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Protein and glucose metabolic responses to hyperinsulinemia, hyperglycemia, and hyperaminoacidemia in obese men

2014; Wiley; Volume: 23; Issue: 2 Linguagem: Inglês

10.1002/oby.20943

1930-739X

Stéphanie Chevalier, Sergio A. Burgos, José A. Morais, Réjeanne Gougeon, Maya Bassil, Marie Lamarche, Errol B. Marliss,

Muscle metabolism and nutrition

AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Objective In insulin-resistant states, resistance of protein anabolism occurs concurrently with that of glucose, but can be compensated for by abundant amino acid (AA) provision. This effect and its mechanism were sought in obesity. Methods Pancreatic clamps were performed in 8 lean and 11 obese men, following 5-h postabsorptive, 3-h infusions of octreotide, basal glucagon, and growth hormone, with clamped postprandial-level insulin, glucose, and AA. Whole-body [1-13C]-leucine and [3-3H]-glucose kinetics, skeletal muscle protein (2H5-phenylalanine) fractional synthesis rates, and insulin signaling were determined. Results Clamp Δ insulin and Δ branched-chain AA did not differ; fasting glucagon and growth hormone were maintained. Glucose uptake was 20% less in obese concurrent with less AktSer473, but also less IRS-1Ser636/639 phosphorylation. Stimulation of whole-body, myofibrillar, and sarcoplasmic protein synthesis was similar. Whole-body protein catabolism suppression tended to be less (P=0.06), resulting in lesser net balance (1.09 ± 0.07 vs. 1.31 ± 0.08 μmol [kg FFM−1] min−1, P = 0.048). Increments in muscle S6K1Thr389 phosphorylation were less in the obese, but 4E-BP1Ser65 did not differ. Conclusions Hyperaminoacidemia with hyperinsulinemia stimulated protein synthesis (possibly via nutrient signaling) normally in obesity, but suppression of proteolysis may be compromised. Whether long-term high protein intakes could compensate for the insulin resistance of protein anabolism remains to be determined. Introduction Alterations of glucose and lipid metabolism in obesity are well documented, but those of protein metabolism only recognized more recently (1-3). Insulin plays a pivotal role in these alterations and resistance to its action could contribute to insufficient protein anabolism, resulting in altered tissue functions and gradual tissue (muscle) loss. Insulin resistance of protein anabolism accompanies that of glucose metabolism in obesity (4) and type 2 diabetes (T2D) (5) during hyperinsulinemic, euglycemic clamps that maintained postabsorptive circulating amino acid (AA) levels (6). Clamp studies without maintained AA induced a paradoxical reduction of protein synthesis (7) despite in vitro (8) and in vivo (9-11) evidence of insulin stimulation of muscle protein synthesis. This occurred because insulin inhibited proteolysis and lowered AA availability for protein synthesis. Elevation of AA above fasting concentrations stimulates protein synthesis at “permissive” baseline insulin levels (12), and further suppresses protein catabolism (13, 14). Thus, isolating insulin effects on anabolism requires AA maintenance. Testing of responses relevant to the postprandial state requires elevation of insulin, AA and glucose concentrations. Prior studies of protein metabolism have differed on alterations in obesity (1-3), for many reasons, including differences in subjects studied, experimental protocols and, inclusion of both sexes (15). One study reported normal suppression of protein breakdown and stimulation of synthesis with hyperaminoacidemia alone, with basal insulin and euglycemia (16). With combined hyperinsulinemia, hyperaminoacidemia and euglycemia, the suppression of breakdown was blunted (17). Hyperglycemia also affects protein metabolism (18, 19). Thus we tested protein anabolism with combined hyperinsulinemia, hyperaminoacidemia and hyperglycemia (Hyper-3). As this simulated fed-state clamp in lean persons stimulated endogenous insulin secretion (20), to assure comparable hyperinsulinemias, we developed the pancreatic clamp protocol. Most studies of insulin effects on protein metabolism in obesity examined whole-body changes (4, 16, 21, 22). In obese men, lower postabsorptive turnover, and during a hyperinsulinemic, hyperaminoacidemic, euglycemic clamp, less stimulation of synthesis and inhibition of breakdown occurred (17). Muscle mitochondrial protein synthesis was not stimulated, and that of total protein was subnormal. However, insulin concentrations were 1.7-fold higher. Our Hyper-3 approach mitigates such higher insulin levels, thereby facilitating intergroup comparisons. Insulin and AA signals converge at the mammalian target of rapamycin complex 1 (mTORC1) to regulate protein synthesis (23). Enhanced protein anabolism and insulin signaling was found in normal men during the simulated fed steady-state (Hyper-3) clamp (20). The same protocol but with decreased rates of clamp insulin infusion to obviate the amplified hyperinsulinemia was applied in insulin-resistant type 2 diabetic men (18) and elderly women (24). Normal anabolic responses occurred, in the whole body in both, and in muscle in the latter. Similar normalization occurred in lung cancer patients (25). We postulated that the same would occur in obesity. Hence, our aim was to assess the effect of obesity on Hyper-3 responses of whole body protein kinetics and skeletal muscle protein synthetic rates and signal transduction. As we have found sex differences in protein metabolism (26), this study includes only men. Methods Eight lean and eleven obese men were screened with electrocardiogram, chest X-ray, 24-h dietary recall, and complete laboratory and physical examinations for hepatic, hematologic, renal, pulmonary, thyroid, and cardiovascular diseases and unconventional diets. Obese men had normal oral glucose tolerance. Exclusion criteria included smoking, unstable weight (>±3 kg) for 6 months, cancer in the previous 5 years, and medications affecting metabolism. Written informed consent was obtained as prescribed by the Ethics Review Board of the McGill University Health Centre. During the 4- to 6-day admission to the Clinical Investigation Unit, they consumed an isoenergetic, protein-controlled diet, in five equal meals from 8:00 to 20:00 h (54.5% of energy from carbohydrate, 30.0% fat, and 15.5% protein; 1.7-1.8 g [kg FFM−1] day−1). It included a commercial formula (Ensure®, Abbott Laboratories, St. Laurent, QC, Canada) and bran cereal with 2% milk. Energy requirements were calculated from resting energy expenditure by indirect calorimetry (TrueOne® 2400 Canopy System, Parvo Medics, Sandy, UT), multiplied by an activity factor of 1.7 in lean and 1.5 in obese and corroborated by 24-h food recall and daily weights. Body composition was determined by dual energy X-ray absorptiometry (DXA) in 12 (9 obese and 3 lean; Lunar Prodigy Advance, GE Healthcare, Madison, WI), and by bioimpedance analysis (BIA; RJL-101A Systems, Detroit, MI) in all, using equations for lean and obese persons (27). DXA data were used because of a 6.0 ± 1.0 kg fat free mass (FFM) overestimation from BIA vs. DXA in obese. There was no such difference in lean participants. The clamp protocol is shown in Figure 1. On the experiment day at 08h00, catheters were inserted in an antecubital vein for infusions, and a contralateral dorsal hand vein retrograde for arterialized blood sampling. A primed infusion of [3-3H]-glucose (22 μCi bolus then 0.22 μCi min−1 constant infusion; PerkinElmer Inc., Boston, MA) was used for estimation of glucose kinetics. For leucine kinetics, an oral bolus of 0.1 mg kg−1 of NaH13CO2 and 0.5 mg kg−1 intravenous [1-13C]-leucine bolus were followed by a constant infusion at 0.008 mg [kg−1] min−1. [2H5]phenylalanine was started with a bolus of 1.22 mg kg−1 FFM, then infusion rate at 0.0204 mg [kg FFM−1] min−1. All stable isotopes were from Cambridge Isotope Laboratory, Andover, MA. The [2H5]phenylalanine infusion started 1.5 h before the other isotopes, then all continued for 6 h, with clamp from 4.5 to 7.5 h with: human insulin (primed, then 0.98-1.3 mU [kg FFM−1] min−1, Humulin R; Eli Lilly Canada Inc, Toronto, ON, Canada); octreotide (35.3 ng [kg FFM−1] min−1, Omega, Montreal, QC, Canada); glucagon (0.94 ng [kg FFM−1] min−1, Eli Lilly); and human growth hormone (hGH; 3.53 ng [kg FFM−1] min−1, Saizen, EMD Serono, Canada). Concurrently, 20% low-enriched 13C-glucose (Avebe b.a., Foxhol, Netherlands) and 10% AA solution (TrophAmine, B. Braun Medical, Irvine, CA) were infused at variable rates to clamp glucose at 8 mmol l−1 and branched-chain amino acids (BCAA), at postprandial concentrations (18), both based on 5-min interval measurements. Blood was collected hourly for substrates, hormones, and isotopic enrichment and at 10-min intervals from 2.5-3 h to 5.5–6 h. L-[1-13C] leucine kinetics were calculated using plasma 1-[13C] α-ketoisocaproic acid (KIC) enrichment (reciprocal model) (28). Figure 1Open in figure viewerPowerPoint Time course of the clamp study protocol. Tracer infusions began at times required for achieving appropriate isotopic enrichment and steady-state conditions for kinetics calculations. Twenty percent glucose and ten percent amino acid infusions began 4 min after the hormones, and rates were adjusted to achieve and maintain the targets specified. BCAA, branched-chain amino acids; HGH, human growth hormone. Indirect calorimetry was performed before and during the clamp and expired air collected for calculation of leucine oxidation. Factors of 10.0 and 7.0% for lean and obese, respectively, were used as adjustment to 13CO2 enrichment for dilution of background enrichment by glucose and AA infusions (4, 6). Vastus lateralis muscle biopsies were performed with sterile technique using a Bergstrom needle under local anesthesia, immediately prior to and at 2 h during the clamp (24). Enrichment of plasma [13C]-α-KIC was determined by gas chromatography-mass spectrometry (6890N and 5973, Agilent Technologies, Santa Clara, CA). Expired air 13CO2 enrichment was measured by isotope-ratio mass spectrometry (Micromass 903D, Vacuum Generators, Winsforce, UK). Muscle was homogenized, centrifuged to separate myofibrillar and sarcoplasmic proteins, then the myofibrillar fraction was precipitated, and AA liberated from both fractions, modified from (29). The phenylalanine tracer/tracee ratio was determined by liquid chromatography-tandem mass spectrometry (Agilent 1290 LC system with 6460 Triple Quadrupole MS). Detection and quantification of AA were performed using multiple reaction monitoring in positive ion mode with transitions of m/z 166 > 120 for phenylalanine and m/z 171 > 125 for [2H5]phenylalanine. The 3-nitro-L-tyrosine was an internal standard used to monitor data quality and reproducibility for LC/MS analysis. Fractional synthesis rates (FSR) were calculated from rates of [2H5]phenylalanine incorporation: FSR = (Epb2 – Epb1)/EFAA (Epb1 and Epb2 are enrichment of proteins pre- and at 2 h of clamp, respectively, and EFAA is the enrichment of the precursor muscle free AA pool). Postabsorptive FSR was calculated from the single biopsy approach in these tracer naive participants (30). Clamp plasma glucose and total BCAA were measured as in (6). Serum insulin, C-peptide, and glucagon concentrations were determined by radioimmunoassay (Millipore Corporation, Billerica, MA), nonesterified fatty acids (NEFA) by colorimetric assay (NEFA C; Wako Chemicals, Richmond, VA) and individual plasma AA by reverse phase HPLC (24). Analysis of phosphorylation state of muscle signaling proteins was as detailed (31). Samples (25 ± 2 mg) were homogenized and processed. Protein was measured (bicinchoninic acid assay), and 25 μg were resolved by SDS-PAGE and transferred onto polyvinylidine fluoride membranes (GE Healthcare, Piscataway, NJ) that were blocked, blotted with phospho-specific primary antibodies (Cell Signaling Technology, Danvers, MA) against AktSer473, IRS-1Ser636/639, S6K1Thr389, rpS6Ser240/244, 4E-BP1Thr37/46, 4E-BP1Ser65, and AMPKThr172. After detection, secondary antibody membranes were reprobed with antibodies against total proteins. Ratios of phosphorylated to total protein are presented. Results are presented as means ± SEM. Data were verified for normality with the Shapiro-Wilk test. Non normally distributed data (concentrations of Asp, Ser, Thr, Tau, Ala, Tyr, Orn) were log-transformed before analysis. Differences in baseline characteristics and variables during the clamp were compared by independent t tests. Clamp responses were analyzed by repeated measures ANOVA with the clamp effect as a within-subject factor, group as a between-subject factor and clamp-by-group interaction indicating a different response to the clamp. Although age was significantly different between groups, it was not used in the ANOVA because of co-linearity with adiposity, the variable of interest. Analyses were done using PASW (SPSS) version 19.0 (Chicago, IL). Results The obese group was heavier, had higher BMI, greater % body fat, waist circumferences and waist/hip ratios than lean, but FFM did not differ (Table 1). They were older, driven by the demographics of our volunteers for such studies. Postabsorptive and Hyper-3 clamp substrate and hormone concentrations are presented in Table 2 and Supporting Information Table 1. Postabsorptive glycemia, essential, non-essential and total AA, NEFA, C-peptide, hGH and glucagon were not different, but the obese had higher BCAA and insulin concentrations. Both groups were clamped at the targets of glycemia (8 mmol l−1) and 1.78-fold increased BCAA. The increment in BCAA was similar in both, to higher concentrations in the obese. Essential, non-essential and total AA increased similarly. Only postabsorptive plasma leucine, valine (not isoleucine) and glutamic acid were greater in the obese group, but none of the other AA differed. All essential and 8 of 12 non-essential AA were elevated during the clamp. Leucine, valine, and glutamic acid increased similarly, thus remaining higher in the obese. Isoleucine increased slightly more in the obese. Citrulline and glutamine did not change. Asparagine and tyrosine decreased. Table 1. Participant characteristics Characteristic Lean Obese n 8 11 Age (years) 21.3 ± 0.6 44.3 ± 4.0a Height (cm) 180.6 ± 1.8 178.2 ± 2.5 Weight (kg) 73.8 ± 2.2 107.0 ± 4.7a BMI (kg m−2) 22.6 ± 0.6 33.7 ± 1.2a FFM (kg) 60.8 ± 1.8 66.5 ± 2.1 Body fat (%) 17.3 ± 2.7 37.3 ± 2.1a Waist circumference (cm) 78.7 ± 1.8 110.1 ± 3.1a Waist/hip ratio 0.81 ± 0.01 0.96 ± 0.02a Energy intake (kcal [kg FFM]−1 d−1) 47.9 ± 1.7 44.8 ± 0.8 Protein intake (g [kg FFM]−1 d−1) 1.84 ± 0.06 1.73 ± 0.03 REE (kcal [kg FFM]−1 d−1) 29.3 ± 0.5 29.8 ± 0.8 Data are means ± SEM. a P < 0.05 from independent samples t test. Table 2. Circulating substrate and hormone concentrations Lean Obese P Postabsorptive Hyper-3 clamp Postabsorptive Hyper-3 clamp Clamp effect Group effect Clamp × group Glucose (mmol l−1) 5.3 ± 0.1 8.0 ± 0.1 5.4 ± 0.1 7.9 ± 0.0 <0.001 0.450 0.342 Branched-chain AA (μmol l−1) 378 ± 19 671 ± 21 438 ± 12a 781 ± 20a <0.001 0.001 0.154 Essential AA (μmol l−1) 940 ± 53 1547 ± 58 1012 ± 37 1688 ± 68 <0.001 0.125 0.473 Non-essential AA (μmol l−1) 1674 ± 92 1849 ± 89 1660 ± 83 1959 ± 94 <0.001 0.689 0.296 Total AA (μmol l−1) 2614 ± 132 3395 ± 127 2672 ± 117 3646 ± 157 <0.001 0.378 0.357 NEFA (μmol l−1) 522 ± 52 62 ± 13 488 ± 42 82 ± 9 <0.001 0.857 0.399 Insulin (pmol l−1) 52 ± 6 515 ± 44 90 ± 9a 566 ± 30 <0.001 0.128 0.802 C-peptide (pmol l−1) 459 ± 46 233 ± 60 645 ± 75 371 ± 55 <0.001 0.059 0.562 Growth hormone (μg l−1) 0.51 ± 0.23 0.48 ± 0.03 0.42 ± 0.15 0.44 ± 0.02 0.997 0.655 0.835 Glucagon (pmol l−1) 16 ± 2 19 ± 3 23 ± 3 24 ± 3 0.093 0.129 0.640 Glucagon/insulin ratio 0.35 ± 0.07 0.04 ± 0.00 0.26 ± 0.04 0.04 ± 0.01 <0.001 0.339 0.700 Data are means ± SEM, analyzed by repeated measures ANOVA. a P < 0.05 vs. lean, by independent samples t tests. NEFA were lowered to the same levels (Table 2), more slowly in the obese (higher at 30 min, P = 0.029, data not shown). Insulin was raised to the same level, and C-peptide was reduced similarly. Glucagon and hGH remained at postabsorptive concentrations and glucagon/insulin ratios were much lower. Thus the clamped hormone and substrate concentrations (increments of BCAA) were well matched between groups, and endogenous hormone secretion stimulation was blunted. Postabsorptive glucose kinetics did not differ between groups (Table 3). Clamp endogenous Ra was significantly (P < 0.001) but incompletely suppressed, to the same extent in both groups. The increments in glucose Rd and in metabolic clearance rate were less in the obese, as indicated by clamp × group interactions. Consequently, glucose infusion rates were lower in the obese group. Table 3. Postabsorptive and Hyper-3 clamp glucose turnover Lean Obese P Postabsorptive Hyper-3 clamp Postabsorptive Hyper-3 clamp Clamp effect Group effect Clamp × group Endogenous Ra, mg [kg FFM]−1 min−1 2.69 ± 0.06 0.35 ± 0.23 2.65 ± 0.10 0.60 ± 0.10 <0.001 0.456 0.237 Infusion rate, mg [kg FFM]−1 min−1 na 6.71 ± 0.24 na 5.11 ± 0.31* na na na Total Rd, mg [kg FFM]−1 min−1 2.68 ± 0.05 7.09 ± 0.27 2.68 ± 0.09 5.74 ± 0.29* <0.001 0.005 0.007 MCR, ml [kg FFM]−1 min−1 2.83 ± 0.03 4.96 ± 0.19 2.76 ± 0.08 4.04 ± 0.21* <0.001 0.007 0.013 Data are means ± SEM. P values from repeated measures ANOVA; *P < 0.05 vs. lean, by independent samples t tests. na, not applicable. Postabsorptive endogenous leucine Ra (breakdown), oxidation, nonoxidative Rd (synthesis) and net balance rates did not differ (Table 4). During the clamp, both groups responded comparably, with increased flux, oxidation and synthesis, and decreased breakdown. The magnitude of suppression of breakdown (lean: −0.82 ± 0.05, obese: −0.65 ± 0.07 μmol [kg FFM−1] min−1) tended to be less in obese men (P = 0.06, but P = 0.003, in μmol min−1). The increase in synthesis was similar between groups (0.47 ± 0.07 vs. 0.44 ± 0.18 μmol [kg FFM−1] min−1). Thus, the AA infusion rates required to raise plasma leucine and total BCAA by the same magnitude were less in the obese men. Principally because of the breakdown response, the increase in net balance was less in the obese group (1.31 ± 0.08 vs. 1.09 ± 0.07 μmol [kg FFM−1] min−1). Table 4. Postabsorptive and Hyper-3 clamp leucine kinetics Lean Obese P Postabsorptive Hyper-3 clamp Postabsorptive Hyper-3 clamp Clamp effect Group effect Clamp × group Total Ra (flux) 2.39 ± 0.10 3.89 ± 0.11 2.54 ± 0.06 3.95 ± 0.14 <0.001 0.481 0.444 Oxidation 0.49 ± 0.03 1.51 ± 0.09 0.52 ± 0.02 1.49 ± 0.10 <0.001 0.932 0.689 Endogenous Ra (breakdown) 2.39 ± 0.10 1.56 ± 0.11 2.54 ± 0.06 1.89 ± 0.10* <0.001 0.063 0.064 Infusion rate na 2.33 ± 0.05 na 2.06 ±0.09* na na na Nonoxidative Rd (synthesis) 1.90 ± 0.09 2.38 ± 0.07 2.02 ± 0.06 2.46 ± 0.07 <0.001 0.285 0.673 Net balance (synthesis – breakdown) −0.49 ± 0.03 0.83 ± 0.10 −0.52 ± 0.02 0.57 ± 0.06* <0.001 0.039 0.048 Data are means ± SEM, in μmol [kg FFM]−1 min−1. P values from repeated measures ANOVA; *P < 0.05 vs. lean, by independent samples t tests. na, not applicable. Postabsorptive skeletal muscle protein FSR was not different between groups nor between myofibrillar vs. sarcoplasmic proteins (Figure 2). The clamp stimulated both significantly to rates that did not differ by group. Figure 2Open in figure viewerPowerPoint Skeletal muscle protein fractional synthesis rates (FSR) in lean and obese men. (A) Myofibrillar and (B) sarcoplasmic proteins. Means ± SEM. There is a significant effect of the hyperinsulinemic, hyperglycemic, and hyperaminoacidemic (Hyper-3) clamp on myofibrillar and sarcoplamic protein FSR, P < 0.05 by repeated measures ANOVA, without group differences. Postabsorptive AktSer473 phosphorylation (Figure 3B) was less in the obese; all other phospho-proteins did not differ. The Hyper-3 clamp increased phosphorylation of all proteins shown (Figure 3A-F), but less for AktSer473 in the obese. AMPKThr172 phosphorylation did not change in either group (not shown). Phospho-S6K1Thr389 increased less in the obese. Phospho-rpS6Ser240/244 was elevated >10-fold, with a trend to less in the obese (interaction P = 0.068). Phospho-4E-BP1Thr37/46 (not shown) and 4E-BP1Ser65 increased similarly. The increment in IRS1Ser636/639 phosphorylation was less in the obese. Figure 3Open in figure viewerPowerPoint Phosphorylation of signaling proteins in skeletal muscle of lean and obese men. (A) AktThr308, (B) AktSer473, (C) S6K1Thr389, (D) rpS6 Ser240/244, (E) 4E-BP1Ser65, and (F) IRS-1Ser636/639. Means ± SEM for n = 6/group. There is a significant effect of the hyperinsulinemic, hyperglycemic, and hyperaminoacidemic (Hyper-3) clamp on all signal proteins illustrated, P < 0.05 by repeated measures ANOVA; † = significant clamp × group interaction, P < 0.05. * = P < 0.05 vs. lean at the same period by independent t test. Discussion The simulated fed steady-state pancreatic clamp achieved the goal of the same circulating hormone levels in lean and glucose-tolerant obese men. It revealed the expected resistance of insulin-mediated glucose disposal in the latter, concurrent with impaired Akt signaling. It demonstrated borderline (P = 0.06) impairment of suppression of whole-body protein catabolism (though P = 0.003 expressed as μmol min−1), but normal stimulation of synthesis in the obese. The former contributed to a significant 32% smaller increment in whole-body net protein balance. The stimulating effect of combined hyperinsulinemia, hyperglycemia and hyperaminoacidemia (Hyper-3) on protein synthesis is consistent with our previous findings in insulin-resistant persons with T2D (18), aging (24), and lung cancer (25), and suggests it may be a characteristic of other such states. Normal increments of muscle protein FSR occurred as well in myofibrillar and sarcoplasmic protein, despite lower S6K1, but with normal 4E-BP1 phosphorylation. This suggests sufficient nutrient signaling via mTORC1 contributing to normal stimulation of translation initiation and the normal protein-synthetic responses, given sufficient AA availability. Taken together, these results raise the question of whether abundant protein provision and/or specific AA may be able to maintain/improve protein homeostasis in insulin-resistant states. As with glucoregulation, there was no obesity effect on any postabsorptive leucine kinetic parameter expressed per FFM. However, this occurred with significantly higher fasting serum insulin (75%), leucine (19%), and BCAA (16%) concentrations, all consistent with fasting insulin resistance. Recent metabolomic studies emphasize the association of this fasting AA “signature” with insulin resistance of glucose metabolism (32). The importance of studying protein metabolism is the clear demonstration of its insulin resistance concurrent with that of glucose (1-5, 17, 33-35). However, the extent to which there is long-term anabolic resistance in the fed state that impairs quantitative resynthesis of the amount lost in the fasted state remains to be defined. As there are differences among the insulin-resistant states in their magnitudes of abnormal protein metabolism, it is essential that it be studied in each. In our obese participants, the antiproteolytic action of insulin was somewhat blunted, consistent with previous studies (16, 22), but not all (4, 33). The inclusion of hyperaminoacidemia, which has varied in level, with different AA solutions, differing protocols, analyses, participant characteristics, and other factors may explain some discrepancies among studies. In our obese men, it did not fully compensate for the defective response, as also reported by Guillet et al. (17). Because of our BCAA feedback loop clamp system, contrasting with constant infusions used by others, this lesser breakdown suppression prompted lesser AA infusion rates to result in an equivalent increment in BCAA levels. Despite this, whole-body protein synthesis was stimulated equally. This differs from our study in obese women showing blunted synthetic response to hyperinsulinemia during isoaminoacidemia that provided ∼10 g of intravenous AA over 3 h (4). The present response to the ∼33 g of AA infused was normal, indicating that higher AA doses can compensate for the underlying resistance of protein synthesis, as we found in other conditions. Likewise, the stimulation of skeletal muscle myofibrillar and sarcoplasmic protein synthesis by insulin and AA was normal. In contrast, Guillet et al. (17) found reduced mitochondrial protein synthesis and no stimulation by higher clamped insulin and leucine (vs. lean), but a similar stimulation of total muscle protein synthesis. These data are complementary and indicate differential regulation of muscle protein fractions by insulin and AA in obesity, as in other situations (29). Indeed, the abundance of mitochondrial compared to myofibrillar proteins is low, but their rapid turnover rate contributes significantly to the total protein FSR. It is thus possible that synthesis of mitochondrial protein be specifically impaired in obesity as has been shown in other insulin-resistance states such as T2D (36), but without effect on myofibrillar and sarcoplasmic proteins. Structural myofibrillar proteins form the bulk of the muscle tissue and are involved in contraction. Their synthesis may be partly enhanced by the weight-bearing stimulus imposed by the excess weight. Sarcoplasmic protein synthesis was not only stimulated normally by insulin and AA, but suggestively more (Figure 1, P = 0.071). If confirmed, this could indicate a compensation mechanism for the lesser suppression of postprandial breakdown rates. Interestingly, data on 24-h muscle protein FSR, integrating fasting and fed periods, in obese Zucker rats support our findings (34). The muscle signaling results are consistent with this tissue being an important contributor to the whole-body insulin resistance of glucose metabolism. The hyperinsulinemic activation of AktThr308 and AktSer473 was markedly attenuated in the obese participants in association with reduced glucose uptake. Downstream of mTORC1, the attenuated phospho-S6K1Thr389 signal was not compensated by AA infusion and elevated plasma leucine concentrations. This was associated with lesser clamp increment in IRS-1Ser636/639 phosphorylation. This site is implicated in the AA-induced negative feedback regulation via S6K1 in healthy lean men (37). Myofibrillar and sarcoplasmic protein synthesis was normal in obese despite lesser S6K1 activation and a trend toward lesser rpS6 phosphorylation (P = 0.06). The other downstream target of mTORC1 measured was 4E-BP1, whose dissociation from eIF4E promotes translation initiation. The 4E-BP1 phosphorylation was not different between groups, consistent with sufficient hyperaminoacidemic stimulation of pathways that compensate for the impaired upstream insulin signaling to protein synthesis. Although impaired stimulation of S6K1 was observed in the obese group, this may be less important for the protein synthetic response than preserved fed-state induced 4E-BP1 phosphorylation, which is considered the master effector of mTORC1-mediated translational regulation (38). The expected total suppression of endogenous glucose Ra did not occur in the lean, and was not different in the obese. A possible explanation is that octreotide in doses comparable to those used in this study decreases splanchnic blood flow in fasting (39, 40). As there would be no portal-peripheral gradient of insulin concentrations, in contrast to normal physiology, less would be presented to the liver during the clamp (and even less than in the absence of octreotide, with the rise in insulin and C-peptide) (20). The concurrent absence of decrease in portal glucagon concentrations might have contributed, though with the absence of a portal-peripheral gradient, it would have been lower than without octreotide. Likewise, the clamp glucagon/insulin ratios were the same in the two groups. Thus a role for glucagon in the failure to fully suppress Ra seems unlikely. Octreotide appears not to have blood flow effects in the periphery (39, 40). Furthermore, the sustained hyperinsulinemia likely increased muscle blood flow (11). Thus, while the physiologic state during the pancreatic clamp was not identical to that of our experiments without octreotide, the hormonal milieu in both groups was comparable, permitting intergroup comparisons not possible in its absence. The Hyper-3 clamp is a simulated fed steady-state necessitated in order to use steady-state kinetic calculations. Therefore, extrapolations to responses of the physiologic state with nutrients absorbed from the gastrointestinal tract must be conditional. We clamped insulin, glucose and BCAA at peak levels following mixed meals that were in the ranges previously reported. Furthermore, total amounts infused over 3 h were similar to a medium-sized meal, though without lipids. However, since NEFA decline postprandially despite absorption from meals, this response is physiological in the clamp. In the present study, there was also a slower rate of decline in the obese, to the same nadir as in the lean, thus likely exerting little confounding effect on the primary outcomes. As we were unable to recruit obese and lean men of the same age, and could not statistically correct for this difference due to co-linearity between age and adiposity, we acknowledge a potential age effect on our findings. A blunted suppression of leg protein breakdown by low hyperinsul