mL/kg FFM/min (C) Obese subjects N Age (years) BMI (kg/m2) Percent fat VO2peak(liters/min) mL/kg TBM/min mL/kg FFM/min

AAW

CW

5 46 ⫾ 4.4 24.5 ⫾ 0.7

7 44 ⫾ 3.4 24.0 ⫾ 0.7

6 25.2 ⫾ 1.4 22.0 ⫾ 0.9 20.0 ⫾ 1.6 1.6 ⫾ 0.2 27.8 ⫾ 1.4 31.0 ⫾ 3.1

7 27.9 ⫾ 2.5 23.6 ⫾ 0.7 24.0 ⫾ 0.8* 1.8 ⫾ 0.1 24.8 ⫾ 2.2 36.8 ⫾ 1.8

9 35.0 ⫾ 1.8 36.6 ⫾ 1.2 38.0 ⫾ 1.3 1.7 ⫾ 0.4 17.1 ⫾ 1.3 27.9 ⫾ 2.0

7 35.1 ⫾ 2.9 39.9 ⫾ 1.3 42.3 ⫾ 1.6† 1.9 ⫾ 0.1 17.8 ⫾ 1.1 33.0 ⫾ 2.1

FAO, fatty acid oxidation; AAW, African-American women; CW, white women; VO2peak, peak oxygen uptake; TBM, total body mass; FFM, fat free mass.(A) depicts data from subjects recruited for rectus abdominus muscle strip experiments. Subjects were individuals undergoing voluntary abdominal surgery (e.g., hysterectomy). Subject’s percent fat and VO2peak were not assessed. (B) and (C) depict data from subjects recruited for vastus lateralis homogenate oxidation studies. Within groups, the subjects were not significantly different from each other in age, BMI, or fitness level. Fat mass was significantly greater in lean CW vs. AAW subjects. The body mass of AAW and CW did not change post- vs. pre-exercise training. Values are mean ⫾ standard error of the mean. * Significantly different between groups, within measurement (p ⬍ 0.05). † Not statistically significant between groups (p ⫽ 0.06).

(energy content and macro nutrient composition) during the 24 hours before the pre- and post-training biopsy. Table 1B shows the characteristics for the lean subjects who underwent exercise training. Lean AAW and CW had no significant differences observed when comparing mean age, weight, BMI, fat-free mass, or VO2peak expressed in either absolute terms (liters per minute) or relative to total or fat free mass (milliliters per kilogram per minute). There was a statistically significant difference in the body fat


Figure 1: Rates of FAO in muscle strips of rectus abdominus from non-obese AAW (N ⫽ 5) and CW (N ⫽ 7) women. FAO rates are the sum of 1-14CO2 plus aqueous fatty acid metabolites. * p ⬍ 0.03.

percentages between the AAW and CW; however, all participants showed values (for their age group) characteristic of healthy, lean individuals. Table 1C shows the characteristics for the obese subjects who underwent exercise training. All subjects had a BMI ⬎30 kg/m2, and there were no significant differences between the mean ages or BMI of the two groups. Body composition analysis revealed that mean differences in the percentage fat mass between the obese groups were not statistically different. Likewise, there also was not a statistically significant difference between fat-free mass between the groups. Similarly, mean differences in the relative (expressed per kilogram body weight and per kilogram fat-free mass) and absolute VO2peak measured through indirect calorimetry were not significantly different between the obese subjects. Rates of In Vitro FAO in Intact Rectus Abdominus Muscle from Lean AAW and CW Earlier experiments from our laboratory indicated that non-obese AAW have depressed rates of whole body FAO during an acute episode of physical activity compared with CW (20). Therefore, we wished to assess whether this impairment in lipid use was evident at the level of the skeletal muscle and whether it persisted in the non-contracting muscle. Figure 1 indicates that skeletal muscle palmitate oxidation rates in intact rectus abdominus strips are significantly (p ⬍ 0.03) lower (64.2 ⫾ 5.4 nmol/g tissue/hr) in lean AAW vs. CW (82.1 ⫾ 5.0). This represents a 22% reduction in oxidative capacity and suggests that skeletal muscle from non-obese AAW possess biological differences relative to CW in the capacity to oxidize exogenous fatty acids, which may predispose this racial group toward fat accumulation (other energy and socioeconomic variables being the same).

Rates of FAO in Vastus Lateralis Muscle Homogenates from Exercise-Trained Lean AAW and CW Earlier, we noted that the rates of in vitro FAO were greater in trained vs. sedentary individuals (16). These data, however, were obtained from white subjects, and the study design was cross-sectional vs. investigating oxidation rates before and after endurance exercise training in the same individuals. Therefore, we measured rates of palmitate oxidation in healthy, but sedentary lean AAW and CW before and after endurance exercise training. Figure 2 (top) shows that as little as 10 days of aerobic training significantly (p ⫽ 0.05) increases the skeletal muscle’s capacity to oxidize exogenous fatty acids in previously sedentary individuals. Also important was the finding that exercise training positively affects the skeletal muscle’s capacity to oxidize palmitate in both AAW (pre: 37.4 ⫾ 10; post: 58.2 ⫾ 16.0 nmol/g protein/min) and CW (pre: 52.5 ⫾ 10.4; post: 74.0 ⫾ 10.7 nmol/g protein/min). This represents a 56% increase from the pre-training condition for AAW and a 41% increase for CW. In the pre-trained condition, mean oxidation rates for palmitate oxidation from whole homogenates were observed to be lower for the AAW, but the differences did not reach statistical significance. Rates of FAO in Vastus Lateralis Muscle Homogenates from Exercise-Trained Obese AAW and CW We showed earlier that the rates of in vitro FAO are lower in skeletal muscle from obese CW (25). However, it has not been shown that exercise training would improve the lipid oxidative capacity in skeletal muscle from these subjects. It is also unknown whether obese AAW would respond similarly to training. Therefore, in separate experiments, we asked obese individuals of both races to aerobically train with the same protocol as used earlier by the lean subjects in this study. As shown in Figure 3 (top), we confirmed earlier findings that sedentary, obese AfricanAmericans have a suppressed oxidative capacity for palmitate vs. CW (p ⫽ 0.05). We also observed the novel finding that, similar to non-obese subjects, both obese AAW (pre: 30.0 ⫾ 6.2; post: 60.7 ⫾ 6.9 nmol/g protein/min) and obese CW (pre: 45.7 ⫾ 6.5; post: 73.2 ⫾ 5.8 nmol/g protein/min) increase their capacity to oxidize exogenous fatty acids after 10 days of exercise training. These increases represented a 100% improvement in skeletal muscle oxidative capacity from the pre-training condition for AAW and a 60% increase in CW. Our experimental system for assessing FAO allows several isotope forms of fatty acids to be tested on a single muscle biopsy. Therefore, we also incubated the same homogenates with [1-14C] labeled palmitoyl-CoA (does not require activation by acyl CoA synthetase) and palmitylcarnitine (does not require conversion by carnitine palmitoyl transferase-1 for transport to the mitochondrial matrix). This allowed us to indirectly evaluate other key regulatory sites of FAO before and after endurance exercise training. OBESITY Vol. 14 No. 7 July 2006

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Figure 2: FAO rates in vastus lateralis muscle homogenates from lean CW (N ⫽ 7) and AAW. Palmitate oxidation rates were assessed as captured 14CO2 using 1-14C-palmitate as substrate. Data are expressed as means ⫾ standard error. * Statistically significant (p ⫽ 0.05) post- vs. pre-exercise training.

Also shown in Figure 3, unlike suppressed rates of palmitate oxidation in obese AAW, differences in palmitoyl-CoA (middle) or palmityl-carnitine (bottom) oxidation were not seen between the groups. However, similar to palmitate, endurance exercise training increased the oxidation rates for both palmitoyl-CoA and palmityl-carnitine, suggesting that 1206


Figure 3: Effects of training on rates of FAO in obese AAW and CW. Values are means ⫾ standard error. # Different from CW pre-training (p ⫽ 0.05). * Represents significant increases from pre- to post-exercise (p ⬍ 0.01).

the transport and subsequent intramitochondrial oxidation of fatty acids, respectively, are improved in both obese AAW and CW after aerobic exercise training. In contrast, in lean subjects, the rates of palmitoyl-CoA (Figure 2, middle) or palmityl-carnitine (Figure 2, bottom) oxidation were similar between the races in the untrained state and were not increased by exercise training.


Discussion The major findings of this study are 1) that, based on data from intact muscle strips, the capacity to oxidize fatty acids by skeletal muscle seems to be reduced in sedentary, lean AAW vs. CW, perhaps predisposing this racial group toward obesity; 2) that, compared with CW, obese AAW have a reduced capacity to oxidize exogenous long-chain fatty acids, which seems to be caused (at least in part) by reduced activation of fatty acyl units to their CoA derivatives; and 3) that both lean and obese AAW and CW are able to significantly increase their rates of long-chain FAO after 10 days of endurance exercise training. The latter finding suggests that the prescription for endurance exercise is extremely important to combat problems in muscle lipid metabolism that manifest as obesity, especially for AAW. Whereas obesity is a multifactorial metabolic disorder that involves environmental factors, inheritable metabolic dysfunctions may also contribute to the development of the disease (27). For example, skeletal muscle oxidation of fatty acids has been shown to be reduced in obese individuals compared with lean individuals (25,28). Earlier in a crosssectional study, we showed that obese subjects have lower in vitro rates of palmitate oxidation compared with nonobese individuals (25). Similarly, we observed a reduced fatty acid oxidative capacity in skeletal muscle from extremely obese subjects (24). Recently, Kelley et al. (27) have identified reductions in the number and size of mitochondria in skeletal muscle from obese and obese-diabetic subjects, and further work suggests that the dysfunction is impaired to a greater extent in the subsarcolemmal subpopulation of mitochondria (29). Studies from our laboratory support these observations, as Kim et al. (25) have shown that reductions in skeletal muscle FAO is accompanied by reduced carnitine palmityl-transferase 1 and citrate synthase activity also suggesting reductions in mitochondrial oxidative function in the obese state. These findings are important for several reasons. First, fatty acids provide a majority of the basal energy needs of skeletal muscle in healthy individuals. Thus, it is conceivable that a reduction in the rate of fatty oxidation by skeletal muscle could result in abnormally high blood lipid levels and shunting of fats toward storage. Second, reductions in oxidation could also result in excess accumulation of skeletal muscle triglycerides and other bioactive lipids. This is important because excess intramyofibrillar lipid content is strongly associated with insulin resistance in this tissue (30 –33). In accordance with this association, an hypothesis has been advanced linking lipid activation of particular protein kinase C (PKCs) with reductions in insulin receptor tyrosine kinase activity (through serine/threonine phosphorylation) and impaired down stream signaling for glut-4 recruitment to the sarcolemmal membrane (30). In support, our laboratory has shown that PKC ␪ is significantly higher in muscle from obese individuals (34). Additional studies by Cortright et al.

(35) show the contrast in that inhibition of PKC restores skeletal muscle insulin stimulated glucose transport in muscle from insulin-resistant, obese subjects. As skeletal muscle is the major site of glucose disposal after a meal (36), the hypothesis has emerged that impairment of skeletal muscle lipid oxidation may represent the link between obesity, insulin resistance, and the development of type 2 diabetes. Whereas other studies have shown lower rates of fat oxidation in African Americans using in vivo methodologies (9), this study supports the single past study showing that obese AAW possess a greater reduction in their in vitro skeletal muscle’s capacity to oxidize fatty acids (10). Similar to the findings in rectus abdominus by Privette et al. (10), we show greater reductions in the basal rate of palmitate, but not palmitoyl-CoA or palmityl-carnitine, oxidation in vastus lateralis from obese AAW vs. CW. Cytosolic fatty acids must gain access to the mitochondrial matrix for oxidation. This requires transport through the outer and inner membranes of the mitochondria, the process that is regulated by CPT-1. CPT-1 requires fatty acyl units to be activated by a synthetase enzyme (acyl-CoA synthetase) to acyl-CoA derivatives to convert the fatty acid to acylcarnitine units, which only then can translocate to the matrix for reconversion back to fatty acyl-CoA species for subsequent oxidation. The lower rate of palmitate oxidation and the lack of differences in the rates of palmitoyl-CoA or palmityl-carnitine oxidation suggest that there is a lower capacity to activate fatty acids in the muscle of obese AAW vs. CW. These oxidative data are supported by an independent laboratory that showed a reduced activity of acyl-CoA synthetase in skeletal muscle from obese AAW vs. CW (10). In contrast, Privette et al. (10) found no decrement in the activities of citrate synthase or the ␤-oxidative enzyme ␤-hydroxyacyl dehydrogenase in obese AAW vs. CW. Thus, the reduced activity of acyl-CoA synthetase is in addition to known reductions in matrix oxidative enzyme activity with obese individuals in general. It should be noted that neither the study by Privette et al. nor this study showed the underlying cause or effect of reduced long-chain fatty acid activation/oxidation in obese (and possibly lean) AAW vs. CW. Further studies are needed to identify whether cellular site–specific reductions exist (e.g., mitochondrial vs. endoplasmic reticulum acyl-CoA synthetase) and whether the potential racial differences are caused by decreased expression of the acyl-CoA synthetase or other factors such as specific defects in the protein per se in AAW. It is noteworthy, however, that these findings replicate similar results in another muscle group (vastus lateralis in this study vs. rectus abdominus in the study of Privette et al.), suggesting that reductions in palmitate oxidation is a global metabolic phenomenon in skeletal muscle from obese AAW. Both this study and that by Privette et al. extend the findings by Tanner et al. (37). Tanner et al. determined the OBESITY Vol. 14 No. 7 July 2006

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muscle fiber type composition from lean and obese individuals. Results showed that obese women possessed fewer type I and more type IIb fibers than lean women. Because type I fibers are associated with a higher oxidative capacity than either type II fibers, it was concluded that obesity is associated with a reduction in the oxidative capacity of skeletal muscle. In addition to these findings, however, when ethnicity was accounted for, the percentage of type IIb fibers in obese AAW was significantly higher than in obese CW. Collectively, the present and past findings indicate that, not only are there racial differences in the amount of oxidative fibers that could account (at least in part) for the greater prevalence and severity of obesity in AAW (37), but the quality of the mitochondria within each fiber type also seems to be dissimilar in this racial group. This is the first report, to our knowledge, to show that reductions in FAO seems to pre-exist in skeletal muscle from lean AAW compared with lean white counterparts. When skeletal muscle was studied as intact strips from rectus abdominus of lean subjects, rates of palmitate oxidation were significantly lower in AAW vs. CW. In contrast, although mean differences were lower in African Americans, rates of basal palmitate oxidation from vastus lateralis homogenates were not statistically different between the races. Although this study did not investigate the specific mechanisms that may account for these differences, results do suggest that compared with lean CW, lean AAW may possess dysfunction(s) in the sarcolemmal membrane transport capacity for fatty acids, reducing uptake and oxidation. This in turn would lead to enhanced partitioning of circulating lipids toward storage in adipocytes, thus promoting additional progression toward fat mass gain. In addition, further examination of subject data revealed that individuals from whom vastus lateralis muscle samples were obtained were younger than those women from which muscle strip fatty acid oxidation was assessed. Thus, further experiments are needed to test whether an age threshold may exist before which reductions in mitochondrial fat oxidation are not observed in skeletal muscle from lean AAW. Other groups have compared the effects of acute exercise on lipid use between AAW and CW (20). To our knowledge, however, this is the first study to report on the effects of endurance exercise training on skeletal muscle FAO between these races. Earlier, Chitwood et al. (9) showed that obese AAW have suppressed rates of fat use during exercise in the untrained state. Additional insights were gained later from Hickner et al. (20), in that they also showed that lean AAW were similar to obese women in that they were metabolically inflexible in their ability to shift toward fat use during endurance exercise. From our results, several conclusions can be made to extend these earlier studies. First, with obese individuals in general, endurance exercise training can elevate the mitochondrial’s capacity to oxidize fatty acids. Second, although obese AAW manifest lower 1208


rates of FAO in the sedentary state, they are equally responsive to endurance exercise training as obese CW. Third, lean AAW show a similar metabolic flexibility to endurance exercise training as their lean white counterparts and significantly increase their skeletal muscle’s capacity to oxidize fatty acids after training. Thus, similar to the known positive effects of endurance exercise training on the cardiovascular–pulmonary systems, these findings support the prescription of endurance exercise training for enhancement of the lipid oxidative capacity in skeletal muscle from both lean and obese subjects of African-American and white descent. As a final note, it is always possible that differences in body composition or physical activity levels between subject groups could influence these conclusions. However, several lines of reasoning make this unlikely. First, statistical differences in BMI were not noted between lean groups or between the obese groups studied. Second, although statistically different, mean values between our lean groups (Figure 2; Table 1B) recruited for the exercise training study indicated that both possessed optimal levels of body fat for their age and, thus, may be considered clinically healthy and physiologically similar with respect to body composition. Third, based on the negative relationship between obesity and skeletal muscle fatty acid oxidative capacity described above, the lean AAW would be predicted to show a greater capacity to oxidize lipids because they possessed lower percentage body fat; however, the data in this study do not support this because the mean oxidative rates were actually lower in the African Americans studied. Finally, differences in physical fitness most likely was not a confounding factor in this study because data received from our physical activity questionnaire revealed similar lifestyles and extent of physical activity. In addition, data obtained on peak oxygen consumption capacity between the groups were similar, whether expressed on an absolute basis or relative to total body or fat-free mass (Table 1B and C). In summary, these findings support previous literature (10) in that obese AAW possess reduced rates of long-chain FAO by skeletal muscle. Reductions in oxidation rates of palmitate, but not palmitoyl-CoA or palmityl-carnitine, suggest an additional biological difference in obese AAW at the site of activation of long-chain fatty acids. In addition, we report the novel finding that lean AAW also manifest reduced rates of long-chain FAO, with comparisons between intact vs. homogenate rates of oxidation suggesting potential differences in membrane transport of fatty acids into the cell cytosol. These findings support the hypothesis of inherent physiological or biochemical differences between AAW and CW that may place the former race at greater risk for developing obesity and related diseases. Despite the reduced rates of FAO in the basal state, both lean and obese AAW and obese CW were metabolically flexible in response to endurance exercise training and showed signifi-


cant improvements in fatty acid oxidative capacity. These findings support an important role for exercise as a therapeutic intervention to reduce the progression toward obesity and type 2 diabetes for individuals at risk for developing these disease states. This recommendation seems extremely relevant for AAW. As such, training studies of longer duration are warranted to determine whether greater and long-lasting improvements in FAO can be realized and whether increases in oxidative capacity are similar in AAW vs. CW. Future studies should also be directed toward understanding possible inherent defects in long-chain FAO at the levels of transport at the cell surface and activation at the mitochondrial outer membrane in the obese state and between these races.

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