The effect of obesity on regional skeletal muscle and adipose tissue amino acid metabolism is not known. We evaluated systemic and regional (forearm and abdominal subcutaneous adipose tissue) amino acid metabolism, by use of a combination of stable isotope tracer and arteriovenous balance methods, in five lean women [body mass index (BMI) <25 kg/m2] and five women with abdominal obesity (BMI 35.0–39.9 kg/m2; waist circumference >100 cm) who were matched on fat-free mass (FFM). All subjects were studied at 22 h of fasting to ensure that the subjects were in net protein breakdown during this early phase of starvation. Leucine rate of appearance in plasma (an index of whole body proteolysis), expressed per unit of FFM, was not significantly different between lean and obese groups (2.05 ± 0.18 and 2.34 ± 0.04 μmol · kg FFM−1 · min−1, respectively). However, the rate of leucine release from forearm and adipose tissues in obese women (24.0 ± 4.8 and 16.6 ± 6.5 nmol · 100 g−1 · min−1, respectively) was lower than in lean women (66.8 ± 10.6 and 38.6 ± 7.0 nmol · 100 g−1 · min−1, respectively;P < 0.05). Approximately 5–10% of total whole body leucine release into plasma was derived from adipose tissue in lean and obese women. The results of this study demonstrate that the rate of release of amino acids per unit of forearm and adipose tissue at 22 h of fasting is lower in women with abdominal obesity than in lean women, which may help obese women decrease body protein losses during fasting. In addition, adipose tissue is a quantitatively important site for proteolysis in both lean and obese subjects.
- stable isotope tracers
- protein metabolism
obesity is associated with altered hormone production, lipolytic activity, and glucose metabolism in adipose tissue (17, 20). However, little is known about the effect of obesity on adipose tissue amino acid metabolism. The relationship between adiposity and amino acid metabolism may have important physiological and clinical implications. For example, differences in protein metabolism between lean and obese subjects are probably responsible for conserving muscle protein and improving survival during fasting in obese persons (6).
The effect of obesity on whole body amino acid kinetics is not clear because of conflicting data from different studies, which have reported that postabsorptive whole body protein breakdown is either increased (15, 16, 24, 36) or the same (22, 25) in obese compared with lean subjects. These studies measured leucine rate of appearance (Ra) into the systemic circulation, as an index of whole body protein breakdown, and did not investigate individual organ- or tissue-specific amino acid metabolism. By use of arteriovenous balance techniques, studies conducted in rats (18,19) and in humans (3, 8) have shown that adipose tissue makes an important contribution to systemic amino acid release during postabsorptive conditions (8, 19). However, the relative importance of muscle and adipose tissues to whole body amino acid kinetics and the effect of obesity on regional amino acid metabolism are not known.
The present study was performed to examine whole body and regional (forearm and adipose tissue) amino acid metabolism in lean and obese human subjects. Subjects were studied at 22 h of fasting to ensure the presence of net protein breakdown during this early phase of starvation. We hypothesized that obesity is associated with a decreased rate of amino acid release (protein breakdown) from skeletal muscle and adipose tissue, which could help explain the greater conservation of body protein observed during fasting in obese than in lean persons (7). A combination of stable isotope tracer infusion and arteriovenous balance techniques, which involved forearm and adipose tissue blood flow measurements and radial artery, deep forearm vein, and abdominal vein blood samples, was used to quantify whole body and regional amino acid kinetics.
Five lean women [body mass index (BMI) <25 kg/m2] and five women with class II abdominal obesity (BMI 35.0–39.9 kg/m2; waist circumference >100 cm) participated in this study. These subjects also participated in studies evaluating the effect of fasting on lipid and glucose metabolism that were reported previously (12, 13). Lean and obese women were matched on fat-free mass (FFM). Although there was a trend [nonsignificant (NS)] for the obese subjects to be older than the lean subjects (37 ± 4 and 29 ± 3 yr, respectively), all subjects were premenopausal and were studied within the first 2 wk of the follicular phase of their menstrual cycle. After completing a comprehensive medical evaluation including an oral glucose tolerance test, all subjects were considered to be in good health except for the presence of obesity. All subjects were weight stable for ≥2 mo before the study, and none had been involved in any regular exercise program for ≥6 mo before the study. No subjects were taking any medications. This study was approved by the Human Studies Committee and the General Clinical Research Center at Washington University School of Medicine, and all subjects gave informed consent before their participation.
Body composition analysis.
Body fat and FFM values were assessed by dual-energy X-ray absorptiometry (DEXA; Hologic QDR 1,000/W, Waltham, MA) in all subjects as outpatients within 3 days of the isotope infusion study. Windows were set on the forearms to estimate fat and lean tissue forearm content.
Subjects were admitted to the General Clinical Research Center at Washington University School of Medicine in the evening before the study. At 1800, they ingested a meal consisting of 12 kcal/kg body wt for lean subjects and 12 kcal/kg adjusted body weight for obese subjects [adjusted body weight = ideal body weight + (actual body weight − ideal body weight) · 0.25]. Carbohydrate, fat, and protein represented 55, 30, and 15%, respectively, of total energy intake. At exactly 2000, all subjects ingested a snack containing 40 g carbohydrate, 6.1 g fat, and 8.8 g protein (Ensure; Abbott Laboratories, Columbus, OH). The subjects then remained fasted until completion of the study on the following day.
On the following morning, catheters were placed in a forearm vein for isotope infusion and in a contralateral deep forearm vein, a superficial abdominal vein, and a radial artery for blood sampling. The abdominal vein catheter was positioned so that the tip was just inferior to the inguinal ligament to help ensure that blood draining subcutaneous abdominal adipose tissue would be sampled (7). The deep forearm vein catheter was inserted in a retrograde fashion, usually in the medial cubital vein (1, 3,34), to sample blood draining primarily the forearm muscle tissue. After all catheters were inserted, an arterial blood sample was withdrawn to determine background [2H3]leucine enrichment. The subjects remained in bed for the duration of the study, and the catheters were kept patent by intravenous infusion of normal saline. After 19 h of fasting, a primed-constant infusion ofl-5,5,5-[2H3]leucine (Cambridge Isotope Laboratories, Andover, MA) was initiated (4.2 μmol/kg priming dose; 0.07 μmol · kg−1 · min−1continuous infusion) and continued for 180 min by use of a calibrated syringe pump (Harvard Apparatus, South Natick, MA). Blood samples were obtained simultaneously from the artery, abdominal vein, and deep forearm vein at 165, 170, 175, and 180 min (from 21.45 to 22 h of fasting). Blood samples were collected in prechilled tubes containing EDTA and were centrifuged immediately. Plasma was stored at −70°C until analysis.
Blood flow measurements.
Abdominal adipose tissue blood flow was measured by the xenon washout technique (21). Approximately 120 μCi of133Xe, dissolved in normal saline, was injected into subcutaneous adipose tissue, ∼3 cm lateral to the umbilicus (21). A cesium iodide detector (Oakfield instruments, Eynsham, UK) was placed directly over the site of injection (28) to measure radioactive decay in adipose tissue over a 15-min period.
Forearm blood flow was measured by venous occlusion plethysmography (37) while subjects remained supine with their arms extended at heart level. Changes in forearm circumference in response to occlusion of forearm venous drainage were measured as a change in voltage with a mercury-in-Silastic strain gauge (Hokanson, Bellevue, WA). To eliminate the influence of venous return from the hand, blood flow from the hand was occluded for 2 min before measurement by increasing the pressure in a wrist cuff to 200 mmHg. Five blood flow measurements were made every 5 min between 2145 and 22 h of fasting. The strain gauge was electrically calibrated (11) before and after each set of five blood flow measurements. The average of the 15 blood flow measurements over a 15-min period was used to represent forearm blood flow.
Plasma amino acid concentrations were determined by using a fluorometric HPLC method involving derivatization witho-phthaldialdehyde, as previously described (39). Amino acids in samples were quantified on the basis of known amounts of authentic standards with a Waters model 810 baseline work station (Waters, Milford, MA).
The tracer-to-tracee ratio (TTR) of plasma leucine was measured by electron impact ionization gas chromatography-mass spectrometry of thetert-butyldimethylsilyl derivative after isolation of leucine from deproteinized plasma (27). The measured instrument response (m+3/m+0 isotopomer area ratio) was calibrated against the measured ratio for [2H3]leucine standards of known isotopic TTR.
Local net amino acid arteriovenous differences were calculated as the amino acid concentration in arterial plasma minus the concentration in venous plasma. Local net fluxes were calculated as the arteriovenous difference multiplied by local plasma flow.
Whole body plasma leucine Ra was determined from the steady-state isotope dilution equation Ra = I/TTRA (29), where I is the tracer infusion rate (in μmol · kg−1 min−1) and TTRA is the TTR of [2H3]leucine in the arterial sample. The endogenous production rate (P, in nmol · min−1 · 100 ml volume of tissue−1) of leucine within the forearm and adipose tissues was estimated from the tracer balance equation (38) where F is the tissue plasma flow rate (in ml · min−1 · 100 ml volume tissue−1), [A] is the arterial plasma leucine concentration (in nmol/ml), and TTRV is the TTR of [2H3]leucine in deep forearm venous and abdominal venous plasma samples. This calculation quantifies the release of unlabeled leucine from tissue into plasma, but it underestimates the total intracellular leucine release rate, because intracellular leucine TTR was not measured (38).
Several assumptions were made to estimate the contribution of whole body muscle mass (WBMM) and whole body fat mass (WBFM) to whole body leucine Ra: 1) 50% of whole body FFM was comprised of muscle tissue (5), 2) the density of muscle was 1.10 g/ml (2), 3) deep forearm muscle tissue was representative of WBMM and subcutaneous abdominal fat was representative of WBFM, 4) skin and bone made only minor contributions to forearm amino acid kinetics (33), and5) the proportion of deep forearm that was comprised of lean tissue was the same as that measured in whole forearm by DEXA.
Adipose tissue blood flow was calculated as the product of the slope of radioactive decay of 133Xe and the partition coefficient for 133Xe between adipose tissue and blood (21). The partition coefficient used in the present study was 10 g/ml, as described by Yeh and Peterson (40).
The significance of differences between lean and obese groups was evaluated by a two-tailed Student's t-test for independent samples. The statistical significance of arteriovenous amino acid concentration differences was determined by Wilcoxon's test, because the data were not normally distributed. A probability value ofP < 0.05 was considered to be statistically significant. Results are presented as means ± SE.
Body composition characteristics of the study subjects are listed in Table 1. Total fat mass was more than threefold greater in obese than in lean subjects, but FFM was similar in both groups. At 22 h of fasting, arterial plasma insulin concentrations were higher in obese than in lean subjects (11.2 ± 0.6 and 4.5 ± 0.7 μU/ml, respectively; P ≤ 0.001), but arterial plasma glucagon concentrations were the same in both groups (78.4 ±7.9 and 78.3 ±8.3 ng/ml). Plasma flow rate was slower in obese than in lean subjects, both in forearm (0.7 ±0.1 and 1.7 ±0.2 ml · 100 ml−1 · min−1, respectively) and in adipose (1.4 ± 0.2 and 3.1 ± 0.6 ml · 100 g−1 · min−1, respectively) tissues (both P < 0.05).
Arterial plasma leucine concentrations were 135 ± 8 μM in lean and 124 ± 4 μM in obese subjects (NS). Systemic leucine Ra, expressed per kilogram FFM, tended to be greater in obese than in lean subjects, but the difference was not statistically significant (Table 2). However, leucine Ra, expressed per kilogram body weight, was >30% lower in obese than in lean subjects (P < 0.05; Table 2).
Leucine release from both deep forearm and subcutaneous abdominal tissues was significantly lower in obese than in lean subjects (bothP ≤ 0.05; Table 2). The local production rate of leucine per 100 ml of tissue was significantly higher in deep forearm tissue than in subcutaneous abdominal tissue for both groups (P < 0.05). The contribution of WBMM to whole body leucine Ra was less in obese than in lean subjects (Table2). The contribution of WBFM to whole body leucine Ra was similar in lean and obese subjects and accounted for 5–10% of systemic leucine release in both groups (Table 2).
Regional amino acid net balance.
Arterial plasma amino acid concentrations and net arteriovenous differences and fluxes of amino acids across subcutaneous abdominal adipose tissues are shown in Table 3. Data obtained from lean and obese subjects were similar, so values from both groups were pooled. In general, net arteriovenous differences across adipose tissue were small except for alanine release, glutamate uptake, and glutamine release.
Although it is known that the regulation of lipid and carbohydrate metabolism is altered in persons with abdominal obesity, the effect of obesity on the regulation of protein metabolism is less clear. To our knowledge, this is the first report to detail regional (i.e., deep forearm muscle and abdominal subcutaneous adipose tissue) amino acid metabolism in obese subjects. A major finding of this study was that leucine release from both forearm and subcutaneous adipose tissue was reduced (per unit of tissue) in our obese compared with our lean subjects. Furthermore, our findings indicate that adipose tissue is a quantitatively important site for proteolysis in both lean and obese subjects; ∼5–10% of total whole body leucine Ra was derived from adipose tissue (Table 2). Muscle tissue tended to make a greater contribution to systemic leucine Ra in lean than in obese women, but the contribution of subcutaneous adipose tissue was similar in the two groups.
The arteriovenous difference data for the amino acids listed in Table 3showed only small differences across subcutaneous adipose tissue. One previous study of human adipose tissue (8), which measured net arteriovenous differences for some amino acids, showed that adipose tissue took up glutamate and released glutamine and alanine. A detailed in vivo study of rat adipose tissue showed release of several amino acids (19), but the arteriovenous differences tended to be small. Our measurements of net arteriovenous differences generally confirm those findings and demonstrate net fluxes for several amino acids for the first time in humans. However, the rate of adipose tissue amino acid uptake and release can be underestimated or missed by evaluating only arteriovenous concentration balance and blood flow, particularly when arteriovenous concentration differences are small, because active uptake and release can cancel each other out. Therefore, the true rate of release of amino acids from adipose tissue (local rate of appearance into plasma) requires the infusion of isotopically labeled amino acid tracers, which can detect unlabeled amino acids released from tissue into the local circulation by the isotope dilution principle (23, 35). The arteriovenous balance technique cannot determine which cell types within a tissue are responsible for a net metabolic action. For example, we cannot determine whether amino acid flux across adipose tissue occurred within adipocytes, stromal cells, vascular endothelia, or interstitial white blood cells.
Leucine Ra is often used as an index of whole body proteolysis (4, 15, 16, 22-25, 36). Leucine Ra values seen in this study are similar to those reported previously (31). Although our values are slightly lower than those of some reports, this is perhaps due to the sex of our volunteers (all female) and the fact that we measured leucine kinetics in plasma rather than in whole blood (33). It is less likely that leucine Ra was reduced because the subjects were being studied after fasting for 22 h. Leucine Ra, expressed per kilogram FFM, tended to be greater in our obese than in our lean subjects, which is consistent with several (15, 16, 24,36) but not all (22, 25) previous studies. However, in the present study, the differences between lean and obese groups did not achieve statistical significance, which may represent a type II statistical error because of small sample size.
Our data on deep forearm leucine release rate and body composition suggest that whole body muscle mass accounts for ∼35% of whole body leucine Ra in lean subjects. This value is in close agreement with previous attempts to extrapolate regional data to whole body muscle mass, even though others measured arteriovenous differences across the leg (30). Nonetheless, conclusions made by extrapolating data from a single muscle bed to whole body muscle mass should be treated with caution because of the possibility of regional heterogeneity between different muscles.
Extrapolating from the depots studied, we estimated that leucine release from adipose tissue accounted for 6% of whole body leucine Ra in our lean women. A higher value (12%) has been reported during postabsorptive conditions (a 12-h fast) in lean men on the basis of infusion of a phenylalanine tracer (3). The discrepancy between studies might reflect sex differences or duration of fasting (12 vs. 22 h). It is also possible that leucine release underestimated protein breakdown in adipose tissue. Leucine can be transaminated and oxidized in adipose tissue (9), thereby preventing its release into the abdominal venous blood, whereas phenylalanine is not metabolized in extrahepatic tissues (10).
The mechanism responsible for the lower rates of leucine release in forearm muscle and abdominal adipose tissues in obese than in lean subjects is not known. Although adipose tissue and forearm blood flows were lower in our obese than in our lean subjects, as has been shown previously (14, 17, 26), it is unlikely that differences in blood flow are responsible for differences in regional amino acid catabolism, because amino acid release from either forearm or adipose tissues should not be limited by blood flow. However, it is possible that the higher plasma insulin concentration observed in our obese than in our lean subjects may have contributed to the differences in proteolysis between groups, because insulin inhibits protein breakdown in lean (15, 22, 31-33) and obese (15,22) persons.
The reduced leucine release per kilogram of muscle is presumably responsible for the reduced contribution from whole body muscle to systemic leucine Ra, because our lean and obese subjects were matched on FFM. Although absolute leucine release rate per unit of adipose tissue was lower in our obese than in our lean subjects, the contribution of total body fat mass to systemic leucine Rawas greater in the obese group, because adipose tissue mass was much greater in obese than in lean subjects.
Leucine rate of appearance in plasma that was not derived from muscle or subcutaneous adipose tissue tended to be greater in our obese than in our lean subjects. This observation suggests that protein turnover at other sites (e.g., splanchnic bed and kidneys) (30) may be higher in obese than in lean persons. However, it is possible that protein metabolism is heterogeneous within tissues at different locations and that the individual muscle and fat depots that we evaluated in the present study were not representative of whole body muscle and fat masses. It is also possible that the depots studied represented whole body tissue in lean but not in obese subjects. Although deep forearm tissue may be a valid model for whole body muscle mass in lean subjects, its validity in obese subjects is more questionable because of possible “contamination” from an uncertain amount of forearm fat. We attempted to “correct” for this problem by assuming that the relative decrease in deep forearm muscle content in our obese subjects was the same as the relative decrease in total forearm lean tissue, as measured by DEXA.
In conclusion, the present study demonstrates that the rate of release of amino acids per unit of forearm and adipose tissue at 22 h of fasting is lower in abdominally obese than in lean women. Moreover, our data suggest that obesity is associated with a lower fractional contribution from skeletal muscle to systemic leucine Ra. These findings help explain why obese persons are more effective than lean persons in preserving body protein during fasting.
We thank the nursing staff of the General Clinical Research Center for help in performing the studies and the study subjects for participation.
This study was supported by National Institutes of Health Grants DK-37948, RR-00036 (General Clinical Research Center), RR-00954 (Biomedical Mass Spectrometry Resource), and DK-56341 (Clinical Nutrition Research Unit) and The Wellcome Trust.
Address for reprint requests and other correspondence: S. Klein, Washington Univ. School of Medicine, 660 S. Euclid Ave., Box 8031, St. Louis, MO 63110-1093.
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- Copyright © 2002 the American Physiological Society