Lower mitochondrial respiration does not lead to decreased fat oxidation in non-obese young African American Women

Abstract

Objective.

The prevalence of type 2 diabetes in African American women (AAW) is nearly twice that of Caucasian women (CW). Lower insulin sensitivity and decreased mitochondrial function may be contributing factors. The purpose of this study was to compare fat oxidation in AAW and CW.

Methods.

Participants were 22 AAW and 22 CW, matched for age (18.7-38.3 years) and body mass index ([BMI]<28 kg/m²). Participants completed two submaximal (50% VO₂max) exercise tests with indirect calorimetry and stable isotope tracers to assess total, plasma, and intramyocellular triglyceride [IMTG] fat oxidation.

Results.

The RQ during the exercise test was nearly identical in AAW and CW (0.813±0.008 vs. 0.810±0.008, p=0.83). Although absolute total and plasma fat oxidation were lower in AAW, adjusting for the lower workload in AAW eliminated these racial differences. There was no racial difference in plasma and IMTG source of fat for oxidation. No racial differences were observed in rates of ex vivo fat oxidation. Exercise efficiency was lower in AAW when adjusted to leg fat free mass.

Conclusions.

The data suggest that fat oxidation is not lower in AAW compared to CW, but additional studies are needed across exercise intensity, body weight and age to confirm these results.

Introduction

African-American women (AAW) have an increased risk of developing T2DM compared to Caucasian women (CW) (1-5). Data presented in the recent National Diabetes Report 2020 reveals that the prevalence of diabetes in AAW is nearly twice that observed in CW (12% vs 6.6%) (6). Reasons behind this racial disparity are not entirely understood, but lower insulin sensitivity (IS) has been observed in AAW (7-10).

We recently demonstrated no difference in hepatic IS, but 26% lower peripheral IS in young, healthy, non-obese AAW compared to matched CW (11). Total and central adiposity, VO₂max, and physical activity were not associated with the lower IS in AAW. However, there was an association between mitochondrial respiration and peripheral IS. One proposed mechanism is that low mitochondrial function leads to decreased skeletal muscle fat oxidation which plays a role in the increased adiposity, as well as reduced IS (12). Skeletal muscle characteristics including fiber composition, mitochondrial oxidative capacity and lipid accumulation have been determined to be associated with insulin resistance (13, 14). There is data indicating a lower percentage of type I oxidative muscle fibers in AAW (15) and lower oxidative capacity in the calf muscle (16). These observations suggest that differences in skeletal muscle characteristics may lead to low fat oxidation in AAW.

Low fat oxidation has been observed in young (21 y), lean (22% fat) AAW with family history of obesity compared to matched CW (17). In another study, fat oxidation during low intensity exercise (15W; ~40% VO₂max) and higher intensity exercise (65% VO₂max) was shown to be lower in AAW than in CW (18). There was no difference in fat oxidation in obese AAW compared to obese CW. However, lower fat oxidation has been observed in obese, postmenopausal AAW compared to CW matched for fat mass and fat free mass (19).

We recently described lower peripheral insulin sensitivity, lower mitochondrial function, and oxidative capacity in skeletal muscle from young non-obese AAW matched for BMI and age to CW (11). We further established that aerobic capacity and resting energy expenditure were lower in this cohort of AAW and these factors were positively correlated to skeletal muscle mitochondrial function (20). We subsequently reported that several genes associated with skeletal muscle mitochondrial function were decreased in a subset of AAW and were correlated to insulin sensitivity, energy expenditure, and mitochondrial function (21). Together these data support the notion of an inherent differences in skeletal muscle mitochondria in AAW leading to attenuated fat oxidation, insulin resistance, and the development of type 2 diabetes. However, whether or not these early differences in mitochondrial gene expression and function translates into impaired fat oxidation during physical activity is unknown.

The purpose of this investigation was to examine rates of fat oxidation (free fatty acids vs. non-plasma fatty acids, presumed to arise from plasma or intramuscular triglyceride) in a cohort of well matched (BMI and age) AAW and CW during physical activity. To achieve this goal, stable isotope tracer methodologies were used during submaximal exercise tests. In addition, rates of ex vivo fat oxidation were determined in skeletal muscle homogenates derived from biopsy samples.

Methods

Subjects.

Twenty-two non-obese (body mass index [BMI]<28 kg/m²), young (18.7-38.3 years) AAW and 22 CW matched for body weight, BMI, and age were recruited using print advertisements in the Pittsburgh Area. Inclusion criteria were, stable body weight over the previous 3 months (< ±3kg in the previous 6 mo.), non-pregnant or lactating, and sedentary (<20 min of activity, 3x/week) by self-report. Exclusion criteria included significant disease or unstable medical condition, diabetes mellitus, and use of medications that alter glucose metabolism or elevated lipid levels (cholesterol >250mg/dL, triglycerides >300mg/dL). The protocol was approved by the University of Pittsburgh Institutional Review Board and participants gave written informed consent.

Study protocol.

Details related to the study protocol have been described elsewhere (11). Briefly, study subjects were enrolled, medically screened and cleared, and completed a series of procedures separated by at least 3 days (Figure 1). Subjects reported to the University of Pittsburgh Endocrinology and Metabolism Research Center where body composition was assessed by dual energy x-ray absorptiometry (Lunar iDXA, GE Healthcare, Madison, WI) followed by a maximal aerobic capacity (VO₂max) test. VO₂max was measured by graded exercise testing using an electronically-braked cycle ergometer (Lode, Groningen, the Netherlands). Subjects warmed up for ~2 minutes with no load. At the start of the exercise, the intensity was increased to 50W. Subjects pedaled at 60 RPMs for 3 minutes at which point the intensity was increased by 25W. Three-minute stages followed by a 25W increase in intensity was repeated until the subject reached volitional fatigue. Maximal effort was confirmed using three criteria: 1) plateau of O₂ consumption despite increased workload, 2) respiratory exchange ratio (RER, VCO₂/VO₂) >1.1, and 3) heart rate within 10 beats of the age predicted maximum (22). The ventilatory anaerobic threshold (VT1) and respiratory compensation point or VT2 were determined from standard VE/VO2 and VE/VCO2 break points during the VO2max test (23).

Figure 1. Schedule of study procedures.

Study procedures were completed at least 3 days after the previous visit. DXA, dual energy x-ray absorptiometry.

Skeletal muscle biopsy.

Participants were admitted to the Clinical Translational Research Center in the evening for an overnight stay where they received a standardized meal (10 kcal/kg; 50% CHO, 15% protein, 35% fat) and then fasted until completion of the studies the following morning. Briefly, a percutaneous muscle biopsy (vastus lateralis) was performed under local anesthesia using the Bergstom method (24). Tissue samples were cleaned of visible adipose tissue and partitioned for high resolution respirometry, histochemical analysis, and transmission electron microscopy (TEM) as previously described (11). An additional portion of the sample was used for ex vivo fatty acid oxidation.

Ex vivo fatty acid oxidation of skeletal muscle.

Rates of fatty acid oxidation on muscle homogenates were determined using a modified method from Dohm et al (25). Briefly, tissue samples (∼50 mg) were placed in ice-cold homogenization buffer (250 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA, and 2 mM ATP, pH 7.4). Samples were minced with surgical scissors, and homogenization buffer added to yield a 20-fold diluted (wt:vol) homogenate. The muscle suspension was transferred to a 3-mL Potter-Elvehjem tissue homogenizer and subsequently homogenized on ice with a Teflon pestle. Aliquots (40 μl) of the 20-fold diluted muscle homogenates were plated in quadruplet into a modified 48-well cell culture plate (Costar, Cambridge, MA). CO₂ freely diffused between the incubation and trap wells through a small groove that was cut between adjacent wells. The reaction was started through the addition of 160 μL of a reaction mixture yielding final concentrations of 0.2 palmitate ([1-¹⁴C]palmitate at 0.5 μCi/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 (all in mM, pH 7.4). A silicone rubber gasket was added to the top of the plate and the entire unit was sealed with parafilm. Plates were incubated in a shaking water bath at 37°C for 60 minutes and the reactions terminated by the addition of 100 μL 70% perchloric acid to the incubation wells. The plate was transferred to an orbital shaker for 1 hour where the ¹⁴CO₂ was trapped in the adjoining well containing 200 μL of 1 N NaOH representing complete oxidation. Radioactivity was determined by liquid scintillation counting using 4 mL ScintiSafe (Thermo Fisher Scientific, Pittsburgh, PA). Incomplete oxidative products (acid-soluble metabolites, ASM) were also determined and the ratio of ASM to complete oxidation calculated as a marker of β-oxidative efficiency (25).

In vivo fatty acid oxidation during exercise.

Fatty acid oxidation was assessed during a 60-minute submaximal exercise (50% VO₂max) test, combining indirect calorimetry and stable isotope tracers. Participants underwent 2 submaximal exercise tests, one with ¹³C-acetate infusion for calculation of acetate recovery factor and another with ¹³C-palmitate tracer. At least 3 days separated the 2 exercise tests, and participants were instructed to avoid strenuous physical activity for two days prior to exercise studies. After collection of a baseline blood sample, a 0.085 mg/Kg bolus of ¹³C-sodium bicarbonate was infused into an indwelling catheter, and then a constant infusion (0.0736 ¹³C₂-acetate uMol/Kg/min or 0.0067 uMol/Kg/min U-¹³C-palmitate in 5% human serum albumin) was initiated for 30 minutes prior to exercise. The infusion rate of the ¹³C-tracers were then increased (0.1472 uMol/Kg/min for acetate or 0.0134 uMol/Kg/min for palmitate), exercise was started, and blood collected every 15 minutes over the hour exercise test.

Exercise efficiency calculations.

Gross efficiency (GE%) during exercise was calculated as the power output, watts converted to kcal/min, over exercise energy expenditure (kcal/min) and multiplied by 100 for the 1-h submaximal bike test (26, 27). Mean values of VO₂ and VCO₂ were averaged for the last 3-minutes at each 15-minute interval (i.e. 15, 30, 45, and 60 minutes) of the test. To calculate net efficiency (NE%), resting energy expenditure (REE, kcal/min; (20)) was subtracted from exercise energy expenditure (kcal/min). This number was used as the denominator under power output (kcal/min) and multiplied by 100. As part of the design, REE was performed on a separate day.

Calculations.

Systemic net carbohydrate and fat oxidation rates were calculated from indirect calorimetry using standard equations (28). The ¹³C/¹²C ratio in breath CO₂ was determined with a Gas Bench II peripheral device interfaced with a Thermo Finnegan Deltaplus XL Isotope Ratio Mass Spectrometer. Plasma ¹³C-palmitate enrichment was determined by GC-combustion-IRMS (29). Standard equations were applied using the isotope tracer data to quantitate the contribution of plasma and non-plasma sources of FFA oxidized (30-34). The recovery of acetate was calculated as (E_CO2 - E_bkg) x V_CO2 / 2 x F, where E_CO2 is the breath ¹³C/¹²C ratio at a given time and E_bkg is the background ¹³C/¹²C ratio, V_CO2 is CO₂ production at each timepoint, F is the ¹³C₂-acetate infusion rate, and 2 is included to account for the fact that both carbon atoms in acetate are labeled. The percent of infused palmitate tracer oxidized is calculated as V¹³_CO2 = (E_CO2 - E_bkg) x V_CO2 / (16 x F x c ) where V¹³_CO2 is the expired ¹³CO₂ production, E_CO2 is the breath ¹³C/¹²C ratio at a given time and E_bkg is the background ¹³C/¹²C ratio, 16 is included to account for the fact that each carbon in palmitate is labeled, F is the ¹³C-palmitate infusion rate, and c is the acetate recovery factor. The rate of disappearance of palmitate (R_d) was calculated as: (F/E_palm – F)/16, where E_palm is the 13C-enrichment of palmitate. Plasma fatty acid oxidation was calculated by multiplying the palmitate R_d by the percentage of infused palmitate tracer oxidized, divided by the percentage of plasma FFAs as palmitate. Non-plasma fatty acid oxidation (primarily intramyocellular triglyceride, IMTG) was estimated as the difference between total fat oxidation from indirect calorimetry minus plasma FFA oxidation.

Statistical analyses.

Data are presented as mean ± SEM. Differences in baseline subject characteristics were determined using a one-way Analysis of Variance (ANOVA, SAS 9.4, Proc GLM). Data from the ex vivo fatty acid oxidation replicates of complete oxidation and ASM were averaged to create a single data point per subject and then analyzed using a one-way ANOVA. Data from the in vivo fatty acid oxidation experiments were analyzed using a two-way (race x time) repeated measures analysis (Proc Mixed, SAS 9.4: REML estimation method for covariance parameters) as mean ± SEM. Unstructured covariance structure was used, because it resulted in the lowest −2 Res Log Likelihood, and AIC. Covariates are indicated in the results. Racial differences between energy expenditure and watts, and between total, plasma, and intramyocellular lipids at maximal rates of fat oxidation vs. energy expenditure were analyzed using linear regression. Statistical significance was assumed at p≤0.05.

Results

Participant Characteristics (Table 1)

Table 1.

Participant Characteristics

Parameter	African American	Caucasian	p
n	22	22	---
Age, years	22.8 ± 4.0	24.3 ± 5.5	0.29
BMI, kg/m2	22.7 ± 3.1	22.7 ± 3.1	1.00
Weight, kg	62.0 ± 9.2	63.0 ± 9.0	0.73
Fat free mass, kg	44.8 ± 5.8	42.4 ± 5.1	0.16
Fat, kg	17.2 ± 5.8	20.5 ± 6.4	0.08
VO2max, ml/min	2020 ± 96	2320 ± 96	0.03
VO2max, ml/min/kg FFM	45.1 ± 6.9	54.4 ± 7.8	0.0001
Watts at 50% VO2max	49.5±16.1	61.2±19.4	0.04
RQ during exercise	0.813±0.008	0.810±0.008	0.83

Data are mean ± SEM. BMI, body mass index. FFM, fat free mass.

A detailed comparison of the groups has been presented elsewhere (11). By design, no differences in age, weight, and BMI were noted. However, fat free mass tended to be higher in the AAW (~16%, p=0.16) and percent body fat was higher in the CW (~16%, p<0.02). VO₂max was 12.9% lower in AAW (p=0.04) and 18.3% lower when normalized to FFM (p<0.01). There were no racial differences in VT1 (43±2% vs 46±2% of VO2max, p=0.32) or VT2 (73±2% vs 73±2% of VO2max p=0.95).

The exercise workload at 50% VO₂max employed for the submaximal exercise tests was 19% lower in the AAW due to a lower VO₂max in the AAW (Table 1; p<0.04). Based on the expected strong relationship between energy expenditure and watts during the exercise bout (Figure 2; R²=0.88, p<0.0001), the lower workload during the exercise test resulted in a mean energy expenditure that tended to be lower in AAW compared to CW (Table 1). No racial difference was observed for the relationship between energy expenditure and watts (Figure 2) when examining slope (p=0.17), intercept (p=0.26) or when conducting a simultaneous test of slope and intercept (p=0.33) at the 60-minute timepoint.

Figure 2. Energy expenditure vs. watts during submaximal exercise bout.

Triangles, African American women (n=22). Circles, Caucasian women (n=21).

Energy expenditure did not change over the course of the exercise test (4.7±0.2, 4.6±0.2, 4.7±0.2, and 4.7±0.3 kcal/min in AAW and 5.3±0.2, 5.3±0.2, 5.4±0.3 and 5.4±0.3. in CW at 15, 30, 45 and 60 minutes, respectively) and there was no difference by race (race by time interaction, p=0.52).

Ex vivo fatty acid oxidation of skeletal muscle.

Based on the observation that mitochondrial content and function are lower in skeletal muscle from AAW compared to CW (20), rates of basal fatty acid oxidation were determined in skeletal muscle homogenates. No racial differences were observed for complete (Figure 3A, p=0.53), acid-soluble metabolites (Figure 3B, p=0.89), or ASM/complete ratio (Figure 3C, p=0.52).

Figure 3. Ex vivo fatty acid oxidation.

Fatty acid oxidation was determined in homogenates from skeletal muscle biopsy samples as described in Methods. Panel A, rates of complete fatty acid oxidation (nmol/h) normalized to starting tissue weight (mg). Panel B, rates of acid soluble metabolite (ASM) formation representing incomplete fatty acid oxidation (nmol/h) normalized to starting tissue weight (mg). Panel C, rates incomplete (ASM) to complete fatty acid oxidation ratio (nmol/h) normalized to starting tissue weight (mg). Black bars, African American women (n=15). White bars, Caucasian women (n=16).

In vivo fatty acid oxidation during exercise.

The RQ during the exercise bout was nearly identical in the AAW and CW (Table 1), reflecting a similar proportion of calories coming from fat oxidation in AAW and CW (overall 59.0±3.8% vs 59.7±2.8%, p=0.85; Figure 4A). There was a significant time effect (p<0.0001), with the proportion of calories coming from fat increasing from 15 to 30 min (p<0.0001), from 30 to 45 min (p<0.02) and from 45 to 60 minutes (p<0.04) during the exercise test. The increase in fat oxidation over time was similar in AAW and CW (race by time p=0.46). Although the proportion of calories coming from fat was not different, absolute total fat oxidation adjusted for FFM was lower in the AAW (p<0.04) during the submaximal exercise test (Figure 4B). However, to account for the lower exercise intensity in the AAW, adjusting total fat oxidation for either watts (1124±63 vs 1180±65; p=0.54) or energy expenditure during the exercise bout (1089±70 vs 1216±72; p=0.22) eliminated the racial difference.

Figure 4. Total fat oxidation during exercise.

Panel A, Percent of energy expenditure (kcal) as fat during the 1-hour submaximal exercise session. Panel B, Total fatty acid oxidation by indirect calorimetry adjusted for FFM. Mean values ± SEM for 15, 30, 45, and 60 minutes are indicated. AA, African American women. C, Caucasian women. Black bars, African American women (n=22). White bars, Caucasian women (n=21).

To determine the contribution of plasma fatty acids and non-plasma sources of fatty acids (assumed to be primarily from intramyocellular triglyceride [IMTG]) to total fatty acid oxidation during the submaximal exercise test, we combined indirect calorimetry and ¹³C-isotope tracer methodology. The recovery of ¹³CO₂ in breath after administration of ¹³C₂-acetate (Figure 5A), used as a correction factor to calculate ¹³C-palmitate oxidation was not different between AAW and CW (p=0.31). There was a highly significant increase over time (p<0.0001) that was not significantly different by race (p=0.10).

Figure 5. Assessment of plasma fatty acid oxidation.

Panel A, Rates of ¹³C-acetate were recovered from breath samples during the 1-h submaximal exercise session to determine acetate correction factor. Panel B, plasma fatty acid oxidation adjusted for FFM. Panel C, plasma fatty acid oxidation adjusted for expenditure during exercise. Mean values ± SEM for 15, 30, 45, and 60 minutes are indicated. AA, African American women. C, Caucasian women. Black bars, African American women (n=22). White bars, Caucasian women (n=21).

Plasma fatty acid oxidation (umol/min, adjusted for FFM) was lower in AAW compared to CW (Figure 5B; p<0.04). However, plasma fatty acid oxidation was not different when adjusting for the lower intensity of the exercise bout in AAW (624±53 vs 735±55 umol/min; p=0.16) or the lower energy expended during the exercise bout (Figure 5C; p=0.19). There was a significant increase in plasma fatty acid oxidation throughout the exercise bout (p<0.0001) except between 30 and 45 minutes (p<0.13).

There was no racial difference in IMTG fatty acid oxidation adjusted for FFM (465±50 vs 526±52 umol/min; p=0.41), when adjusting for exercise intensity (519±46 vs 470±48; p = 0.46) or adjusting for energy expenditure during the exercise bout (Figure 6; p=0.70). There was a significant increase in IMTG oxidation over time (p<0.01), which was not different by race (p<0.24). Furthermore, the contribution of IMTG to total fatty acid oxidation was similar in AAW and CW (43±3% vs 39±4%; p<0.50).

Figure 6. Intramyocellular triglyceride (IMTG) oxidation during exercise.

Rates of IMTG oxidation adjusted for FFM. Mean values ± SEM for 15, 30, 45, and 60 minutes are indicated. AA, African American women (n=21). C, Caucasian women (n=20).

To further explore fatty acid oxidation by race, we examined the relationship between total, plasma, and IMTG fatty acid oxidation at 60 minutes, when maximal fatty acid oxidation was observed, versus kcal expended at 60 minutes (Figure 7, panels A-C). No racial differences were observed when conducting a simultaneous test of slope and intercept (p=0.85, 0.55, and 0.54), intercept alone (p=0.64, 0.84, and 0.46) or slope alone (p=0.70, 0.66, and 0.36) for total, plasma, or IMTG fatty acid oxidation, respectively.

Figure 7. Association between fat oxidation and energy expenditure.

Rates of total (panel A), plasma (panel B), and intramyocellular triglyceride (IMTG, panel C) fatty acid oxidation were plotted against energy expenditure at 60 minutes. Slope and intercept, alone and together, were assessed for differences between African American women (AAW, solid line) and Caucasian women (CW, dotted line).

An additional measure of fat oxidation was obtained by examining fasting RQ measured before initiation of the hyperinsulinemic euglycemic clamp. Fasting RQ was not different between AAW and CW (0.79±0.04 and 0.77±0.05; p=0.28) indicating similar substrate utilization at rest.

Finally, we examine the possibility of a racial difference in exercise efficiency. Thus, we compared net efficiency, gross efficiency, and delta efficiency in our subjects. Net efficiency, gross efficiency, and delta efficiency were not different when unadjusted, or adjusted for total FFM (all p>0.05) between AAW and CW. However, because the exercise efficiency has been demonstrated to be affected by body mass (35), we normalized our measures of efficiency by leg fat free mass, given the modality of exercise. Normalized values of net efficiency (0.0011±0.00005 vs. 0.0013±0.00004 %/FFM_leg; p=0.018) and gross efficiency (0.0009±0.00003 vs. 0.0011± 0.00003 %/FFM_leg; p=0.006) were both significantly lower in AAW compared to CW, respectively, while no racial differences were observed with normalized delta efficiency (0.0010±0.00004 vs. 0.0010±0.00004 %/FFM_leg; p=0.79, respectively).

Discussion

Racial disparities in the development of type 2 diabetes are evident among African American and Caucasian women. We have previously reported that insulin sensitivity in young non-obese African American women is lower compared to Caucasian women and that this difference may be associated with lower mitochondrial function in skeletal muscle. To further test this hypothesis, we examined rates of fat oxidation using different methodologies. The proportion of calories coming from fat oxidation during the exercise test was nearly identical in the AAW and CW (Figure 4A). Although we found lower absolute fat oxidation in young African American women (AAW) compared to Caucasian women (CW), once we adjusted for the lower workload during the submaximal exercise bout in AAW due to the lower VO₂max, there was no racial difference in fat oxidation.

There is substantial evidence that alterations in skeletal mitochondrial content and function are related to impaired insulin action and decreased energy expenditure, and fat oxidation, leading to weight gain, the development of insulin resistance, and transition to type 2 diabetes. To further our previous observations of racial differences in mitochondria, we first explored rates of fatty acid oxidation in skeletal muscle biopsy homogenates. In contrast to our demonstration of impaired skeletal muscle mitochondrial function using intact muscle fibers, we did not observe any differences in fatty acid oxidation from muscle homogenates. These data are comparable to those of Cortright et al. (36), who demonstrated lower rates of fatty acid oxidation in muscle strips, yet no differences in fat oxidation using muscle homogenates. It should be noted that the muscle strips were obtained from older (~45 years) subjects, while the muscle homogenate study used subjects comparable in age to our subjects. Thus, rates of fatty acid oxidation using muscle homogenates does not appear to be different in young lean AAW compared to CW.

To further explore the possibility of racial differences in fatty acid oxidation, we examined substrate metabolism under submaximal exercise conditions. We found that substrate utilization during the exercise test was similar based on the RQ during the exercise test. However, we also found that absolute fat oxidation was lower in AAW compared to CW at an exercise capacity equivalent to 50% of their VO₂max. These data are in line with those of Hickner et al. (18), who reported lower rates of fat oxidation, both absolute and normalized to fat free mass, at 40% and 65% of VO₂peak in lean AAW compared to CW of similar age. Similarly, Chitwood et al. (17), using treadmill exercise at 65% of VO₂max, demonstrated higher respiratory exchange ratio (RER) during exercise, suggesting a greater reliance on carbohydrate oxidation and lower rates of fat oxidation in AAW compared to CW. Our finding that fat oxidation was not different when adjusting for workload to account for the lower VO₂peak in AAW is in line with a report by Melby et al. (37), who reported no differences in RER values between young lean AAW and CW during exercise at fixed power outputs (25W, 50W, and 75W) for 15 minutes each.

Energy production during submaximal exercise relies on the contribution of increased circulating free fatty acids (FFAs) derived through lipolysis of subcutaneous adipose tissue (38) and intramyocellular lipids (IMCLs) (39). Using tracer methodology, we found that the contribution of fatty acids form plasma and IMTG was similar in the AAW and CW.

To our knowledge, this is the first report to use tracers in the examination of source specific fat oxidation in AAW and CW. Our data demonstrating that absolute fat oxidation from plasma FFAs during an exercise bout at 50% VO₂peak was lower in AAW compared to CW agrees with previous studies demonstrating lower plasma fat oxidation in obesity (40, 41). However, when adjusted for FFM and workload, racial differences in fat oxidation during exercise are eliminated. These data seem to suggest that there is a tendency for rates of fat oxidation, in absolute terms, to mirror insulin sensitivity (42) and insulin action (25), in support of the link between mitochondrial function and insulin sensitivity. Further, similar rates of IMTG fat oxidation in the current study suggest that lipolytic enzyme (i.e., adipose triglyceride lipase, hormone sensitive lipase) action is not attenuated in skeletal muscle from non-obese AAW compared to CW during exercise.

Exercise efficiency, the ability to perform a given amount of work per unit of energy expended (43), is lower in obese subjects (44) with certain types of exercise. Conversely, a higher proportion of slow-oxidative type I fibers is linked to higher exercise efficiency and insulin sensitivity (45). These data are in line with our observation of lower exercise efficiency in lean AAW who also had lower proportions of type I fibers, VO₂max, and insulin sensitivity. We acknowledge that absolute exercise efficiency was not different between AAW and CW leading to interesting questions related to fat oxidation during moderate intensity cycling exercise, an activity that primarily utilizes lower limb muscles, versus treadmill walking, an activity that incorporates the activation of additional core and upper limb muscles. This is likely related to the differences in biomechanics between the two exercise modalities (44). Nevertheless, our data are supportive of the link between the oxidative profile of skeletal muscle, maximal aerobic fitness, mitochondrial capacity, and exercise efficiency.

This study was not without limitations. Due to lack of biopsy sample and issues with the tracer studies, we were missing data for ex vivo (7 AAW and 6 CW) and tracer measures of fatty acid oxidation (up to 1 AAW and 3 CW depending on timepoints). Nevertheless, the phenotypic data in this report mirror that in our initial study. It is uncertain if the exercise efficiency data is generalizable to other modes of exercise (i.e., treadmill walking). This requires further investigation.

In summary, our findings are somewhat equivocal. We provide evidence that rates of total and plasma fat oxidation are lower in lean AAW compared to CW during an exercise bout at 50% VO₂max. These findings are in line with observations of a lower skeletal muscle oxidative profile and decreased mitochondrial capacity found in this cohort. However, when examining the proportion of calories coming from fat during the exercise bout, or when adjusting for the lower workload in the AAW due to a lower VO₂max, we find no racial difference in fat oxidation. This data suggesting no racial difference in fat oxidation during an exercise bout corresponds to our observations of similar ex vivo fat oxidation and fasting RQ. Future studies will need to confirm these observations at differing exercise intensities and modalities in lean, overweight, and obese subjects, as well as the effectiveness of various interventions to improve mitochondrial function in AAW.

Study Importance.

What is already known about this subject?

• Decreased mitochondrial function in African American women (AAW) compared to Caucasian women (CW) has been shown to be related to lower insulin sensitivity.

• Low mitochondrial function could lead to lower fat oxidation and ectopic fat accumulation.

What does this study add?

• Fat oxidation at the same exercise intensity was not lower in AAW.

• The source of fatty acids, plasma vs IMTG, utilized during an exercise bout was not different between AAW and CW.

How might these results change the direction of research or the focus of clinical practice?

• Additional studies are needed across exercise intensity, body weight and age to confirm these results.

• Future research should examine changes in mitochondrial function and fatty acid oxidation in AAW following interventions designed to improve insulin sensitivity and determine if the magnitude of change is similar between AAW and CW.