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 primary 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. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) 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 (μmol·kg −1 ·min −1 ), indicating suppression of muscle proteolysis, in both 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.

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), whereas others do not (10, 20). In all of 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 well-characterized 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 an 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 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/m2, 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 3 days preceding the study. For the experimental part of the study, subjects arrived at the CRU at ∼7 AM after an overnight fast of ∼10 h. 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-2H 5 ]phenylalanine (constant rate: 0.05 μmol·kg−1·min−1; 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 presence of increased plasma BCAA, a solution consisting of equimolar amounts of leucine, isoleucine, and valine (4% BranchAmin; Baxter, Deerfield, IL) was started (constant rate: 5.0 μmol·kg−1·min−1; prime: 15.0 μmol·kg−1·min−1during the first 30 min) in one half of the study participants and continued throughout the experimental protocol. The other half received an infusion of saline. Insulin stimulation 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 3 h of the experiments. Insulin was infused at a rate of either 40 (n = 4 per BCAA and control experiments) or 80 mU·m2·min−1 (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 Insruments, 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 constantly 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 Fig. 1. Blood samples were collected for the determinations of plasma insulin, amino acids, and glucose concentrations as well as blood d 5 -phenylalanine enrichment. Muscle biopsies were collected to determine mixed-muscle protein-bound amino acid enrichment with d 5 -phenylalanine. Fig. 1.Design of the control and branched-chain amino acids (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 BCAA concentrations, respectively, at those measured in the “basal” period. Download figureDownload PowerPoint

Analyses of samples. For isolating blood amino acids for determination of d 5 -phenylalanine enrichment, we used procedures that we have described previously (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 g for 15 min at 4°C, and the supernatant was collected. The supernatant was then processed through cation-exchange columns (AG 50W-8x 100-200-mesh; Bio-Rad Laboratories, Hercules, CA) to isolate/purify blood amino acids. Before the addition of the samples, the cation-exchange columns were conditioned with 3 ml of 2 N NH 4 OH and 3 ml of 1 N 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 d 5 -phenylalanine enrichment, we followed standard procedures that we have also described previously (16, 33). Briefly, ∼15 mg of muscle tissue was homogenized after it was combined with 500 μl of 5% SSA. After the muscle/SSA homogenate was centrifuged at 2,500 g for 45 min at 4°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°C to dry. Mixed-muscle proteins in the dried muscle pellet were hydrolyzed in 6 N HCl at 110°C for 24 h. The protein hydrolysate, corresponding to 1 mg of dry tissue weight, was processed through cation-exchange columns (AG 50W-8x 200-400-mesh; Bio-Rad Laboratories) to isolate the amino acids, and as we have described above for blood. Samples were processed for determination of the enrichments in blood and mixed-muscle protein-bound d 5 -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 described previously (22, 28). Briefly, phenylalanine isolated by ion exchange chromatography from muscle hydrolysates and the blood samples was derivatized to its t-butyldimethylsilyl 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 eight-point enrichment standard curve. Insulin was determined using a commercially available kit (ALPCO Diagnostics, Windham, NH). Plasma amino acid concentrations were determined using high-performance-liquid-chromatography (7). Calculations. Fractional synthesis rate (%/h−1) of mixed-muscle protein was calculated as described previously (14): FSR = Δ IE mmp IE p × T × 60 × 100 where ΔIEmmp is the increment in mixed-muscle protein 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 R a ) in blood, a reflection of whole body muscle protein breakdown, was calculated using the single pool model from the d 5 -phenylalanine infusion rate (F) and the average blood d 5 -phenylalanine enrichment (E b ) during the last 30 min of the basal and insulin infusion experimental periods: Phe R a = F E b Plasma clearance of each of the infused BCAAs 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 × 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·m2·min−1 insulin infusion rates on any of the measured variables within either the BCAA or control experiments, data within each experimental group were pooled and analyzed together. Results are expressed as means ± SE. Statistical significance was evaluated at P < 0.05. The statistical analyses were performed using the Minitab 16.2.4 statistical software (Minitab, 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 over time after the initiation of the insulin infusion in both experiments to maintain the plasma BCAA 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 and 7.9 ± 0.9 μmol·kg−1·min−1 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 and increased by ∼150% compared with fasting levels in the BCAA experiment (Table 2). BCAA infusion in the latter experiment significantly increased all three BCAAs 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 nonessential amino acids did not change during the insulin infusion (P > 0.05). Table 1. Subject characteristics Control BCAA Males/females 6/1 4/3 Age, yr 23 ± 2 19 ± 1 Weight, kg 71 ± 2 67 ± 4 BMI, kg/m2 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 ± 5 152 ± 14 HDL 45 ± 3 51 ± 4 LDL 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 2.3 ± 0.4 Table 2. Plasma concentrations of amino acids prior to the initiation of the infusions (fasting) and during the last 30 min of the basal and insulin infusion periods Fasting Basal Insulin Infusion ANOVA P Value BCAA Control 336 ± 42 282 ± 40 310 ± 41 0.647 BCAA 386 ± 66 1,059 ± 140* 933 ± 91* <0.001 NonBCAA EAA Control 109 ± 10 98 ± 10 70 ± 10*† 0.030 BCAA 121 ± 13 100 ± 10 69 ± 11*† 0.012 NEAA Control 225 ± 29 175 ± 21 177 ± 27 0.329 BCAA 244 ± 27 233 ± 15 186 ± 7 0.079 Arginine Control 68 ± 9 65 ± 11 54 ± 10 0.600 BCAA 81 ± 13 81 ± 10 64 ± 9 0.499 Asparagine Control 59 ± 9 52 ± 6 44 ± 5 0.274 BCAA 60 ± 4 55 ± 4 43 ± 3*† 0.023 Isoleucine Control 66 ± 8 56 ± 8 74 ± 11 0.386 BCAA 71 ± 10 281 ± 36* 240 ± 26* 0.001 Leucine Control 94 ± 15 73 ± 13 72 ± 9 0.404 BCAA 112 ± 24 264 ± 47* 241 ± 33* 0.016 Methionine Control 45 ± 4 35 ± 4* 28 ± 5*† 0.049 BCAA 51 ± 7 42 ± 5 30 ± 6 0.082 Phenylalanine Control 64 ± 7 63 ± 6 42 ± 5*† 0.033 BCAA 70 ± 8 58 ± 5 38 ± 4* 0.007 Serine Control 106 ± 19 74 ± 9 79 ± 15 0.275 BCAA 104 ± 15 97 ± 9 79 ± 6 0.257 Valine Control 176 ± 21 153 ± 21 164 ± 22 0.748 BCAA 202 ± 33 513 ± 59* 513 ± 41* 0.001 In the control experiments, insulin infusion significantly increased (P < 0.05) the plasma insulin concentrations both at 40 mU·m2·min−1 (33 ± 6 μIU/ml vs. fasting: 6 ± 2 μIU/ml; and basal: 5 ± 2 uIU/ml) and 80 mU·m2·min−1 (88 ± 12 μIU/ml vs. fasting: 3 ± 1 μIU/ml; and basal: 4 ± 1 μIU/ml). In the BCAA experiments, insulin infusion also increased (P < 0.05) the plasma insulin concentrations significantly at both 40 (39 ± 10 μIU/ml vs. fasting: 5 ± 2 μIU/ml; and basal: 4 ± 1 μIU/ml) and 80 mU·m2·min−1 (113 ± 8 μIU/ml vs. fasting: 2 ± 1 μIU/ml; and basal: 5 ± 1 μIU/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 Fig. 2. Blood phenylalanine enrichments were not different over time within either the basal or the insulin infusion periods or in either the BCAA or the control experiments (P > 0.05). Average responses for fractional synthesis rate of muscle protein are shown in Fig. 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; Fig. 3B). Fig. 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. Download figureDownload PowerPoint

Fig. 3.A: 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. B: change in FSR between the basal and insulin infusion periods. Download figureDownload PowerPoint

Whole body protein breakdown. Time course and average responses for Phe R a in blood are shown in Fig. 4, A and B, respectively. With respect to the average responses for Phe R a , ANOVA analyses indicated significant main effects for both treatment (i.e., a BCAA effect) and time (i.e., an insulin effect) on blood phenylalanine R a (P < 0.05). Pairwise comparisons between experiments showed no significant difference in blood Phe R a in the basal period (P > 0.05), but Phe R a was different during the insulin infusion period. The Phe R a decreased in both experiments during the insulin infusion period (P < 0.05). However, the insulin-stimulated change in blood Phe R a was not different between the control and BCAA experiments (P > 0.05; Fig. 4C). Fig. 4.A: time course of whole body phenylalanine rate of appearance (R a ) in the control and BCAA experiments before (basal) and after (insulin infusion) the initiation of the insulin infusion. B: average values for the whole body phenylalanine R a corresponding to the last 30 min of the basal and insulin infusion periods. C: change in whole body blood phenylanine R a between the basal and insulin infusion periods calculated from the data presented in Fig. 3B. *P < 0.05 vs. basal; #P < 0.05 vs. control. Download figureDownload PowerPoint

Plasma clearance of infused branched-chain amino acids. The infusion rate of each of the BCAAs in the last 30 min of the insulin infusion period and during steady-state plasma BCAA levels was 3.3 ± 1.0 and 9.8 ± 1.1 mmol/h 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 Because of its role in inhibiting protein catabolism, insulin suppresses the availability of circulating amino acids for protein synthesis, including the BCAAs, which are primary regulators of the protein synthesis machinery. In the present study, we evaluated whether preventing the drop or specifically increasing 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 are 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 BCAAs are maintained at their postabsorptive levels. We show that maintaining the concentration of plasma BCAAs 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 BCAAs. 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 EAA (i.e., non-BCAA). 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 BCAAs 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 compared with the non-EAA pool. The decrease in non-BCAA EAA appears to be the limiting factor restricting the stimulation of muscle protein synthesis in the present studies and is 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 BCAAs (36). Interestingly, and as opposed to the lack of stimulation of muscle protein synthesis by increasing the overall concentration of plasma BCAAs (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 EAAs other than BCAAs 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 R a 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 R a in response to the BCAA infusion. However, the change in blood Phe R a 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 that of either leucine or isoleucine. Since the plasma clearance of the infused amino acids was not higher in the BCAA experiment compared with 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 in line with the lack of an effect of the increased plasma BCAA availability on increasing disposal of plasma amino acids specifically toward muscle protein synthesis (Fig. 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 Ref. 21). Our findings show that even the addition of the anabolic stimulus of insulin in the presence of the elevated plasma BCAA levels is still not sufficient to stimulate muscle protein synthesis. Therefore, these overall findings explain why neither BCAAs nor leucine supplementation improve muscle protein synthesis or enhance muscle protein accretion (2, 31). Furthermore, neither BCAA (24) nor leucine (2) supplementation enhances 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). However, when 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 EAAs 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 EAAs. The extent by which insulin suppresses proteolysis is observed independently by the presence of increased plasma BCAA concentrations.

GRANTS This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-094062 (C. S. Katsanos), the Mayo Clinic Metabolomics Resource Core through Grant No. U24-DK-100469 from the NIDDK, and Mayo Clinic CTSA Grant UL1-TR-000135 from the National Center for Advancing Translational Sciences.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS S.E., C.M., and C.S.K. performed experiments; S.E., L.T., N.H., C.C.C., W.L.D., and C.S.K. analyzed data; S.E., C.M., C.C.C., and C.S.K. interpreted results of experiments; S.E. and C.S.K. prepared figures; S.E. and C.S.K. drafted manuscript; S.E., C.M., L.T., C.C.C., and C.S.K. edited and revised manuscript; S.E., C.M., L.T., C.C.C., and C.S.K. approved final version of manuscript; C.S.K. conception and design of research.