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American Journal of Clinical Nutrition, Vol. 82, No. 2, 355-365, August 2005
© 2005 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Whole-body protein anabolic response is resistant to the action of insulin in obese women1,2,3

Stéphanie Chevalier, Errol B Marliss, José A Morais, Marie Lamarche and Réjeanne Gougeon

1 From the McGill Nutrition and Food Science Centre, McGill University Health Centre–Royal Victoria Hospital, Montreal, Canada

2 Supported by research grants no. MOP-15487 (to RG) and MOP-42500 (to EBM) from the Canadian Institutes of Health Research, a research grant from the Canadian Diabetes Association (to JAM), salary awards from the McGill University Health Centre Research Institute (to RG) and from the Fonds de la recherche en santé du Québec (to JAM), and a fellowship from the Canadian Diabetes Association (to SC).

3 Reprints not available. Address correspondence to R Gougeon, McGill Nutrition and Food Science Centre, MUHC–Royal Victoria Hospital, 687 Pine Avenue West, Montreal, PQ H3A 1A1, Canada. E-mail: rejeanne.gougeon{at}muhc.mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Obesity is associated with insulin resistance of glucose and lipid metabolism.

Objective: We sought to determine the effects of obesity on the insulin sensitivity of protein metabolism.

Design: Whole-body [13C]leucine and [3H]glucose kinetics were measured in 9 lean and 10 obese women in the postabsorptive state and during a hyperinsulinemic, euglycemic, isoaminoacidemic clamp.

Results: In the postabsorptive state, the leucine endogenous rate of appearance (catabolism), normalized for fat-free mass, was 11% greater and the nonoxidative rate of disappearance (synthesis) was 8% greater in the obese than in the lean women, but net balance was 29% more negative (P < 0.05). Clamp amino acid and glucose infusion rates were significantly lower in the obese women than in the lean women (0.65 ± 0.02 compared with 0.85 ± 0.04 and 5.7 ± 0.3 compared with 9.1 ± 0.5 mg · kg fat-free mass–1 · min–1, respectively; P < 0.0001 for both), and their rates correlated positively (r = 0.635, P = 0.005). During hyperinsulinemia, synthesis was stimulated less and net leucine balance was much lower in the obese women than in the lean women (–0.08 ± 0.06 and 0.30 ± 0.03 µmol · kg fat-free mass–1 · min–1, respectively; P < 0.0001). The percentage change in net leucine balance correlated negatively with all adiposity indexes. Plasma free fatty acids were less suppressed and the respiratory quotient was lower in the obese women than in the lean women.

Conclusion: Obese women show a blunted protein anabolic response to hyperinsulinemia that is consistent with resistance to the action of insulin on protein concurrent with that on glucose and lipid metabolism.

Key Words: Hyperinsulinemic clamp • leucine kinetics • resting energy expenditure • glucose kinetics • amino acids • women


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Obesity is associated with insulin resistance of glucose and lipid metabolism (1), but its influence on protein metabolism remains a subject of controversy. Postabsorptive protein kinetics have been reported to be significantly higher in obese than in nonobese subjects (2–4) or similar in the 2 groups (5–9). Because insulin is an anabolic hormone, its action in suppressing protein breakdown and stimulating synthesis would be predicted to be impaired in obesity. Whereas some studies in obese persons showed that protein metabolism during a hyperinsulinemic, euglycemic clamp was normal (6, 9), other studies showed a blunted antiproteolytic effect (2, 8). One limitation in interpretating such kinetic results in both lean and obese persons is that the elevated plasma insulin concentrations were accompanied by reduced plasma amino acid (AA) concentrations (2, 6, 9), which were due to the suppression of proteolysis. Consequently, insulin stimulation of protein synthesis cannot be assessed in the face of decreased substrate availability (10). Luzi et al (8) found normal stimulation of protein synthesis in obese persons during the infusion of AAs but found it to be associated with hyperaminoacidemia. Because AAs themselves stimulate protein synthesis (8, 11–13), quantifying the relative roles of insulin and of the increases in AA is precluded. One way of dissecting out the role of insulin, and thus of showing insulin resistance, is to "clamp" plasma AAs at their postabsorptive concentrations during a hyperinsulinemic clamp. We recently showed both inhibition of protein catabolism and stimulation of synthesis in healthy male subjects by using this technique (14). Thus, we tested 1) whether increased adiposity interferes with the protein anabolic responses to insulin of suppressing protein breakdown, stimulating protein synthesis, or both, and 2) whether the AA infusion rates during a clamp could serve as an index of insulin resistance of protein metabolism, much as glucose infusion rates define insulin resistance of glucose metabolism. Preliminary results were presented in abstract form (15).


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and diet
Nine lean and 10 nondiabetic, normotensive obese women were screened with the use of medical history, physical examination, and laboratory investigation as previously detailed (16). No women were taking medications. They were admitted to the McGill University Health Centre–Royal Victoria Hospital Clinical Investigation Unit.

All subjects provided written informed consent. The study protocol was approved by the Human Ethics Review Committee of McGill University Health Centre.

The subjects were classified according to their body mass index (BMI; in kg/m2) as lean (BMI: <25) and obese (BMI: >30). Subjects received for 7 d an individualized, formula-based isoenergetic diet that was based on resting metabolic rate obtained by indirect calorimetry (Deltatrac; Sensor Medics, Yorba Linda, CA) and multiplied by a physical activity factor of 1.7 for the lean and 1.6 for the obese subjects. The diet was recently described fully (14). A 24-h food recall was performed by a registered dietitian to estimate usual protein intake for exclusion of subjects whose intakes would differ quantitatively and qualitatively from our diet protocol of 1.8 g · kg fat-free mass (FFM)–1 · d–1. Nitrogen balance was measured during the last 3 d of the diet (17). Body composition was assessed by bioelectrical impedance analysis (RJL-101A; RJL Systems, Detroit, MI) by using equations validated for lean (18) and obese (19) subjects. With the use of these equations, the FFM values in each of our groups were close to those of published studies of protein kinetics in subjects who resembled ours with respect to BMI, body weight, or both (6, 7, 18).

Hyperinsulinemic, euglycemic, isoaminoacidemic clamp protocol
The hyperinsulinemic clamp was performed in the postabsorptive state as recently detailed (14), with plasma glucose at 5.5 mmol/L and at each subject’s own plasma branched-chain AA (BCAA) concentrations. Glucose turnover was studied by using a primed [22 µCi (814 kBq)] continuous infusion [0.22 µCi/min (8.14 kBq/min)] of [3-3H]glucose that was begun 180 min before insulin. Concurrently with the tritiated glucose infusion and after an oral bolus of 0.1 mg/kg NaH13CO3, leucine kinetics were studied by using a primed (0.5 mg/kg), constant (0.008 mg · kg–1 · min–1) infusion of [13C]leucine (20). A primed infusion of biosynthetic regular human insulin (Humulin R; Eli Lilly Canada Inc, Toronto, Canada) was started at 0 min and maintained at a rate of 40 mU · m–2 · min–1 for ≥210 min. At 4 min, sterile 20% (wt:vol) potato starch–derived glucose (AVEBE BA, Foxhol, Netherlands) in water with added [3-3H]glucose was infused [ie, the "hot GINF" method (21)] at variable rates to achieve euglycemia based on measurements every 5 min. We have verified by isotope ratio–mass spectrometry that this glucose has negligible 13C content, whereas commercial dextrose solutions have corn-derived glucose with 13C enrichment sufficiently high that it would have to be quantified and adjusted for in calculations of leucine kinetics. Plasma BCAAs were kept constant with a variable infusion of a 10% AA mixture (10% TrophAmine without electrolytes; B Braun Medical Inc, Irvine, CA) based on measurements of BCAA every 5 min. This approach maintains most individual AAa within the normal postabsorptive range in healthy subjects (14).

Blood samples were collected for analyses at baseline and every 10 min for 40 min before the insulin infusion and then every 30 min until the last 40 min, at which time they were again drawn at 10-min intervals. Indirect calorimetry was performed for 20 min before the insulin infusion and during the last 30 min of the infusion (22). Glucose turnover was calculated as specified by Saad et al (23), and substrate oxidation was calculated as specified by Bogardus et al (22). Expired air samples were collected and then transferred to evacuated tubes (Vacutainer; Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Leucine kinetics were calculated according to Matthews et al (20) by using plasma {alpha}-keto isocaproic acid ({alpha}-KIC) as an index of the precursor pool enrichment (reciprocal model). Protein kinetic data are presented as units per minute, units per kg body weight, and units per kg FFM. The latter measurement represents the denominator most closely related to the tissues responsible for most of the protein turnover. Data are presented as mean values for the baseline period (at 2 points) before insulin and for the steady state of the hyperinsulinemic period (at 4 points) for all variables except free fatty acids (FFAs).

Assays
Plasma glucose was measured by the glucose oxidase method (GM7 Micro-Stat; Analox Instruments USA, Lunenberg, MA). Assays have been detailed previously for immunoreactive insulin and glucagon (24) and glucose specific activity (21, 24). Plasma total BCAA concentrations were measured by using an enzymatic fluorometric assay (14). Individual plasma AA concentrations were measured by using ion-exchange HPLC with postcolumn ninhydrin detection (25). FFAs were measured by using an enzymatic colorimetric method (NEFA C test kit; Wako Chemicals USA Inc, Richmond, VA). The 13C enrichment of plasma {alpha}-KIC, reduced to hydroxyisocaproate by NaBH4, was analyzed by gas chromatography–mass spectrometry (GCMS 5988A; Hewlett-Packard, Palo Alto, CA) after derivatization with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (tBDMS; Regis Technologies Inc, Morton Grove, IL) to yield a tBDMS derivative of hydroxyisocaproate. Expired air was analyzed for 13CO2 enrichment by isotope ratio–mass spectrometry on a Micromass 903D (Vacuum Generators, Winsforce, United Kingdom).

Validation studies of background enrichment of expired 13CO2 and plasma [13C]KIC, and of recovery of 13C bicarbonate
We reported a 10.1 ± 1.6% dilution in the background enrichment of expired 13CO2 in lean young men, mainly due to the infusion of the potato starch–derived, low-13C glucose (14). Therefore, this effect was sought in representative lean and obese women, to test for sex or obesity differences, which required the performance of identical clamp studies without tracer infusions. The mean age, BMI, percentage body fat (%BF), and glucose infusion rates in the representative women did not differ significantly from those in their respective whole groups. In the lean women (n = 4), the percentage of dilution was the same as that in men, so leucine oxidation rates were corrected by using the same 10.1% factor. In the obese women (n = 4), 7.0 ± 1.7% dilution was found and used in the oxidation rate calculations for this group. Because plasma [13C]{alpha}-KIC is a key endpoint, we verified its enrichment in these control experiments as well. No dilution in plasma [13C]{alpha}-KIC was found in either lean or obese subjects (data not shown). The recovery of 13C from the bicarbonate pool was also assessed in 2 lean and 4 obese women and compared with that in 6 lean men (14). This comparison involved the continuous infusion of 13C sodium bicarbonate without other tracers throughout the clamp experiment (26). Recovery factors did not differ significantly between men and women or between lean and obese subjects (data not shown). Factors of 0.671 (postabsorptive) and 0.799 (clamp) were thus used for the calculation of leucine oxidation in both groups of women.

Statistical analysis
Results are presented as means ± SEMs. Different variables, including leucine kinetics, were analyzed after normalization per kg FFM by repeated-measures analysis of variance, to identify time (baseline or clamp) and group (lean or obese) effects, as well as possible interactions indicating a different response to the hyperinsulinemic clamp between lean and obese women. Leucine kinetics were also analyzed with age as a covariate. For certain variables, differences between groups at a given time were assessed by using an unpaired t test. Pearson’s coefficient was used for all correlations. Stepwise linear regression analysis was used to test the possible variables predicting the anabolic response to hyperinsulinemia. The analyses were performed with SPSS for WINDOWS software (version 10.0; SPSS Inc, Chicago, IL). Significance was defined as P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The obese women were significantly older and had significantly higher FFM than did the lean women (Table 1Go). They also differed significantly in indexes of adiposity: greater body weight, BMI (by design), %BF, waist and hip circumferences, and waist-hip ratio. They had significantly higher total energy intake but not significantly higher intake per kg FFM. Protein intake was controlled per kg FFM and thus did not differ significantly between groups. Nitrogen balance was slightly positive only in lean women but did not differ between groups. Fasting plasma glucose was normal in both groups (Table 2Go) but was higher 2 h after an oral-glucose-tolerance test (OGTT) in the obese women. Four obese women had impaired glucose tolerance (IGT) (ie, 2 h value > 7.8 mmol/L). Serum total cholesterol and triacylglycerol were significantly higher and HDL cholesterol was significantly lower in the obese women than in the lean women, but none of the values was outside the reference range.


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  		TABLE 1. Subject characteristics1

 

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  		TABLE 2. Metabolic responses of the lean and obese women to the hyperinsulinemic, euglycemic, isoaminoacidemic clamp1

 
Hormonal and metabolic data are reported in Table 2Go and Figure 1Go. Plasma total BCAA determined by HPLC were significantly higher in the obese women at baseline and during the clamp (Table 2Go). There was no change in BCAAs between basal and clamp periods. Obese women required significantly less exogenous AA per kg FFM (Figure 1AGo, Table 2Go) to maintain BCAA concentrations than did the lean women. Glucose infusion rates to maintain euglycemia per kg FFM (Figure 1BGo, Table 2Go) were significantly lower in the obese women than in the lean women. When the data for all of the subjects were combined, a significant positive correlation was found between rates of glucose and AA infusion per FFM (Figure 2Go). In the obese women, the rate of appearance (Ra) of glucose per kg FFM was not completely suppressed during hyperinsulinemia, as it was in the lean women. The rate of disappearance (ie, utilization) (Rd) of glucose per kg FFM was also significantly lower in the obese women than in the lean women and was increased in both groups during the clamp, but less so in the obese women, a finding that was concurrent with the lower glucose infusion rates. Among the obese women, glucose Rd was significantly less in the women with IGT than in the women with normal glucose tolerance (5.5 ± 0.2 and 6.3 ± 0.2 mg · kg FFM–1 · min–1, respectively; P = 0.017). Both postabsorptive and clamp plasma insulin concentrations were significantly higher in the obese women than in the lean women, and there was a greater increment in the obese than in the lean (627 ± 48 and 504 ± 25 pmol/L, respectively). Therefore, the obese subjects were exposed to endogenous fasting insulin that was twice that in the lean women and to a 33% higher steady state insulin concentration than were the lean women; these differences represented a 24% greater increment in data reported during the clamp in the obese women than in the lean women. Plasma glucagon did not differ significantly between the 2 groups and decreased to the same extent during the clamp. Therefore, the ratio of glucagon to insulin was significantly lower in the obese women than in the lean women and decreased in the obese women to a level similar to that in the lean women during the clamp. Postabsorptive FFA concentrations did not differ significantly (Figure 3Go) between the groups, but maximal suppression required 30 min in the lean women compared with 60 min in the obese women. From 30 to 180 min after the start of insulin, FFA remained higher in the obese women.



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  		FIGURE 1.. (A) Amino acid (AA) infusion rates and plasma branched-chain AA (BCAA) concentrations during the hyperinsulinemic clamp in lean and obese women. Plasma BCAA concentrations were significantly different between the groups at baseline and during the steady state period (at the end of the infusion, to the right of the interruption of each line), P < 0.01. AA infusion rates were significantly different between the groups during the steady state period, P < 0.0001. (B) Glucose infusion rates and plasma glucose concentrations during the hyperinsulinemic clamp in lean and obese women. Glucose infusion rates were significantly different between the groups during the steady state period, P < 0.0001.

 


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  		FIGURE 2.. Correlation between amino acid (AA) and glucose infusion rates during the hyperinsulinemic clamp. Pearson’s coefficient: r = 0.635; P = 0.005. FFM, fat-free mass (in kg).

 


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  		FIGURE 3.. Serum free fatty acid (FFA) concentrations before and during insulin infusion in lean and obese women. Time and group effects and time x group interactions were assessed by repeated-measures ANOVA. There was a significant time effect (P < 0.001) and a significant time x group interaction for maximal suppression (P = 0.030). {dagger}Significantly different from lean women, P < 0.05 (unpaired t test at each time point).

 
Whereas oxygen consumption (O2) did not change during the clamp, carbon dioxide production increased significantly in both groups (Table 3Go). These 2 variables were higher in obese than in lean women except when adjusted for differences in FFM (not shown). Nonprotein respiratory quotient increased significantly in both groups during hyperinsulinemia and was significantly lower in the obese women than in the lean women. Resting energy expenditure was significantly higher in the obese women than in the lean women except when adjusted for FFM, and it increased significantly in all of the women during the clamp. The obese women oxidized significantly more lipids than did the lean women. During the clamp, lipid oxidation represented 21.6 ± 4.7% of the total nonprotein oxidation in the obese women and 7.5 ± 2.1% in the lean women (P = 0.018); as a percentage of energy expenditure, it was 27.0 ± 4.5% compared with 11.4 ± 3.0%, respectively (P = 0.012). Clamp rates of lipid oxidation correlated with serum FFA concentrations (r = 0.609, P = 0.006). In both postabsorptive and hyperinsulinemic states, rates of lipid oxidation correlated positively with leucine oxidation (r = 0.520, P = 0.022) and negatively with glucose oxidation (r = –0.689, P = 0.001).


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  		TABLE 3. Nonprotein substrate utilization in lean and obese women at baseline and during the hyperinsulinemic clamp1

 
Four individual plasma AA values (ie, isoleucine, leucine, ornithine, and glutamate) were significantly higher in obese than in lean women, whereas serine was lower at baseline (Table 4Go). In both lean and obese subjects, not only were total BCAAs (Figure 1AGo, Table 2Go) clamped at baseline concentrations, but so were total AAs. These findings were the result of the small, oppositely directed changes listed being equivalent when totaled. Although the significant percentage changes varied from {approx}5% to 31% (highest in those with low concentrations), it is noteworthy that none of the clamped values was outside the range of reported reference postabsorptive concentrations (14). There were significant time x group interactions for 4 AAs: serine decreased in the lean women only, alanine increased in the lean women and decreased in the obese women, and threonine and asparagine decreased less in the obese women than in the lean women. It is notable, because [13C]leucine was the tracer employed, that the mean increments were small—11 µmol/L in both groups, an amount that represented changes of 9% in the lean women and 10% in the obese women. Quantitatively, glutamine changed the most, decreasing 58 µmol/L in both, but it was not present in the infusate.


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  		TABLE 4. Individual plasma amino acid (AA) concentrations at baseline and during the hyperinsulinemic clamp1

 
Although lean tissues are the site of most protein turnover, because our estimation of FFM includes many individual tissues with different rates and depends on regression equations that were developed by using different reference populations and methods, we present data as rates per unit of time, per kg body weight/min, and per kg FFM/min (Table 5Go). In the baseline postabsorptive state, total Ra (total flux and endogenous breakdown) and nonoxidative Rd (synthesis) were significantly greater per min and per kg FFM but significantly less per kg body weight in the obese women (Table 1Go). Oxidation was significantly greater but net balance was more negative per min and per kg FFM in the obese women than in the lean women. Because age correlated with all postabsorptive and clamp leucine kinetics it was introduced as a covariate in the analysis of variance, which resulted in the disappearance of the difference in postabsorptive oxidation and net balance between the groups. However, when we tested these correlations by control for indexes of adiposity (ie, BMI and waist and hip circumferences), they were no longer significant, and age did not correlate with responses in leucine kinetics, which indicates that, in our cohort, the effects of age, when seen, were due to higher adiposity. Thus age was not introduced as a covariate.


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  		TABLE 5. Leucine kinetics in lean and obese women at baseline and during the hyperinsulinemic clamp1

 
During the hyperinsulinemic clamp, rates of total Ra and nonoxidative Rd increased from baseline and were significantly greater per min but less per body weight in the obese women than in the lean women; per kg FFM, they did not differ between groups. Oxidation also increased and was significantly greater per min and per kg FFM in the obese women than in the lean women, but there was no difference between the groups in measurements per kg body weight. Endogenous Ra decreased in both groups; however, the rates remained significantly higher per min and per kg FFM but significantly lower per kg body weight in the obese women than in the lean women. Most important, net balance became positive in the lean women but not in the obese women, and therefore it was significantly lower by all denominators. The effects of the clamp, when expressed as change from baseline, were as follows: the increases in nonoxidative Rd and net balance irrespective of denominator were significantly less in the obese women than in the lean women, as were those of total Ra per body weight and FFM (indicated by a significant time x group interaction). Expressed as the percentage of change (Figure 4AGo), which removed the effect of choice of denominator, increases in total Ra, nonoxidative Rd, and net balance were significantly less in the obese women than in the lean women. The results show that endogenous Ra (catabolic) responses to hyperinsulinemia were not proportionally less, although absolute rates were higher, in the obese women than in the lean women (Table 5Go). Although the change in plasma insulin differed significantly between the 2 groups, it did not correlate with the anabolic response to insulin and consequently was excluded as a covariate in the analysis of covariance.



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  		FIGURE 4.. (A) Changes in leucine kinetics and net balance during the hyperperinsulinemic clamp in lean and obese women. Ra, rate of appearance; Rd, rate of disappearance. {dagger},{ddagger}Significantly different from the lean group (unpaired t test): {dagger}P < 0.05, {ddagger}P < 0.001. The difference in the percentage change between the groups was also significant, P < 0.05 (analysis of covariance with fat-free mass as a covariate). The anabolic response was not correlated with the change in plasma insulin, and, therefore, it was not included as a covariate in the analysis of covariance model. (B) Correlation between the anabolic response (percentage change in net leucine balance) to the hyperinsulinemic clamp and BMI (in kg/m2). Pearson’s coefficient: r = –0.839; P < 0.001.

 
With all subjects combined, the percentage change in net leucine balance correlated negatively with all indexes reflecting adiposity: weight, BMI, fat mass, %BF, waist and hip circumferences, and waist-hip ratio (r from –0.582 to –0.849, P < 0.01). In a stepwise linear regression model, BMI was the single independent variable predicting the percentage change in anabolic response to hyperinsulinemia; the effect was a lesser response with higher BMI (R2 = 0.704, F value = 40.0, P < 0.001) (Figure 4BGo). Only BMI correlated significantly with the absolute change in net leucine balance when controlled for FFM (r = –0.535, P = 0.022). Glucose disposal rates correlated with the percentage changes in nonoxidative leucine Rd (r = 0.655, P = 0.002) and net leucine balance (r = 0.687, P = 0.001). Results of OGTT (2 h glycemia) correlated positively with baseline flux and oxidation and negatively with net balance in absolute rates and per kg FFM (r from 0.485 to 0.533, P < 0.05) and positively with clamp flux and endogenous Ra in absolute rates and per kg FFM (r from 0.477 to 0.533, P < 0.05). Similarly, OGTT (area under the curve) correlated positively with baseline flux and oxidation and negatively with net balance per kg FFM and with clamp endogenous Ra per kg FFM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our clamp protocol showed that, in obese women, the whole-body protein anabolic response is resistant to the action of insulin, that this resistance occurred along with that of glucose and FFA, and that it is associated with adiposity. This blunted anabolic response results mainly from impaired stimulation of protein synthesis, whereas suppression of protein breakdown in obese women is not significantly different from that in lean women. These interpretations are based principally on the data per kg FFM, the compartment where most protein turnover occurs and that includes the tissues that are most responsive to insulin. Furthermore, postabsorptive turnover was highly correlated with FFM with an intercept that was not different from zero (27). Other studies of protein turnover in obesity during hyperinsulinemia have not compensated for the hypoaminoacidemia due to the suppression of breakdown (4, 6, 8, 9). The fact that we obtained comparable inhibition of breakdown, as in these studies, further supports the hypothesis that, without exogenous AAs, insulin could not stimulate protein synthesis because of limited substrate availability.

Hypoaminoacidemia decreases the initiation of protein synthesis via decreased activity of eukaryotic initiation factor 2B (28). The stimulation of protein synthesis by AAs in vivo requires substantial increases in plasma AA (leucine: >200%) by infusion (8, 11, 29) or by oral ingestion equivalent to a meal (13, 30, 31), ie, a "fed state." Although we infused a total of 6 g protein in both groups, with 70 g glucose in the lean and 60 g glucose in the obese, plasma AA changes were trivial (leucine ≤10%), and O2 was constant, which only partially mimicked the fed state. Whereas much is known about insulin stimulation of protein synthesis, studies that isolate its effects from those of AAs are difficult to design (13, 32, 33). We produced a state in which, with minimal changes in AA concentrations, an effect on synthesis that was almost exclusively attributable to insulin could be unmasked.

Obesity is associated with higher postabsorptive plasma AAs, especially BCAAs, phenylalanine, and tyrosine, in some (6, 34) but not all (8, 35) studies. Decreases in these AAs (36, 37), which are thought to reflect protein breakdown suppression, have been used as an index of the resistance of whole-body protein response to the action of insulin in obese persons (38, 39) and in patients with type 2 diabetes (40). However, AA concentrations reflect not only protein breakdown but also the balance among protein breakdown, synthesis, and AA oxidation. Moreover, BCAAs are closely related to FFM (r = 0.630 and P = 0.004 in the present study), which is greater in obesity.

Isotopic studies to define insulin resistance of protein by its failure to suppress protein breakdown (Ra) have reached different conclusions. Significantly less Ra suppression was reported in upper- than in lower-body obesity (2) and at low (8) but not high (6, 8, 9, 40; also in the current study) insulin infusion rates. This suggests that the insulin dose-response of Ra suppression is shifted to the right but that the maximum effect is not altered. Alternatively, by the infusion of sufficient AAs to maintain plasma concentrations without inhibiting insulin action on glucose metabolism (41–44), enough substrate may have been available to contribute to catabolism suppression, which prevented the unmasking of an effect of obesity on catabolism (45). Our finding of significantly higher insulin concentrations in obese than in lean subjects was also found in numerous hyperinsulinemic clamp studies (2, 4, 6, 9). That we nevertheless found insulin resistance of protein synthesis and net leucine balance made this an even more robust observation.

We propose that clamp AA infusion rates may be an index of the sensitivity of whole-body protein anabolism to the action of insulin. As in the euglycemic clamp, rates of substrate infusion are determined by increased uptake, increased oxidation, decreased endogenous release, or all 3. Whereas glucose production was fully suppressed in the lean women and submaximally suppressed in the obese women, endogenous protein breakdown was not completely blocked. Therefore, the rate of AA infusion required resulted from partial endogenous Ra suppression plus stimulation of (oxidative plus nonoxidative) disposal. Because the 2 groups had similar breakdown suppression and increase in leucine oxidation, the lower AA infusion rates must be attributable to the lesser stimulation of synthesis in the obese. The time required to reach maximum AA infusion rates was significantly longer in the obese women than in the lean women (130 and 90 min, respectively; Figure 1AGo). This difference probably also reflects a resistance to activation of the pathways involved. A longer time was also required for suppressing FFAs in the obese women than in the lean women (Figure 3Go). FFA availability is reported to influence whole-body protein metabolism (46). We observed less suppression of lipid oxidation in the obese than in the lean subjects (27% and 11% of energy production, respectively). This greater availability of lipid had no effect on leucine oxidation in the obese (9). Their higher lipid oxidation was "glucose sparing"—48% of energy production compared with 66% in the lean women—but is probably due to muscle insulin resistance of glucose metabolism (47). It is of interest that resting energy expenditure increased only in the lean in whom the increase in synthesis was greater. This could be due to the difference in energy cost of protein synthesis (48).

The obese women in the current study showed typical anthropometric (Table 1Go) and metabolic (Tables 1Go-4Go) features of obesity. We clamped BCAAs at each person’s postabsorptive values because of expected higher concentrations in the obese women than in the lean women (6) and also to avoid a bias toward low AA infusion rates by clamping the BCAAs at lower concentrations. That the obese subjects still required less AA to maintain higher plasma BCAAs than did the lean subjects supports even more convincingly the fact that the whole-body protein anabolic response is resistant to the action of insulin. That the method used is robust is supported by reproducibility in the validation studies. Intraindividual variation (n = 5) did not differ significantly from 0: P = 0.419 and 0.711 for AA and glucose infusion, respectively. Furthermore, we tested for an effect of the absence of 13C enrichment in our glucose infusate on leucine oxidation, and we verified appropriate bicarbonate recovery factors. The calculations based on these control experiments thus minimize any possible bias from these variables.

All women were selected on the basis of comparable usual protein intakes before admission, and diet was controlled to ensure nitrogen (N) equilibrium. N balance did not differ significantly between the lean and the obese subjects, although it tended to be more negative in the subgroup of obese women with IGT. It correlated with postaborptive net leucine balance in the whole group, but, when the 4 women in the subgroup were removed from the calculations, N balance no longer correlated with net leucine balance. There were no significant correlations between N balance during the week before the study and leucine kinetic responses to the clamp, which strongly suggests that the different responses between groups were due to differences in body composition and could not be influenced by the slight positive N balance in some.

The reduction by half in the anabolic response to the even greater degree of hyperinsulinemia reinforces the possibility that postprandial insulin secretion is elevated in obesity, not only to maintain glucose homeostasis but also to compensate for the resistance of protein (and lipid) metabolism. This blunted anabolic response appears to be aggravated by IGT. Indeed, we found a positive correlation between OGTT 2-h glucose or area under the curve and postabsorptive leucine flux and oxidation and a negative correlation with net leucine balance. The parallel between the resistance of glucose and that of protein was further supported by a correlation between clamp glucose Rd and the percentage change in net leucine balance and in synthesis. These findings suggest that the continuum of insulin insensitivity of glucose in progressing from glucose tolerance to intolerance and to diabetes also applies to protein.

We (49) and others (50) showed that a greater absolute and percentage of endogenous glucose production are due to gluconeogenesis in obesity. Our work showed a correlation between indexes of increased protein turnover and gluconeogenesis (49), which implicates the liver as well as muscle in protein insensitivity to insulin. The obligate postabsorptive hypersecretion of insulin is accompanied by augmented postprandial responses, not only for glucose and lipid homeostasis but also to promote anabolism from dietary amino acids. Because this hypersecretion of insulin can be maintained only until the beta cell is no longer able to compensate, when diabetes occurs, in both fasting and postprandial states, protein turnover could become sufficiently impaired to lead to a loss of FFM (51). Further studies are needed in subjects with IGT and with type 2 diabetes to quantify these effects.


   ACKNOWLEDGMENTS
 
We acknowledge the assistance of Mary Shingler, Josie Plescia, Madeleine Giroux, Ginette Sabourin, Concettina Nardolillo, and Paul Meillon and statistical consultation with James Hanley (Division of Clinical Epidemiology, McGill University Health Centre).

All authors contributed to the study design, data collection, analyses and interpretation. RG and SC wrote the manuscript, and all authors read and approved it. None of the authors had financial or personal conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication December 1, 2004. Accepted for publication March 31, 2005.




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