Insulin does not Stimulate Muscle Protein Synthesis during Increased Plasma Branched-chain

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1 Articles in PresS. Am J Physiol Endocrinol Metab (August 16, 2016). doi: /ajpendo Insulin does not Stimulate Muscle Protein Synthesis during Increased Plasma Branched-chain Amino Acids Alone but Decreases Whole-body Proteolysis in Humans Sarah Everman, Christian Meyer, Lee Tran, Nyssa Hoffman, Chad C Carroll, William L Dedmon, and Christos S Katsanos Center for Metabolic and Vascular Biology, Arizona State University, Tempe, AZ 85297, Mayo Clinic in Arizona, Scottsdale, AZ 85259, and Midwestern University, Glendale, AZ Running Title: Insulin, BCAA, and Muscle Protein Metabolism Address correspondence to: Christos S. Katsanos, Ph.D. Center for Metabolic and Vascular Biology Mayo Clinic Collaborative Resarch Building East Shea Boulevard, Scottsdale, AZ Phone: (480) christos.katsanos@asu.edu 1 Copyright 2016 by the American Physiological Society.

2 ABSTRACT Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primarily role in stimulating muscle protein synthesis, and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy, young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. Half of the subjects were studied in the presence of postabsorptive BCAA concentrations (Control), while the other half in the presence of increased plasma BCAA (BCAA). When compared to that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/hour) did not change (P > 0.05) during insulin in either the Control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs 0.05 ± 0.01) experiments. Insulin decreased (P < 0.01) whole-body phenylalanine rate of appearance (umol/kg/min), indicating suppression of muscle proteolysis, both in the Control (1.02 ± 0.04 vs 0.76 ± 0.04) and the BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin s suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. Keywords: BCAA, protein synthesis, protein breakdown, BCAA and insulin, BCAA clearance 2

3 INTRODUCTION Insulin exerts anabolic effects in human skeletal muscle. Although there is substantial evidence that plasma insulin suppresses whole-body and muscle proteolysis (1, 5), the role of plasma insulin in achieving muscle anabolism by increasing the rate of protein synthesis is still not clear (25). Some studies show stimulation of muscle protein synthesis by increased plasma insulin concentrations (4, 11), while others do not (10, 20). In all these studies, insulin was infused locally (i.e., intra-arterially) to prevent a drop in plasma amino acid levels, secondary to suppressed proteolysis, and thus maintain adequate amino acid delivery in muscle. The latter is believed to be necessary for insulin-stimulated protein synthesis in muscle (5). Generally, circulating insulin exerts its effects on muscle protein synthesis at higher concentrations and via an amino acid delivery-dependent manner. That is, protein synthesis increases only when amino acid delivery to muscle increases (11). Insulin most evidently stimulates muscle protein synthesis when infused in the presence of increased plasma concentrations of all amino acids (9, 12, 29), or essential amino acids (EAA) (26). With regard to the stimulation of muscle protein synthesis by EAA (15, 34), the delivery specifically of branched-chain amino acids (BCAA) is considered of particular importance given their wellcharacterized role in regulating signaling elements of the muscle protein synthesis machinery (6, 17, 35). Currently, however, there is no evidence of whether insulin can stimulate muscle protein synthesis in the presence of increased plasma concentrations of BCAA alone in humans. This knowledge is important, considering the prevalent use of BCAA supplements to enhance muscle anabolism, and which also elicit increase in plasma insulin concentrations (37). It is reasonable to hypothesize that stimulation of muscle protein synthesis by insulin in humans can be enhanced by the anabolic stimulus provided by increased availability of plasma BCAA. Therefore, we sought to measure stimulation of muscle protein synthesis by insulin in young healthy 3

4 subjects when plasma BCAA concentrations alone are either maintained or increased relative to their concentrations in the postabsorptive state. METHODS Subjects Prior to subject recruitment, the experimental procedures were approved by the Institutional Review Board at Arizona State University. All subjects recruited in the study were healthy as determined by initial screening over the phone. Exclusion criteria included smoking, body mass index > 30 kg/m 2, hypertension, diabetes, heart disease, peripheral vascular disease, history of liver or kidney disease and use of either prescription or over-the-counter medications. Further screening performed in the Clinical Research Unit (CRU), included medical history, physical examination, resting electrocardiogram, and standard blood and urine tests. None of the subjects admitted to the study were taking any supplements. Risks associated with participation in the study were described to each study participant prior to obtaining written consent. Experimental protocol Subjects that qualified for the study (N=14) were instructed to avoid any form of exercise, maintain their regular diet, and avoid alcohol consumption during the three days preceding the study. For the experimental part of the study, subjects arrived at the CRU at approximately 7:00 AM after an overnight fast of approximately 10 hours. Before any experimental procedures were performed, compliance with the instructions given to the subjects was confirmed with each study participant. Experimental procedures were initiated by the placement of one catheter into a hand vein for blood sampling, and another into an antecubital arm vein for infusions. L-[ring- 2 H 5 ]phenylalanine 4

5 (constant rate, 0.05 umol/kg/min; prime, 2.5 umol/kg) was infused throughout the experimental protocol to track muscle protein metabolism. To determine the effects of insulin on muscle protein synthesis in the present of increased plasma BCAA, a solution consisting of equimolar amounts of leucine, isoleucine and valine (4% BranchAmin ; Baxter, Deerfield, IL) was started (constant rate, of 5.0 umol/kg/min; prime: 15.0 umol/kg/min during the first 30 mins) in half of the study participants and continued throughout the experimental protocol. The other half received an infusion of saline. Insulinstimulation of muscle protein synthesis in the presence of increased blood BCAA concentrations (BCAA) or not (Control) was determined by the intravenous infusion of insulin during the last three hours of the experiments. Insulin was infused at a rate of either 40 (N=4 per BCAA and Control experiments) or 80 mu/m 2 /min (N=3 per BCAA and Control experiments). During the insulin infusion we measured plasma glucose concentrations at regular intervals using an automated glucose analyzer (YSI 2300, Yellow Springs, OH) and infused 20% dextrose at a rate needed to maintain the plasma glucose concentrations at the Basal levels. In addition, and during the insulin infusion in both experiments, we infused BCAA at a rate needed to maintain the plasma BCAA concentrations at those measured at the Basal period (i.e., prior to the initiation of the insulin infusion). This variable rate of BCAA infusion during the insulin infusion was adjusted/increased based on measurements of the total plasma BCAA concentrations performed at regular intervals during the insulin infusion using a quick spectrophotometric technique (3). BCAA infusion was maintained constant during the last 30 min of the Insulin Infusion period (i.e., steady-state plasma BCAA concentrations). Blood samples and skeletal muscle biopsies from the vastus lateralis muscle (~ 40 mg) were collected at the time points shown in Figure 1. Blood samples were collected for the determinations of plasma insulin, amino acids, and glucose concentrations, as well as blood d5-phenylalanine enrichment. 5

6 Muscle biopsies were collected to determine mixed-muscle protein-bound amino acid enrichment with d5-phenylalanine. Analyses of samples For isolating blood amino acids for determination of d5-phenylalanine enrichment we used procedures we have previously described (16, 33). Briefly, 1 ml of collected blood sample was immediately transferred into tubes containing 1 ml of 15% sulfosalicylic acid (SSA) and the sample was vortexed. The blood/ssa samples were centrifuged at 2,500 x g for 15 min at 4 o C and the supernatant was collected. The supernatant was then processed through cation-exchange columns (AG 50W-8x mesh; Bio-Rad Laboratories, Inc., Hercules, California 94547) to isolate/purify blood amino acids. Before the addition of the samples, the cation-exchange columns were conditioned with 3 ml of 2N NH 4 OH and 3 ml of 1N HCl, and the amino acids were eluted from the columns using 8 ml of 2N NH 4 OH. For the determination of mixed-muscle protein-bound d5-phenylalanine enrichment we followed standard procedures we have also previously described (16, 33). Briefly, approximately 15 mg of muscle tissue was homogenized after combining it with 500 ul of 5% SSA. After the muscle/ssa homogenate was centrifuged at 2,500 x g for 45 min at 4 o C, the supernatant was discarded, and the resulting muscle pellet was collected. The muscle pellet was then washed two more times with 5% SSA, followed by ethanol and ethyl ether washes, and placed in an oven overnight at 50 o C to dry. Mixed-muscle proteins in the dried muscle pellet were hydrolyzed in 6 N HCl at 110 o C for 24 hours. The protein hydrolysate, corresponding to 1 mg of dry tissue weight, was processed through cation-exchange columns (AG 50W- 8x mesh; Bio-Rad Laboratories, Inc.) to isolate the amino acids, and as we have described above for blood. 6

7 Samples were processed for determination of the enrichments in blood and mixed-muscle protein-bound d5-phenylalanine using gas chromatography/mass spectrometry (GC/MS). All GC/MS analyses on the isolated amino acids were performed at the Mayo Clinic Metabolomics Core as previously described (22, 28). Briefly, phenylalanine isolated by ion-exchange chromatography from muscle hydrolysates and the blood samples was derivatized to its t-butyldimethylsilyl (TBDMS) in acetonitrile. The derivatives were analyzed under electron impact condition using selected ion monitoring at m/z 336 and 341 for unlabeled and labeled phenylalanine, respectively. Approximately, equal amounts of unlabeled phenylalanine were injected into the GC/MS across samples and enriched standards. Tracer-to-tracee ratios (t/t) were calculated against an 8-point enriched standard curve. Insulin was determined using a commercially available kit (ALPCO Diagnostics, Windham, NH). Plasma amino acid concentrations were determined using high-performance-liquidchromatography (7). Calculations Fractional synthesis rate (FSR; % hour -1 ) of mixed-muscle protein was calculated as previously IEmmp described (14): FSR , where ΔIEmmp is the increment in mixed-muscle protein IEp phenylalanine isotopic enrichment (i.e., t/t) between two biopsies, IEp is the corresponding average blood phenylalanine enrichment in the Basal or Insulin Infusion experimental periods, and T is the time interval (min) between biopsies. Whole-body phenylalanine rate of appearance (Phe Ra) in blood, a reflection of whole-body muscle protein breakdown, was calculated using the single pool model, from the d5-phenylalanine 7

8 infusion rate (F) and the average blood d5-phenylalanine enrichment (E b ) during the last 30 min of the Basal and Insulin Infusion experimental periods: F Phe Ra. Eb Plasma clearance of each of the infused BCAA was calculated during the last 30 min (i.e., steady-state conditions) of the Insulin Infusion period as its infusion rate divided by its prevailing concentration in plasma. Statistical analyses Data were analyzed using two-factor (treatment x time) ANOVA, where treatment represents the presence (i.e., BCAA) or absence (i.e., Control) of elevated BCAA concentrations and time describes the Basal and Insulin Infusion conditions. One-way ANOVA was used to compare differences within variables of interest over time. Differences between and within groups were compared by unpaired and paired student s t-tests, respectively. Bonferroni correction was used in the case of multiple comparisons. Because there were no differences between the effects of 40 and 80 mu/m 2 /min insulin infusion rates on any of the measured variables within either the BCAA or the Control experiments, data within each experimental group were pooled and analyzed together. Results are expressed as means±se. Statistical significance was evaluated at P < The statistical analyses were performed using the Minitab statistical software (Minitab Inc., State College, PA). RESULTS Subject characteristics, and plasma biochemical parameters Anthropometric characteristics and metabolic profiles were not different between subjects in the Control and BCAA experiments (Table 1). BCAA infusion was increased progressively overtime after the initiation of the insulin infusion in both experiments in order to maintain the plasma BCAA 8

9 concentrations at those observed in the Basal period. The BCAA infusion rate during the last 30 min of steady-state plasma BCAA concentrations was 2.4±0.7 umol/kg/min and 7.9±0.9 umol/kg/min in the Control and BCAA experiments, respectively. Overall, and per study design, total plasma BCAA concentration was maintained at fasting levels in the Control experiment, while increased by approximately 150% compared to fasting levels in the BCAA experiment (Table 2). BCAA infusion in the latter experiment increased significantly all three BCAA in plasma (P < 0.05). The sum of the concentrations of measured EAA when excluding the BCAA, (i.e., non-bcaa EAA) decreased in both experiments during the insulin infusion (P < 0.05), but the sum of the concentrations of measured NEAA did not change during the insulin infusion (P > 0.05). In the Control experiments, insulin infusion increased significantly (P < 0.05) the plasma insulin concentrations both at 40 mu/m 2 /min (33±6 uiu/ml vs fasting: 6±2 uiu/ml and basal: 5±2 uiu/ml), and at 80 mu/m 2 /min (88±12 uiu/ml vs fasting: 3±1 uiu/ml and basal: 4±1 uiu/ml). In the BCAA experiments, insulin infusion also increased significantly (P < 0.05) the plasma insulin concentrations both at 40 mu/m 2 /min (39±10 uiu/ml vs fasting: 5±2 uiu/ml and basal: 4±1 uiu/ml), and at 80 mu/m 2 /min (113±8 uiu/ml vs fasting: 2±1 uiu/ml and basal: 5±1 uiu/ml). Fasting plasma glucose concentrations were not different between the BCAA and Control experiments (Table 1). Plasma glucose concentrations were not different over time (i.e., fasting vs Basal period vs Insulin Infusion period) within either the BCAA or Control experiments (P > 0.05; data not shown). Muscle Protein Synthesis Average blood phenylalanine enrichments, expressed as t/t, in the Control and BCAA experiments are shown in Figure 2. Blood phenylalanine enrichments were not different overtime within either the Basal or the Insulin Infusion periods, and in either the BCAA or the Control experiments (P > 9

10 0.05). Average responses for fractional synthesis rate of muscle protein are shown in Figure 3A. ANOVA analyses indicated no significant main effects for treatment or time on the rate of muscle protein synthesis (P > 0.05). Likewise, insulin-stimulated change in muscle protein synthesis was not different between the BCAA and Control experiments (P > 0.05; Figure 3B). Whole-body protein breakdown Time-course and average responses for Phe Ra in blood are shown in Figures 4A and 4B, respectively. With respect to the average responses for Phe Ra, ANOVA analyses indicated significant main effects for both treatment (i.e., a BCAA effect) and time (i.e., an insulin effect) on blood phenylalanine Ra (P < 0.05). Pairwise comparisons between experiments showed no significant difference in blood Phe Ra in the Basal period (P > 0.05), but Phe Ra was different during the Insulin Infusion period. The Phe Ra decreased in both experiments during the Insulin Infusion period (P < 0.05). However, the insulin-stimulated change in blood phenylalanine Ra was not different between the Control and BCAA experiments (P > 0.05; Figure 4C). Plasma clearance of infused branched-chain amino acids The infusion rate of each of the BCAA in the last 30 min of the Insulin Infusion period, and during steady-state plasma BCAA levels, was 3.3±1.0 mmol/hr and 9.8±1.1 mmol/hr in the Control and BCAA experiments, respectively. However, the plasma clearances (ml/min) of infused isoleucine, leucine and valine were not different between the Control and BCAA experiments (isoleucine: 680±36 vs 689±99; leucine: 690±27 vs 748±160; valine: 315±21 vs 306±56; for all P > 0.05). DISCUSSION 10

11 Because of its role in inhibiting protein catabolism, insulin suppresses the availability of circulating amino acids for protein synthesis, including the BCAA, which are primary regulators of the protein synthesis machinery. In the present study we evaluated if preventing the drop or increasing specifically the concentration of plasma BCAA is sufficient to allow stimulation of muscle protein synthesis by insulin. We found that increased concentrations of plasma BCAA alone is not sufficient to stimulate muscle protein synthesis by insulin. These findings ultimately underline the important role of non-bcaa EAA in the regulation of muscle protein synthesis by insulin. Previous investigations have shown that increased concentrations of plasma insulin alone do not stimulate muscle protein synthesis due to a decrease in the concentration of the plasma amino acids (5, 36). However, when insulin is infused locally (i.e., intra-arterially) to prevent a systemic decrease in plasma amino acids a stimulatory effect of insulin on muscle protein synthesis has been documented in some (4, 11) but not all (10, 20) studies. Our findings extend this body of work to a condition where only the plasma BCAA are maintained at their postabsorptive levels. We show that maintaining the concentration of plasma BCAA alone at the postabsorptive levels is not sufficient to stimulate muscle protein synthesis by insulin. Furthermore, we show that muscle protein synthesis is not stimulated by insulin even in the presence of increased levels of circulating BCAA. Thus, our studies provide clear evidence that plasma hyperinsulinemia cannot stimulate muscle protein synthesis in humans even in the presence of increased plasma BCAA concentrations, an effect that is presumably the result of the insulin-mediated decrease in the other (i.e., non-bcaa) EAA. In line with previous reports (8, 36), we found decreased plasma amino acid concentrations in response to systemic hyperinsulinemia. With the exception of the plasma BCAA that were experimentally increased in the present study, the decrease in plasma amino acids was more evident in the measured plasma non-bcaa EAA pool when compared to the non-eaa pool. The decrease in non- 11

12 BCAA EAA appears to be the limiting factor restricting the stimulation of muscle protein synthesis in the present studies, and based on evidence showing that when the concentrations of non-bcaa EAA are maintained at their postabsorptive levels, muscle protein synthesis is stimulated by insulin in the presence of elevated plasma BCAA (36). Interestingly, and as opposed to the lack of stimulation of muscle protein synthesis by increasing the overall concentration of plasma BCAA (18, 19), large increases in plasma phenylalanine do appear to stimulate muscle protein synthesis (30). Because increased plasma BCAA concentrations stimulate muscle protein synthesis when the concentrations of non-bcaa EAA are maintained (36) or when a single non-bcaa EAA (i.e., phenylalanine) is increased, it is tempting to speculate that EAA other than BCAA have an equal or possibly greater role in stimulating muscle protein synthesis. As expected based on the well-described effects of insulin on suppressing muscle protein breakdown (1), insulin infusion acutely decreased blood Phe Ra in the present study. In addition, consistent with previous studies showing reduced whole-body (13, 23) and muscle (23) protein degradation when leucine is infused alone, we found a main effect describing decreased blood Phe Ra in response to the BCAA infusion. However, the change in blood Phe Ra by insulin observed in the presence of increased plasma BCAA was not different from that resulting from the insulin infusion alone. Therefore, our findings extend those of previous studies by showing that insulin suppresses proteolysis that is quantitatively not affected by the presence of increased plasma BCAA. The clearance of infused amino acids from plasma describes their utilization across tissues. Similar to previous findings (32), the plasma clearance of infused valine was approximately half of that of either leucine or isoleucine. Since the plasma clearance of the infused amino acids was not higher in the BCAA experiment compared to the Control experiment, the presence of increased plasma BCAA levels alone did not enhance the insulin-mediated utilization of the plasma BCAA across tissues. This is 12

13 in line with the lack of an effect of the increased plasma BCAA availability on increasing disposal of plasma amino acids specifically towards muscle protein synthesis (Figure 3). Therefore, greater accumulation of BCAA in plasma largely explains the fate of the infused BCAA in the BCAA experiment. Infusion (26) or ingestion (15, 16) of EAA stimulates muscle protein synthesis, but increasing the concentrations of plasma BCAA alone does not stimulate muscle protein synthesis in humans (as reviewed in (21)). Our findings show that even the addition of the anabolic stimulus of insulin in the presence of the elevated plasma BCAA levels still is not sufficient to stimulate muscle protein synthesis. These overall findings, therefore, explain why neither BCAA nor leucine supplementation improve muscle protein synthesis or enhance muscle protein accretion (2, 31). Furthermore, neither BCAA (24) nor leucine (2) supplementation enhance physical performance. Nevertheless, lack of stimulation of protein synthesis in such circumstances does not exclude previously described beneficial effects of BCAA alone on muscle, such as those related to recovery from exercise (24, 27). When, however, the goal is to stimulate muscle protein synthesis, formulations must likely increase the plasma concentrations of BCAA and insulin while providing sufficient amounts of the other EAA to either maintain or increase their plasma concentrations. In conclusion, increased plasma insulin does not stimulate muscle protein synthesis even in the presence of increased plasma BCAA concentrations. This is presumably because of the insulin s effect on decreasing the EAA (other than BCAA), thus underlying the importance of non-bcaa in the stimulation of protein synthesis by the combined increase in all of the EAA. The extent by which insulin suppresses proteolysis is observed independently by the presence of increased plasma BCAA concentrations. 13

14 ACKNOWLEDGMENTS The authors thank the nurses at the Clinical Research Unit at Arizona State University for help with the conduct of the experiments, and the Mayo Clinic Metabolomics Core in Rochester, Minnesota for contacting the stable isotope enrichment determinations. The study was supported by NIH/NIDDK grant R01DK (CSK), and the Mayo Clinic Metabolomics Resource Core through grant number U24DK from the NIH/NIDDK and the Mayo Clinic CTSA grant UL1TR from the National Center for Advancing Translational Sciences. 14

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19 33. Tran L, Masters H, Roust LR, and Katsanos CS. A new method to measure muscle protein synthesis in humans by endogenously introduced d9-leucine and using blood for precursor enrichment determination. Physiological reports 3, Volpi E, Kobayashi H, Sheffield-Moore M, Mittendorfer B, and Wolfe RR. Essential amino acids are primarily responsible for the amino acid stimulation of muscle protein anabolism in healthy elderly adults. Am J Clin Nutr 78: , Yoshizawa F. Regulation of protein synthesis by branched-chain amino acids in vivo. Biochem Biophys Res Commun 313: , Zanetti M, Barazzoni R, Kiwanuka E, and Tessari P. Effects of branched-chain-enriched amino acids and insulin on forearm leucine kinetics. Clin Sci (Lond) 97: , Zhang Y, Kobayashi H, Mawatari K, Sato J, Bajotto G, Kitaura Y, and Shimomura Y. Effects of branched-chain amino acid supplementation on plasma concentrations of free amino acids, insulin, and energy substrates in young men. J Nutr Sci Vitaminol (Tokyo) 57: ,

20 FIGURE LEGENDS Figure 1 Design of the Control and BCAA experiments. During the Insulin Infusion period, 20% Dextrose and 4% BranchAmin were infused at variable rates to maintain the plasma glucose and total plasma branched-chain amino acid concentrations, respectively, at those measured in the Basal period. Figure 2 Time-course of blood phenylalanine enrichment in the Control and BCAA experiments before (Basal) and after (Insulin Infusion) the initiation of the insulin infusion. Figure 3 Fractional synthesis rate (FSR) of mixed-muscle protein in the Control and BCAA experiments before (Basal) and after (Insulin Infusion) the initiation of the insulin infusion (A). Change in FSR between the Basal and Insulin Infusion periods (B). Figure 4 Time-course of whole-body phenylalanine rate of appearance (Ra) in the Control and BCAA experiments before (Basal) and after (Insulin Infusion) the initiation of the insulin infusion (A). Average values for the whole-body phenylalanine Ra corresponding to the last 30 mins of the Basal and Insulin Infusion periods (B). Change in whole-body blood phenylanine Ra between the Basal and Insulin Infusion periods calculated from the data presented in 3B (C). *P < 0.05 vs Basal; # P < 0.05 vs Control. 20

21 Table 1. Subject characteristics Control BCAA 6M/1F 4M/3F Age, y 23 ± 2 19 ± 1 Weight, kg 71 ± 2 67 ± 4 BMI, kg/m 2 23 ± 1 23 ± 1 Fat free mass, kg 63 ± 2 56 ± 4 Body fat, % 13 ± 2 17 ± 2 Plasma lipids, mg/dl Triglycerides 69 ± 8 77 ± 10 Total Cholesterol 133 ± ± 14 HDL-C 45 ± 3 51 ± 4 LDL-C 76 ± 4 88 ± 14 Plasma glucose, mg/dl 89 ± 2 86 ± 2 Plasma insulin, uiu/ml 5 ± 1 4 ± 1 TSH, mu/l 2.1 ± ± 0.4 Values are means ± SE; BMI, Body Mass Index; HDL-C, High Density Lipoprotein-Cholesterol; Low Density Lipoprotein-Cholesterol; TSH, Thyroidstimulating Hormone; There were no differences between Insulin and BCAA+Insulin subjects.

22 Table 2. Plasma concentrations of amino acids prior to the initiation of the infusions (Fasting) and during the last 30 minutes of the Basal and Insulin Infusion periods Fasting Basal Insulin Infusion ANOVA P Value BCAA Control 336 ± ± ± BCAA 386 ± ± 140* 933 ± 91* <0.001 Non-BCAA EAA Control 109 ± ± ± 10* BCAA 121 ± ± ± 11* NEAA Control 225 ± ± ± BCAA 244 ± ± ± Arginine Control 68 ± 9 65 ± ± BCAA 81 ± ± ± Asparagine Control 59 ± 9 52 ± 6 44 ± BCAA 60 ± 4 55 ± 4 43 ± 3* Isoleucine Control 66 ± 8 56 ± 8 74 ± BCAA 71 ± ± 36* 240 ± 26* Leucine Control 94 ± ± ± BCAA 112 ± ± 47* 241 ± 33* Methionine Control 45 ± 4 35 ± 4* 28 ± 5* BCAA 51 ± 7 42 ± 5 30 ± Phenylalanine Control 64 ± 7 63 ± 6 42 ± 5* 0.033

23 BCAA 70 ± 8 58 ± 5 38 ± 4* Serine Control 106 ± ± 9 79 ± BCAA 104 ± ± 9 79 ± Valine Control 176 ± ± ± BCAA 202 ± ± 59* 513 ± 41* Values are umol/l (means ± SE); BCAA, branched-chain amino acids; Non-BCAA EAA, non-branched-chain essential amino acids; NEAA, non-essential amino acids; *P < 0.05 compared to Fasting; P < 0.05 compared to Basal.

24 Figure 1 Basal Insulin Infusion Insulin 40 (N=4) or 80 (N=3) mu/m 2 /min 20% Dextrose (variable rate) Control Experiment Saline L-[ring- 2 H 5 ]phenylalanine (0.05 umol/kg/min) 4% BranchAmin (variable rate) Insulin 40 (N=4) or 80 (N=3) mu/m 2 /min 20% Dextrose (variable rate) BCAA Experiment 4% BranchAmin (5 umol/kg/min) 4% BranchAmin (variable rate) L-[ring- 2 H 5 ]phenylalanine (0.05 umol/kg/min) Time Muscle Biopsy X X X Blood Sample

25 Figure 2 Phenylalalnine Enrichment (tracer/tracee) Control BCAA Basal Insulin Infusion Time (hrs)

26 Figure 3 A 0.14 B 0.14 FSR (%/hour) Basal Insulin Infusion FSR (%/hour) Control BCAA 0.00 Control BCAA -0.06

27 Figure 4 A 1.6 Control BCAA Phenylalalnine Ra (umol/kg/min) Basal Insulin Infusion Time (hrs) B Basal Insulin Infusion Phenylalanine Ra (umol/kg/min) * * # Control BCAA C 0.00 Phenylalanine Ra (umol/kg/min) Control BCAA