Plasma Amino Acids Stimulate Uncoupled Respiration of Muscle Subsarcolemmal Mitochondria in Lean but Not Obese Humans

Abstract

Context:

Obesity is associated with mitochondrial dysfunction in skeletal muscle. Increasing the plasma amino acid (AA) concentrations stimulates mitochondrial adenosine triphosphate (ATP) production in lean individuals.

Objective:

To determine whether acute elevation in plasma AAs enhances muscle mitochondrial respiration and ATP production in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in obese adults.

Design:

Assessment of SS and IMF mitochondrial function during saline (i.e., control) and AA infusions.

Participants:

Eligible participants were healthy lean (body mass index, <25 kg/m²; age, 37 ± 3 years; n = 10) and obese (body mass index >30 kg/m²; age 35 ± 3 years; n = 11) subjects.

Intervention:

Single trial of saline infusion followed by AA infusion. SS and IMF mitochondria were isolated from muscle biopsies collected at the end of the saline and AA infusions.

Main Outcomes:

Mitochondrial respiration and ATP production.

Results:

AA infusion increased adenosine 5′-diphosphate (ADP)-stimulated respiration and ATP production rates of SS mitochondria in the lean (P < 0.05), but not obese, subjects. Furthermore, AA infusion increased the uncoupled (i.e., non–ADP-stimulated) respiration of SS mitochondria in the lean subjects only (P < 0.05). AA infusion had no effect on any of these parameters in IMF mitochondria in either lean or obese subjects (P > 0.05).

Conclusions:

Increasing the plasma AA concentrations enhances the capacity for respiration and ATP production of muscle SS, but not IMF, mitochondria in lean individuals, in parallel with increases in uncoupled respiration. However, neither of these parameters increases in muscle SS or IMF mitochondria in obese individuals.

Amino acid infusion increased maximal oxygen consumption and uncoupled respiration in subsarcolemmal, but not intermyofibrillar, mitochondria in lean, but not obese, subjects.

Obesity is an underlying cause of diabetes, heart disease, and cancer. The financial burden on the health care system to treat obesity continues to increase as access to food increases in parallel with the adoption of a more sedentary lifestyle. According to some evidence, obese individuals have reduced skeletal muscle mitochondrial function as indicated by lower citrate synthase activity (1), reduced capacity for mitochondrial electron transport (2), and/or adenosine triphosphate (ATP) production (3). However, such differences in mitochondrial function have not been evident when lean and obese subjects were matched for physical activity levels (4). Reduced physical activity in obese individuals (5) might impair muscle mitochondrial function (6) and exacerbate the metabolic consequences of obesity. Regardless of the mechanisms involved, diminished skeletal muscle mitochondrial function in obesity has been linked causally to insulin resistance (7).

Skeletal muscle mitochondria are unique with respect to their arrangement within myocytes: mitochondria near the periphery of the cell are termed subsarcolemmal (SS) mitochondria and those near the myofibrils are termed intermyofibrillar (IMF) mitochondria. These two mitochondrial subpopulations are biochemically distinct (8). Muscle mitochondria have been previously studied relative to their subcellular location and with regard to their content/size (9) and substrate metabolism (10, 11). To the best of our knowledge, limited evidence evaluating the respiratory capacity separately in SS and IMF mitochondria between lean and obese humans is available. In relevant evidence, Ritov et al. (12) have shown that electron transport chain activity is reduced in patients with type 2 diabetes and in obese adults in both SS and IMF muscle mitochondria, in parallel with a greater reduction in SS mitochondrial content. In contrast, SS mitochondria have shown more pronounced improvements in function in response to interventions, such as exercise, than have IMF mitochondria (13). Therefore, studying SS and IMF mitochondria independently will allow for a more detailed exploration of the mechanisms that can lead to alterations in energy metabolism in the muscle of obese individuals. Improving mitochondrial metabolism has been proposed as a novel approach to treating obesity and insulin resistance (7).

Plasma amino acids (AAs), in addition to their well-known effects in stimulating protein synthesis (14), improve mitochondrial ATP production of SS mitochondria of young healthy lean subjects (15). Generally, SS mitochondria have appeared more responsive than IMF mitochondria to the physiological perturbations induced by muscle use (or disuse) (16). To the best of our knowledge, no studies have evaluated the effects of increasing the plasma AAs on the function of IMF mitochondria in humans and, more importantly, how the responses in SS and IMF mitochondria might differ between lean and obese humans. Moreover, the metabolism of certain AAs, such as the branched-chain AAs, is impaired in skeletal muscle in obesity (17), which could differentially influence the effects of plasma AA availability on mitochondrial function.

Our objective was to compare the effects of increased plasma AA concentrations on SS and IMF mitochondrial function, specifically in relation to oxygen consumption and ATP production in lean and obese individuals. We hypothesized that elevated plasma AA concentrations would stimulate oxygen consumption and ATP production in skeletal muscle SS mitochondria only and that this effect would be observed in lean, but not obese, subjects.

Subjects and Methods

Subjects

Healthy sedentary lean (n = 10) and obese (n = 11) individuals participated in the present study. The institutional review board at Mayo Clinic approved the study, and all experimental procedures were performed in the Clinical Studies Infusion Unit (CSIU) at Mayo Clinic in Scottsdale (Scottsdale, AZ). The purpose, design, and risks associated with our study were explained to each subject before obtaining their written consent. The participants were determined to be healthy based on the results of screening procedures that involved medical history, routine physical examination, and standard laboratory tests. The latter included a blood metabolic panel, urinalysis, and a 2-hour oral glucose tolerance test. Body fat was determined using bioelectrical impedance analysis (BIA 310e, Biodynamics Corp., Seattle, WA). The maximal oxygen uptake was measured (Ultima™ Series; MGC Diagnostics Corp., Saint Paul, MN) on a separate day after the screening using an incremental (30 W/min) cycle ergometer (Lode Corival; Lode B.V., Groningen, Netherlands) test to exhaustion. The subject characteristics are presented in Table 1. Participants returned to the CSIU on a separate day after their screening visit for the main experiment, which included intravenous infusion of saline followed by infusion of AAs, and collection of blood and muscle samples.

Table 1.

Subject Anthropometric and Metabolic Characteristics

Characteristic	Lean	Obese	P Value
Sex, n
Male	4	6
Female	6	5
Age, y	37 ± 3	35 ± 3	0.60
Body weight, kg	62 ± 3	103 ± 4	<0.01
BMI, kg/m2	22 ± 1	34 ± 1	<0.01
Body fat, %	24 ± 3	34 ± 2	<0.01
FFM, kg	43 ± 3	68 ± 3	<0.01
VO2max, mL/kg FFM/min	35 ± 7	34 ± 7	0.90
Matsuda indexa	9.2 ± 0.9	4.5 ± 1.2	<0.01
2-h OGTT glucose, mmol/L	4.9 ± 0.1	5.4 ± 0.2	0.05
HbA1c, %	5.3 ± 0.1	5.6 ± 0.1	0.05

Data presented as mean ± standard error of the mean.

Abbreviations: BMI, body mass index; FFM, fat-free mass; HbA1c, glycosylated hemoglobin; OGTT, oral glucose tolerance test; VO₂max, maximal oxygen uptake.

^aInsulin sensitivity index calculated from the plasma glucose and insulin responses during an OGTT.

Experimental design

The participants arrived at the CSIU at ~6:30 am after an overnight fast. All subjects were instructed to refrain from any form of exercise for 3 days before the experiments. An intravenous line was inserted into an antecubital vein for infusions, and a second intravenous line was place in a dorsal hand vein to collect arterialized blood samples (i.e., heated-hand technique). Next, a saline infusion was started (time 0) and continued for 300 minutes. At 300 minutes, an AA mixture infusion (15% Clinisol; Baxter Health Care Corp., Deerfield, IL) was started with a priming dose of 82 mg/kg fat-free mass and maintained at 240 mg/kg fat-free mass/h for 240 minutes (i.e., 540 minutes from the beginning of the experiments). Blood samples were collected at 60 and 300 minutes after the start of the saline infusion and at 30 and 240 minutes after the initiation of the AA mixture infusion. Muscle samples were collected at the end of the saline and AA infusion periods to evaluate skeletal muscle mitochondrial function.

Muscle biopsy samples (~100 mg) were obtained from the vastus lateralis under local anesthesia (lidocaine, 2%). After removing blood, fat, and visible connective tissues, the muscle samples were placed into a premassed beaker containing 2 mL of ice-cold solution (modified Chappell-Perry medium; Solution I): 100 mM KCl, 40 mM Tris-HCl, 10 mM Tris-Base, 5 mM MgCl₂, 1 mM EDTA, 1 mM ATP (pH 7.5). Next, the beaker was reweighed for calculation of the wet muscle mass.

Analytical procedures

Isolation of skeletal muscle mitochondria

The muscle samples were minced with scissors in ice-cold Solution I. An aliquot was stored at −80°C and used at a later time for measurement of mitochondrial DNA content using procedures described in detail in the Supplemental Materials ^{(225.3KB, pdf)} section. Isolation of SS and IMF mitochondria from the skeletal muscle of lean and obese individuals was performed using a differential centrifugation protocol (Supplemental Fig. 1 ^{(225.3KB, pdf)} ). Isolation of the SS mitochondria was performed using procedures we have described previously (18). For isolation of the IMF mitochondria, the pellet resulting from isolation of the SS mitochondria was subjected to incubation with Nagarse (type XXIV; P-8038; Sigma-Aldrich, St. Louis, MO), a bacterial protease. Nagarse was prepared using Solution I and added (5 mg/g wet weight muscle) to the pellet to liberate IMF mitochondria. After a 7-minute incubation, 1 mL of Solution I was added to stop the enzymatic digestion. Further details on the procedure used to isolate the IMF mitochondria are provided in the Supplemental Materials ^{(225.3KB, pdf)} section.

Assays for mitochondrial oxygen consumption and ATP production

Freshly isolated mitochondria were assayed for rates of oxygen consumption (J_O) and ATP production (J_P). Oxygen consumption was measured polarographically in a respiration chamber (Hansatech Instruments, Norfolk, UK) at 37°C following procedures we have previously described (18) (also provided in the Supplemental Materials ^{(225.3KB, pdf)} section). Mitochondrial respiration was measured in the presence of saturating amounts of malate (1 mM), pyruvate (1 mM), and glutamate (10 mM) (MPG), as well as succinate (10 mM) (SUCC). We evaluated state 2 J_O after the addition of the MPG or SUCC substrates into the respiration chamber. The addition of ADP stimulated state 3 (i.e., maximal) J_O. Phosphorylation of this ADP resulted in state 4 J_O. Mitochondrial function was also evaluated using the respiratory control ratio (RCR), which was calculated as state 3 J_O/state 4 J_O.

Plasma hormone, glucose, and AA concentrations

Arterialized blood samples were analyzed for concentrations of plasma insulin and C-peptide using commercially available kits (ALPCO Diagnostics, Salem, NH) and for glucose using an automated glucose analyzer (STAT 2300; Yellow Springs Instruments, Yellow Springs, OH).

Concentrations of the plasma AAs were measured using high-performance liquid chromatography. In brief, plasma samples for the determination of AA concentrations were treated with an equal volume of 10% trichloroacetic acid, followed by the derivatization of the samples and standards with o-phthalaldehyde (19, 20). Derivatized samples and standards were injected onto an Agilent 1100 series high-performance liquid chromatography system (G1312A and G1329A; Agilent Technologies, Santa Clara, CA). Separation of the AAs was achieved using a Shimadzu C18, 50-mm × 4.6-mm column (Shimadzu Scientific Instruments, Columbia, MD). Peaks were monitored at 230 nm excitation/450 nm emission (G1321A; Agilent Technologies, Santa Clara, CA). The concentration of individual AAs was determined by comparison with a standard curve developed from known AA concentrations for each AA.

Statistical analysis

The data are presented as the mean ± standard error of the mean. The areas under the curve (AUCs) for the variables of interest were calculated using the trapezoidal rule. Two-way with repeated measures analysis of variance (ANOVA) was used to test for the main effects of AAs and obesity, and their interactions, on the variables of interest. Bonferroni correction was performed for multiple comparison tests. Nonpaired two-tailed t tests were used for single measurements between the lean and obese groups. Statistical significance was set at P < 0.05. Analyses were performed using the GraphPad Prism7 statistical software (GraphPad Software, San Diego, CA).

Results

AA concentrations

Individual plasma AA (Asp, Glu, Asn, Ser, Gln, Thr, Arg, Tyr, Met, Val, Phe, Ile, Leu) concentrations increased two- to sixfold in response to the AA mixture infusion (P < 0.05) in both lean and obese subjects (data not shown). The sum of the individual plasma AA concentrations, branched-chain AA concentrations, and essential AA concentrations, were all increased (P < 0.001) during the AA infusion compared with the saline infusion in both the lean and the obese subjects (Supplemental Fig. 2 ^{(225.3KB, pdf)} ).

Hormones and glucose concentrations

Insulin

The plasma insulin responses during the saline and AA infusions are shown in Fig. 1(A). ANOVA revealed statistically significant main effects for obesity (P < 0.001) and AAs (P < 0.001) and for their interaction (P < 0.01) on the calculated plasma insulin AUCs. Multiple comparison tests indicated that the plasma insulin AUC was greater in response to AA infusion than that during the saline infusion within the obese group only [Fig. 1(B)].

Figure 1.

Hormone and glucose concentrations. (A, C, E) Measurements were performed at the initiation of the experiments [time (t) = 60 minutes], at the end of the saline infusion period (t = 300 minutes), 30 minutes after the start of the AA infusion (t = 330 minutes), and at the end of the AA infusion period (t = 540 minutes). (B, D, F) The corresponding AUCs describing the hormone and glucose responses during the saline and AA infusion periods. Data presented as the mean ± standard error of the mean. Two-way with repeated measures ANOVA was used to analyze the data. Bonferroni correction was performed for multiple comparison tests (***P < 0.001, **P < 0.01, *P < 0.05 vs saline infusion, unless otherwise stated).

C-peptide

The plasma C-peptide responses during the saline and AA infusions are shown in Fig. 1(C). ANOVA revealed statistically significant main effects for obesity (P < 0.05) and AAs (P < 0.001) and for their interaction (P < 0.05) on the calculated C-peptide AUCs. Multiple comparisons tests showed that the C-peptide AUCs were greater within both the lean and the obese groups during the AA infusion compared with those during the saline infusion [Fig. 1(D)].

Glucose

The plasma glucose responses during the saline and AA infusions are shown in Fig. 1(E). The plasma glucose AUC increased significantly during the AA infusion compared with that during the saline infusion within the obese group only [Fig. 1(F)].

Mitochondrial protein and citrate synthase activity

No statistically significant main effects or interactions were found for the protein concentration in either the SS or IMF mitochondrial fractions (P > 0.05; Table 2). Similarly, no statistically significant main effects or interactions were found for citrate synthase activity, a marker of mitochondrial content (21), or citrate synthase-specific activity in either the SS or IMF mitochondrial fractions (P > 0.05; Table 2). Furthermore, no differences were found in the mitochondrial DNA content between the lean and obese subjects (Supplemental Fig. 3 ^{(225.3KB, pdf)} ). These findings for comparable muscle mitochondrial content between lean and obese subjects are in line with those from other investigations with subjects of similar age (4, 9, 22).

Table 2.

Protein and Citrate Synthase Activity Yields and Citrate Synthase-Specific Activity in SS and IMF Mitochondrial Fractions During Saline (i.e., Control) and AA Infusions

Variable	Lean		Obese
	Saline Infusion	AA Infusion	Saline Infusion	AA Infusion
Protein, mg/g wet muscle
SS	3.1 ± 0.2	3.1 ± 0.2	3.2 ± 0.4	2.8 ± 0.4
IMF	2.1 ± 0.4	2.0 ± 0.3	2.2 ± 0.3	2.4 ± 0.4
CS activity, µmol/min/g wet muscle
SS	2.2 ± 0.2	2.1 ± 0.3	2.2 ± 0.3	1.8 ± 0.3
IMF	1.3 ± 0.2	1.5 ± 0.3	1.5 ± 0.2	1.6 ± 0.2
CS-specific activity, µmol/min/mg
SS	0.8 ± 0.1	0.8 ± 0.1	0.7 ± 0.1	0.6 ± 0.1
IMF	0.8 ± 0.1	0.8 ± 0.1	0.7 ± 0.1	0.7 ± 0.1

Data presented as mean ± standard error of the mean.

Abbreviation: CS, citrate synthase.

Two-way with repeated measures ANOVA was used to analyze the data; no statistically significant effects were detected.

Maximal (state 3) J_O and ATP production

We detected no statistically significant main effects for either obesity or AAs on state 3 J_O in SS mitochondria, and when using either MPG or SUCC substrates (P > 0.05). However, a statistically significant interaction was found between factors (P < 0.01). Multiple comparison tests revealed an increase in state 3 J_O in the lean, but not obese, subjects in response to the AA infusion [Fig. 2(A) and 2(C)]. Similar to the finding in state 3 J_O in SS mitochondria, state 3 J_P increased in the SS mitochondria in the lean, but not the obese, subjects [Fig. 2(E) and 2(G)]. Also, the findings were the same when state 3 J_O and J_P were expressed relative to the muscle wet weight instead of mitochondrial protein (data not shown). No statistically significant differences were detected in state 3 J_O and J_P for the IMF mitochondria within or between groups [P > 0.05; Fig. 2(B), 2(D), 2(F), and 2(H)].

Figure 2.

State 3 oxygen consumption (State 3 J_O) and ATP production (State 3 J_P). State 3 J_O and J_P of (A, C, E, G) SS and (B, D, F, H) IMF mitochondria were measured at the end of the saline (Saline Infusion) and AA (AA Infusion) infusion periods. State 3 J_O and J_P were measured in the presence of MPG (malate, 1 mM; pyruvate, 1 mM; glutamate, 10 mM) or SUCC (10 mM) as substrates, and maximal rates for J_O and J_P were stimulated by the addition of ADP (0.67 mM). Data presented as the mean ± standard error of the mean. Two-way with repeated measures ANOVA was used to analyze the data. Bonferroni correction was performed for multiple comparison tests (**P < 0.01, *P < 0.05 vs saline infusion).

“Resting” (states 2 and 4) J_O

State 2 J_O of SS mitochondria did not differ between the lean and obese subjects before the AA infusion, but it increased in the lean subjects only in response to the AA infusion [Fig. 3(A)]. Also, state 4 J_O of SS mitochondria did not differ between the lean and obese before the AA infusion, but it increased in the lean subjects only in response to the AA infusion [Figs. 3(E) and 4(G)]. No differences were detected for either state 2 J_O or state 4 J_O in the IMF mitochondria either within or between groups [P > 0.05; Fig. 3(B), 3(D), 3(F), 3(H)].

Figure 3.

State 2 J_O and state 4 J_O oxygen consumption. State 2 J_O and state 4 J_O of (A, C, E, G) SS and (B, D, F, H) IMF mitochondria were measured at the end of the saline (Saline Infusion) and AA (AA Infusion) infusion periods. State 2 J_O was measured in the presence of MPG (malate, 1 mM; pyruvate, 1 mM; glutamate, 10 mM) or SUCC (10 mM) as substrates. State 4 J_O was measured following state 3 J_O after depletion of ADP in the medium. Data presented as the mean ± standard error of the mean. Two-way with repeated measures ANOVA was used to analyze the data. Bonferroni correction was performed for multiple comparison tests (***P < 0.001, *P < 0.05 vs saline infusion).

Figure 4.

RCR (state 3 J_O/state 4 J_O) and ADP/O ratio. The RCR and ADP/O ratio were measured in the presence of MPG (malate, 1 mM; pyruvate, 1 mM; glutamate, 10 mM) or SUCC (10 mM) as substrates; Data presented as the mean ± standard error of the mean. Two-way with repeated measures ANOVA was used to analyze the data. Bonferroni correction was performed for multiple comparison tests (**P < 0.01, *P < 0.05 vs saline infusion).

RCR and ADP/oxygen ratio

ANOVA showed a statistically significant main effect of AAs on the calculated RCR (i.e., state 3 J_O/state 4 J_O) in SS mitochondria with MPG (P < 0.01). Multiple comparison tests indicated an ~30% decrease in the RCR in response to the AA infusion in the lean subjects only [Fig. 4(A)]. No statistically significant differences were detected for the RCR in the IMF mitochondria within or between groups (P > 0.05).

The ADP/oxygen (ADP/O) ratios are presented in Fig. 4. ANOVA showed a statistically significant main effect of AAs on the ADP/O ratio in SS mitochondria with MPG (P < 0.01). Multiple comparison tests indicated a substantial increase in the ADP/O ratio in response to AAs in the obese subjects only [Fig. 4(E)]. No statistically significant differences were detected for the ADP/O ratio in the IMF mitochondria within or between groups (P > 0.05).

Discussion

The present study examined the effects of elevated plasma AA concentrations on skeletal muscle mitochondrial function in lean and obese adults. The main findings were that elevated plasma AA concentrations increase the capacity for oxygen consumption and ATP production of SS mitochondria in the muscle of lean, but not obese, subjects. Moreover, only the lean subjects showed an increase in uncoupled respiration in response to the elevation of plasma AAs.

Current evidence suggests that an elevation of plasma AAs enhances the capacity of the SS fraction of muscle mitochondria to produce ATP (15). Our results add further support to this evidence. Furthermore, although previous findings were limited to ATP production (15), we measured mitochondrial respiration to more fully characterize mitochondrial function in skeletal muscle. The increased capacity for ATP production in the presence of elevated plasma AAs in SS mitochondria in the lean subjects was supported by increased state 3 J_O in this mitochondrial subpopulation.

Based on the mitochondrial substrates used, our results have shown that the greater mitochondrial oxygen consumption in the SS mitochondria of the lean subjects when plasma AAs increased was mediated by increased catalytic potential of both complex I (i.e., MPG substrate) and complex II (i.e., SUCC substrate) of the mitochondrial electron transport chain. However, ~85% of muscle mitochondria are IMF (16). Because maximal mitochondrial oxygen consumption (and ATP production) did not improve in the IMF mitochondria under the same conditions, our findings reveal a role for plasma AAs in regulating, specifically, the SS compartment of the muscle mitochondrial reticulum. We have shown that the SS mitochondria in lean subjects are more responsive than are the IMF mitochondria in modifying their catalytic potential in response to increased plasma AA concentrations.

The reduced responsiveness of muscle SS mitochondria in the obese subjects to the elevated plasma AAs could have possibly resulted from reduced transport of plasma AAs into the muscle, secondary to reduced muscle AA transporters in the obese subjects. Although whether such a mechanism is implicated in the present findings remains to be determined, current evidence suggests that the content of skeletal muscle AA transporters does not differ between lean and obese subjects in the basal state or with elevated plasma AAs (23). In contrast, the lack of stimulation of mitochondrial maximal oxygen consumption by the plasma AAs in the obese subjects might, at least in part, result from a trend for an already elevated respiratory capacity in these subjects in the basal state. Specifically, the state 3 J_O in the SS mitochondria of the obese subjects during the saline infusion was already 94% of the AA-stimulated state 3 J_O in the SS mitochondria of the lean subjects. Our results, therefore, during saline infusion did not show mitochondrial dysfunction previously reported in obese subjects (2, 3) but are in agreement with other findings showing no differences in muscle mitochondrial function with obesity (4, 9, 22). In line with our findings in humans, the basal state mitochondrial enzyme activities in rodents were 20% to 50% greater in the presence of increased muscle lipid availability (24), a typical observation in obese humans as well (25). Current evidence suggests that the mitochondrial complexes in skeletal muscle in obese humans are exposed to relative greater amounts of redox cofactors (i.e., reduced form of nicotinamide adenine dinucleotide) (26). Therefore, it is possible that the increased availability of mitochondrial substrate in the basal state in the muscle of the obese subjects provides a constant stimulus that increases the catalytic potential of mitochondrial electron transport chain complexes, minimizing the stimulation of these complexes by the increased plasma AA concentrations.

In addition to the usage of protons to generate ATP via the ATP synthase complex, mitochondria demonstrate proton leakage (i.e., uncoupled respiration) across the mitochondrial inner membrane, running in parallel with the ATP synthesis. Thus, measures of state 4 J_O in isolated mitochondria mainly indicate proton leakage (27). Previous reports have documented increased proton leakage in response to fasting (28) and acute exercise (29). In the present experiments, state 4 J_O increased (~75% with MPG) in response to the AA infusion in the SS mitochondria of the lean subjects only. Because state 4 J_O did not change in the IMF mitochondria, our results have shown that SS mitochondria have greater flexibility than IMF mitochondria to modify uncoupled respiration in response to acute increases in plasma AAs. State 2 J_O also reflects proton leakage in the presence of reducing substrates, but in the absence of ADP (30), and, therefore, is not affected by recycled ADP present during the state 4 J_O measurements. State 2 J_O was also significantly greater in response to the increase in plasma AAs. To the best of our knowledge, the present study has provided the first evidence showing that increased plasma AA concentrations enhance uncoupled respiration in human skeletal muscle mitochondria.

Our finding related to state 4 J_O have shown that the acute elevation of plasma AAs induces “metabolic inefficiency” in the SS mitochondria of lean subjects. From a physiological perspective, this suggests an increased capacity for fuel/energy usage in the SS mitochondria in the muscle of these lean subjects. It has been argued that such a response might have beneficial effects, because it can limit the generation of reactive oxygen species due to excess nutrients (31) and can also prevent the muscle nutrient overload observed in pathophysiological circumstances (32). In line with our finding for increased uncoupled respiration at the mitochondrial level, AA infusion acutely increases whole body oxygen consumption and energy expenditure (33). Therefore, failure to increase uncoupled respiration in obese individuals in response to increased plasma AA concentrations (i.e., during the postprandial period) could affect the ability of these individuals to increase their overall energy expenditure and regulate their body weight.

Because insulin alone stimulates mitochondrial ATP production (34), enhanced mitochondrial function might be mediated by increased plasma insulin concentrations during the AA infusion. However, the plasma insulin concentrations were not significantly greater when the mitochondrial respiratory capacity increased during the AA infusion compared with that after the saline infusion in the lean subjects. Furthermore, insulin decreases proton leakage (35), which is the opposite of what we observed in the lean subjects after AA infusion in the present study. The increased proton leakage in the present study, therefore, would have been the direct result of the increased plasma AA concentrations and in the same manner that fatty acids alone increase mitochondrial uncoupling/proton leakage (36). In a supplemental set of experiments using rodent muscle (experimental details are provided in the Supplemental Materials ^{(225.3KB, pdf)} section), we found that treating isolated mitochondria with AAs increased mitochondrial respiration (Supplemental Fig. 4 ^{(225.3KB, pdf)} ). These findings, together with evidence for improved mitochondrial function in cell lines treated with the AA leucine (37, 38), indicate a possibly independent role of plasma AAs in enhancing mitochondrial function. Thus, protein post-translational modifications appear as a likely mechanism involved in the observed effects of plasma AAs on mitochondria. Future studies are warranted to examine the effects of AAs in modifying directly the activity of individual mitochondrial proteins.

A limitation of the present study was that complex II activity was evaluated without the presence of rotenone. This results in the formation of oxaloacetate that inhibits SUCC dehydrogenase, and can potentially reduce the mitochondrial respiratory capacity when compared to measurements of complex II activity performed in the presence of rotenone (39). However, we still found an increase in respiratory capacity (i.e., state 3 J_O) under our experimental conditions in the lean, but not obese, subjects. It is possible that, as recent evidence has indicated, robust respiration can be measured without rotenone at least up to a certain respiratory state (40). Regardless, our overall findings, and when considering that the same effect of elevated plasma AAs on mitochondrial respiration was observed also for complex I activity, show stimulation of mitochondrial function by plasma AAs in lean, but not obese, humans.

In conclusion, our results have shown specificity of plasma AAs in stimulating the respiration of SS (but not IMF) mitochondria and, specifically, enhancing uncoupled respiration (i.e., state 4 J_O) and sustaining increased capacity for ATP production (i.e., state 3 J_O and J_P). However, none of these effects of plasma AAs were observed in the obese individuals. A reduced response of uncoupled respiration to increased plasma AA concentrations might play a role in attenuating the overall energy expenditure in obese individuals.