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Reduction of Plasma Leptin Concentrations by Arginine but Not Lipid Infusion in Humans Harald Stingl, Wolfgang Raffesberg, Peter Nowotny, Werner Waldha¨usl, and Michael Roden

Abstract STINGL, HARALD, WOLFGANG RAFFESBERG, ¨ USL, AND PETER NOWOTNY, WERNER WALDHA MICHAEL RODEN. Reduction of plasma leptin concentrations by arginine but not lipid infusion in humans. Obes Res. 2002; 10:1111–1119. Objective: We examined short-term effects of arginine infusion on plasma leptin in diabetic and healthy subjects. Research Methods and Procedures: Arginine stimulation tests were performed in C-peptide negative type 1 [DM1; hemoglobin A1c; 7.3 ⫾ 0.3%], hyperinsulinemic type 2 diabetic (DM2; 7.6 ⫾ 0.7%), and nondiabetic subjects (CON; 5.4 ⫾ 0.1%). Results: Fasting plasma leptin correlated linearly with body mass index among all groups (r ⫽ 0.61, p ⫽ 0.001). During arginine infusion, peak plasma insulin was lower in DM1 than in DM2 (p ⬍ 0.05) and CON (p ⬍ 0.01). Plasma leptin decreased within 30 minutes by ⬃11% in DM1 (p ⬍ 0.001), DM2 (p ⬍ 0.01), and CON (p ⬍ 0.005), slowly returning to baseline thereafter. Plasma free fatty acids (FFAs) were higher in DM1 (0.6 ⫾ 0.1 mM) and DM2 (0.6 ⫾ 0.1 mM) than in CON (0.4 ⫾ 0.1 mM, p ⬍ 0.05) and transiently declined by ⬃50% (p ⬍ 0.05) at 45 minutes in all groups before rebounding toward baseline. To examine the direct effects of FFAs on plasma leptin, we infused healthy subjects with lipid/heparin and glycerol during fasting, and somatostatin-insulin (⬃35 pM) -glucagon (⬃90 ng/mL) clamps were performed. In both protocols, plasma leptin continuously declined by ⬃25% (p ⬍ 0.05) during 540 minutes without any difference between the high and low FFA conditions. Discussion: Arginine infusion transiently decreased plasma

Received for review December 31, 2001. Accepted for publication in final form July 30, 2002. Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna Medical School, Vienna, Austria. Address correspondence to Michael Roden, M.D., Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna Medical School, General Hospital of Vienna, Wa¨hringer Gu¨rtel 18 –20, A-1090 Vienna, Austria. E-mail: [email protected] Copyright © 2002 NAASO

leptin concentrations both in insulin-deficient and hyperinsulinemic diabetic patients, indicating a direct inhibitory effect of the amino acid but not of insulin or FFAs. Key words: diabetes, free fatty acids, insulin

Introduction In humans, plasma leptin concentrations positively correlate with body mass index (BMI) and several other indicators of body fat content (1,2). Obesity is associated with insulin resistance, hyperinsulinemia, and type 2 diabetes (3,4). Therefore, leptin could relate to changes in plasma insulin and metabolic parameters such as free fatty acids (FFAs) and amino acids (5). Plasma leptin concentrations corrected for BMI are lower in female type 1 and type 2 diabetic subjects than in healthy subjects, suggesting a defect in leptin production and/or secretion in human diabetes (6). Exogenous insulin administration increases plasma leptin concentrations in healthy lean, obese, and type 2 diabetic patients (7–10), although this effect is delayed by some hours and does not occur acutely. This may explain the divergent results of the relationship between plasma insulin and leptin in the literature (11–17). FFAs represent an important link between obesity, insulin resistance, and type 2 diabetes (3) and are involved in the regulation of insulin secretion (18 –20). However, the role of FFAs on leptin secretion in animals and humans is not clear at present, because no effect (21–23), inhibition (24), or even stimulation (25) is reported. Interpretation of some of these results is limited by the increase in insulin secretion typically occurring during lipid infusion (20). Amino acids such as L-arginine are important stimulators of insulin secretion and widely used to test ␤-cell function. Arginine infusion has been reported to lower plasma leptin concentrations in postmenopausal women (26), but not in healthy, non-obese children (27). At present, no data are available on the time course of plasma leptin concentration during arginine-induced insulin secretion in insulin resistant and/or diabetic subjects. This study examined whether plasma leptin concentrations are affected by infusion of arginine, which stimulates OBESITY RESEARCH Vol. 10 No. 11 November 2002

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Arginine Infusion Decreases Plasma Leptin, Stingl et al.

Table 1. Characteristics and fasting metabolic data in type 1 (DM1), type 2 (DM2), and nondiabetic (CON) subjects following a 12- to 14-hour fast Arginine infusion

n Age (years) Body mass index (kg/m2) Hemoglobin A1c (%) Plasma insulin (pM) Plasma C-peptide (nM) Serum TG (mg/dL) Serum cholesterol (mg/dL) Serum LDL (mg/dL) Serum HDL (mg/dL) Plasma leptin (nM)

Lipid infusion

DM1

DM2

CON

Basal protocol

Pancreatic clamp

9m 31.6 ⫾ 2.0* 21.9 ⫾ 0.4* 7.3 ⫾ 0.3† 46.5 ⫾ 6.2 ⬍0.1*† 90 ⫾ 13* 187 ⫾ 8 106 ⫾ 8† 63 ⫾ 3† 0.20 ⫾ 0.02*

9m 50.1 ⫾ 2.1 25.7 ⫾ 0.7 7.6 ⫾ 0.7† 57.4 ⫾ 8.4 0.82 ⫾ 0.15† 216 ⫾ 49 222 ⫾ 14 139 ⫾ 12 47 ⫾ 3 0.34 ⫾ 0.06

8m 41.1 ⫾ 2.9 24.6 ⫾ 1.0 5.4 ⫾ 0.1 43.0 ⫾ 7.4 0.56 ⫾ 0.07 162 ⫾ 19 227 ⫾ 12 153 ⫾ 9 44 ⫾ 2 0.31 ⫾ 0.05

6 (3 f/3 m) 30.0 ⫾ 2.3 22.8 ⫾ 1.2 5.2 ⫾ 0.1 44.3 ⫾ 3.1 0.53 ⫾ 0.03 104 ⫾ 13 197 ⫾ 9 61 ⫾ 7 115 ⫾ 10 0.33 ⫾ 0.05

6 (1 f/5 m) 30.5 ⫾ 1.9 22.8 ⫾ 0.8 5.3 ⫾ 0.1 44.2 ⫾ 9.3 0.49 ⫾ 0.03 103 ⫾ 7 191 ⫾ 12 61 ⫾ 5 109 ⫾ 16 0.27 ⫾ 0.04

Data are presented as means ⫾ SEM. m, male; f, female; TG, triglycerides; LDL, low-density lipoproteins; HDL, high-density lipoproteins. * p ⬍ 0.05 vs. type 2 diabetic patients. † p ⬍ 0.05 vs. nondiabetic subjects.

endogenous insulin secretion in healthy and type 2 diabetic subjects but not in type 1 diabetic patients. As plasma FFA concentrations are not constant during these protocols, leptin concentrations were examined in a second study during short-term plasma FFA elevation during both fasting and somatostatin-insulin-glucagon clamp tests providing constant plasma concentrations of glucoregulatory hormones.

Research Methods and Procedures Two different protocols were followed. The arginine infusion test was used to stimulate endogenous insulin secretion, whereas the lipid infusion test was employed to increase plasma FFA concentrations. Subjects’ characteristics and baseline (fasting) metabolic data are presented in Table 1. Arginine Infusion Male type 1 (DM1) and type 2 diabetic (DM2) subjects, as well as nondiabetic subjects (CON) who were matched for age and BMI to the respective diabetic groups, were studied (Table 1). All subjects gave informed consent to participate in the protocols. To provide for adequate BMI matching, control subjects were divided into lean (n ⫽ 4; BMI, 22.5 ⫾ 1.3 kg/m2) and moderately obese (n ⫽ 4; BMI, 26.7 ⫾ 0.8 kg/m2) subgroups. Healthy subjects (fasting plasma C-peptide, 0.56 ⫾ 0.07 nM) had neither a family history of diabetes nor evidence for any other disease and did not take any medication on a regular basis. None of the patients suffered from diabetes-related complications. Type 1112

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1 diabetic patients (C-peptide ⬍ 0.1 nM, p ⬍ 0.001 vs. CON) were on an intensified insulin regimen with multiple daily insulin dosage (28). They were instructed not to take neutral protomin Hagedorn or zinc insulin on the evening before the study and to correct plasma glucose with regular insulin only. Type 2 diabetic patients (C-peptide, 0.82 ⫾ 0.15 nM, p ⬍ 0.05 vs. CON) had discontinued their oral hypoglycemic agents at least 3 days before the study. Studies were begun at 8:00 AM after a 12- to 14-hour overnight fast with insertion of Teflon cannulas (Vasofix; B. Braun, Melsungen, Germany) in antecubital veins of the right and left arm for blood sampling and infusion, respectively. After a 15-minute baseline period, L-arginine (30 g; 1 g/min) was infused intravenously over 30 minutes (29). Venous blood samples were taken at ⫺15, 0, 15, 30, 45, 60, 90, and 120 minute(s) for measurement of leptin, insulin, C-peptide, glucagon, FFAs, and glucose. Lipid Infusion Six healthy subjects (Table 1) gave informed and written consent to participate in the protocols, which were reviewed and approved by the local Human Ethics Board. Female subjects were studied in the follicular phase of the menstrual cycle. No changes in diet, weight, and lifestyle were recorded from the time of recruitment until completion of all studies. These protocols were part of another study (20) that aimed to examine effects of FFA on gluconeogenesis (with oral administration of 2H2O) and glucose production (with 2 D-[6,6- H2]glucose). Briefly, subjects were fasted overnight


for 8 hours, and protocols were started with insertion of Teflon cannulas in antecubital veins of the right and left arm for blood sampling and infusions, respectively. During the basal (fasting) protocol, subjects were studied for 9 hours, once in the presence of high-plasma FFA induced by infusing a triglyceride emulsion (0.5 to 1.0 mL/min; Intralipid 20%; Kabi Pharmacia, Uppsala, Sweden) with heparin (bolus: 200 IU; continuously: 0.2 IU/kg 䡠 min) and on a second occasion during infusion of glycerol (0.7 mg/kg 䡠 min in 0.9% saline) to match its plasma concentration to that achieved during lipid/heparin infusion. During the pancreatic clamp, the protocols were repeated in the presence of simultaneous infusions of somatostatin (0.1 ␮g/kg 䡠 min), insulin (0.07 mU/kg 䡠 min), and glucagon (0.65 ng/kg 䡠 min) to maintain their postabsorptive plasma concentrations. Blood samples were taken at timed intervals, immediately chilled, and centrifuged, and the supernatants were stored at ⫺20 °C until determination of hormones and metabolites. Analytical Procedures Plasma leptin concentration was measured with a doubleantibody radioimmunoassay (Linco Research Inc., St. Charles, MO) at within and between coefficients of variance (CVs) of 4.1% and 5.5%, respectively (6). Total plasma FFA (Wako Chemical, Neuss, Germany; CVs ⬍ 6%) and glycerol (Boehringer-Mannheim, Mannheim, Germany; CVs ⬍ 4%) were determined enzymatically. In addition, FFAs were extracted from plasma and separated by thin layer chromatography (30). The fraction containing FFA was separated, and FFAs were converted into their methylesther derivatives (31,32) and analyzed using a Hewlett-Packard 5890 gas chromatograph (column DB-23; J&W Scientific, Folsom, CA) interfaced to a Hewlett-Packard 5971A detector (flame ionization detector). Plasma glucose concentrations were measured with a Beckmann Glucose Analyzer II (Beckman, Fullerton, CA). Plasma insulin, C-peptide, and glucagon were measured by commercial radioimmunoassays with CVs ⬍9%. To avoid any cross-reaction with proinsulin, C-peptide was prepurified by high-performance liquid chromotography as reported previously (33). Hemoglobin A1c was measured after high-performance liquid chromotography separation by using cation exchange columns (6). Measurements of serum cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides were performed by automated enzymatic assays (CHOD-Pap and GPO-Pap; Hitachi, Tokyo, Japan) in the routine laboratory. Calculations and Data Analysis All data are given as means ⫾ SEM. Plasma leptin is presented as total concentration and as corrected for individual BMI (leptin/BMI ratio) (6). Release of hormones and metabolites was calculated from the incremental area under the

concentration–time curves (AUC) corrected for baseline (fasting) concentrations by using the trapezoidal rule. Maximum response to arginine is given as the difference between individual maximum and baseline (fasting) concentrations. One-way ANOVA with Bartlett’s test for equal variances and Bonferroni post hoc testing were used for statistical comparisons between the different groups. Statistical comparisons within the time courses of the experiments were performed using the paired Student’s t test. Correlation between fasting leptin concentrations and BMI was calculated by a linear regression model using the least-squares method. p values ⬍0.05 were considered to indicate statistical significance. All statistical calculations were done with the Sigma Stat software package (V 2.01; Jandel Corp., San Rafael, CA).

Results Arginine Infusion Fasting plasma leptin concentrations correlated with BMI (r ⫽ 0.61, r2 ⫽ 0.37%, p ⫽ 0.001) despite the narrow range of BMI (20.1 to 29.4 kg/m2) within the groups (Figure 1). Within 30 minutes of arginine stimulation, plasma insulin rose ⬃6-fold (maximum response, 223.8 ⫾ 49.1 pM; p ⬍ 0.005) in CON and 4-fold (141.8 ⫾ 31.2 pM; p ⬍ 0.05) in DM2, but not in DM1 (p ⬍ 0.001 vs. other groups; Figure 2A). As the lean and the moderate obese subgroups were not different in any tested parameter, their data are presented as one group (CON). Fasting plasma C-peptide concentrations were higher in DM2 than in CON (p ⬍ 0.05) and below the detection limit of 0.1 nM in DM1 (p ⬍ 0.001 vs. CON; Figure 2B). In parallel to insulin, plasma C-peptide increased ⬃3-fold (1.22 ⫾ 0.21 nM, p ⬍ 0.001) in CON and ⬃2-fold (0.86 ⫾ 0.15 nM, p ⬍ 0.001) in DM2, but not in DM1 (p ⬍ 0.001 vs. other groups). C-peptide concentrations declined to baseline values within 60 minutes in CON, but remained elevated in DM2 until 120 minutes (p ⬍ 0.01; Figure 2B). Incremental release of insulin and C-peptide (AUC, 0 to 60 minutes) was markedly lower in DM1 (p ⬍ 0.01 vs. other groups; Table 2). Plasma glucagon concentrations were comparable at baseline and increased similarly by ⬃85% (p ⬍ 0.005 vs. baseline) in all groups (Figure 2C). Plasma glucose concentrations were markedly higher (p ⬍ 0.0001) in DM1 (0 minutes: 17.0 ⫾ 1.2 mM) and DM2 (10.7 ⫾ 1.1 mM) than in CON (5.4 ⫾ 0.0 mM). In DM2 and CON, plasma glucose increased within 30 minutes (p ⬍ 0.05) by ⬃15% and ⬃35%, respectively, and then returned to baseline values. During arginine stimulation, plasma leptin decreased within 30 minutes by ⬃12% (maximum response, ⫺23 ⫾ 4 pM, p ⬍ 0.001), ⬃11% (⫺38 ⫾ 9 pM, p ⬍ 0.01), and ⬃10% (⫺35 ⫾ 8 pM, p ⫽ 0.012) in DM1, DM2, and CON, respectively, and approached fasting values after 90 minutes (Figure 3A). Plasma leptin concentrations remained lower in DM1 compared with DM2 and CON (⫺42% and ⫺36%, respectively, OBESITY RESEARCH Vol. 10 No. 11 November 2002

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Figure 1: Correlations of (A) body mass index (BMI) and plasma leptin (r ⫽ 0.61; p ⫽ 0.001), (B) plasma insulin and leptin (r ⫽ 0.65; p ⫽ 0.0004), and (C) plasma insulin and BMI-adjusted leptin (r ⫽ 0.61; p ⬍ 0.0009) in type 1 (n ⫽ 9, F), type 2 (n ⫽ 9, Œ), and nondiabetic (n ⫽ 8, 䡺) male subjects during infusion of 30 g arginine.

p ⬍ 0.001 vs. each group). Incremental leptin release (AUC) did not differ among groups (Table 2). BMI-adjusted leptin concentrations (leptin/BMI) were not different between nondiabetic (12 ⫾ 2 pM/kg 䡠 m2) and diabetic subjects (DM1: 9 ⫾ 1 pM/kg 䡠 m2; DM2: 13 ⫾ 2 pM/kg 䡠 m2). Plasma insulin correlated with plasma leptin (r ⫽ 0.65; p ⫽ 0.0004) and BMI-adjusted leptin (r ⫽ 0.61; p ⫽ 0.0009; Figure 1), but not with the decrease of plasma leptin. Plasma FFA were slightly higher in the DM1 and DM2 than in CON (p ⬍ 0.05) and transiently declined during arginine infusion by ⬃41% (p ⬍ 0.05), ⬃62% (p ⫽ 0.005), and ⬃64% (p ⫽ 0.005), respectively, at 45 minutes. At 120 1114


Figure 2: Plasma concentrations (means ⫾ SEM) of (A) insulin, (B) C-peptide, and (C) glucagon in type 1 (n ⫽ 9, F), type 2 (n ⫽ 9, Œ), and nondiabetic (n ⫽ 8, 䡺) male subjects during and after infusion of 30 g arginine. *p ⬍ 0.05 vs. basal; †p ⬍ 0.05 vs. nondiabetic and vs. type 2 diabetic subjects.

minutes, plasma FFA had recovered to baseline values (p ⬍ 0.05; Figure 3B). The decrease in plasma FFA did not correlate with plasma leptin. Lipid Infusion Total plasma FFA increased from 0.05 ⫾ 0.07 mM (0 minute) to 2.76 ⫾ 0.29 mM (p ⬍ 0.001) during lipid/ heparin infusion, and did not change from baseline values during glycerol infusion. Concentrations of individual FFA are shown in Table 3. The rise in total FFA primarily resulted from increased C16:0, C18:0, C18:1(n-9), C18:2(n6), and C18:3(n-3), whereas the changes in the other FFAs were rather small (Table 3).


Table 2. Arginine infusion

Insulin (nM ⫻ 60 minutes) C-peptide (nM ⫻ 60 minutes) Glucagon (pM ⫻ 60 minutes) Leptin (nM ⫻ 60 minutes) FFA (mM ⫻ 60 minutes) N

DM1

DM2

Nondiabetic subjects

0.51 ⫾ 0.18* 2.38 ⫾ 1.15* 1036 ⫾ 136 ⫺1.00 ⫾ 0.17 ⫺9.3 ⫾ 3.9 9

4.92 ⫾ 1.05 32.82 ⫾ 4.99 1271 ⫾ 182 ⫺1.89 ⫾ 0.50 ⫺15.1 ⫾ 3.9 9

7.22 ⫾ 1.62 43.74 ⫾ 7.58 1115 ⫾ 240 ⫺1.47 ⫾ 0.40 ⫺9.4 ⫾ 2.0 8

DM1, type 1 diabetic subjects; DM2, type 2 diabetic subjects; FFA, free fatty acids. Incremental areas under the concentrations–time curves (0 to 60 minutes) corrected for fasting concentrations during arginine stimulation. Data are given as means ⫾ SEM. * p ⬍ 0.05 vs. nondiabetic subjects.

In the basal (fasting) protocol, lipid/heparin infusion induced a continuous rise in plasma insulin from 35 ⫾ 4 to 39 ⫾ 6 (180 minutes), 43 ⫾ 7 (360 minutes), and 44 ⫾ 5 pM (p ⬍ 0.005 vs. basal; p ⬍ 0.05 vs. glycerol infusion:

30 ⫾ 1 pM). Plasma glucose concentrations were similar at baseline (⬃5.1 mM) and declined slightly by ⬃7% (p ⬍ 0.0001) during the last 3 hours of lipid, but not glycerol, infusion. Plasma leptin concentrations were comparable at baseline and continuously declined by ⬃25% (p ⬍ 0.05) and ⬃33% (p ⬍ 0.01) in similar fashion during both lipid/ heparin and glycerol infusion (Figure 4A). During the pancreatic clamp protocol, plasma FFA increased from 0.37 ⫾ 0.09 to 2.05 ⫾ 0.30 mM (p ⬍ 0.01) during lipid/heparin infusion and remained constant during glycerol infusion (0.33 ⫾ 0.07 mM). Plasma insulin was maintained at fasting peripheral concentrations throughout both protocols (lipid/heparin: 33.2 ⫾ 2.9 pM; glycerol: 31.6 ⫾ 3.0 pM). Plasma glucose declined by ⬃24% during the first 90 minutes of both lipid and glycerol infusion. Thereafter, it increased by ⬃50% to 7.9 ⫾ 0.6 mM (540 minutes, p ⬍ 0.005) during lipid infusion, being ⬃1.8-fold higher (p ⬍ 0.005) than during glycerol infusion (4.4 ⫾ 0.7 mM). Plasma leptin concentrations were not different at baseline and similarly declined by ⬃22% (p ⬍ 0.005) and ⬃33% (p ⬍ 0.01) without difference between lipid/heparin and glycerol infusion (Figure 4B).

Discussion

Figure 3: Plasma concentrations (means ⫾ SEM) of (A) leptin and (B) free fatty acid (FFA) in type 1 (n ⫽ 9, F), type 2 (n ⫽ 9, Œ), and nondiabetic (n ⫽ 8, 䡺) male subjects during and after infusion of 30 g arginine. *p ⬍ 0.05 vs. basal; †p ⬍ 0.05 vs. nondiabetic subjects.

Fasting plasma leptin concentrations were 30% to 40% lower in type 1 diabetic than in type 2 diabetic or nondiabetic subjects. Nevertheless, the high variability of individual leptin levels makes it difficult to prove leptin deficiency in type 1 diabetes. Moreover, plasma leptin was no longer different between the diabetic and healthy subjects after adjustment for BMI, which is in line with a recent study reporting similar plasma leptin concentrations in type 1 and type 2 diabetic as well as nondiabetic men, independently of their prevailing metabolic control. (6). During arginine infusion, plasma leptin transiently decreased by ⬃10% in all groups. This small, but significant OBESITY RESEARCH Vol. 10 No. 11 November 2002

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Table 3. Lipid infusion Lipid

Glycerol

Fatty acid

0 minute

180 minutes

360 minutes

540 minutes

0 minute

180 minutes

360 minutes

540 minutes

C14:0 C15:0 C16:0 C16:1(n-7) C18:0 C18:1(n-9) C18:1(n-7) C18:2(n-6) C18:3(n-6) C18:3(n-3) C20:0 C20:1(n-9) C20:2(n-6) C20:3(n-6) C20:4(n-6) C20:5(n-3) C22:4(n-6) C22:5(n-3) C22:6(n-3)

7.9 ⫾ 1.3* 1.0 ⫾ 0.1 63.9 ⫾ 11.9 9.9 ⫾ 3.0 24.3 ⫾ 4.2* 85.3 ⫾ 19.2 5.3 ⫾ 1.4 39.7 ⫾ 5.9* 0.5 ⫾ 0.1 1.99 ⫾ 0.5* 0.21 ⫾ 0.0 0.60 ⫾ 0.1* 0.55 ⫾ 0.1 0.65 ⫾ 0.1 3.2 ⫾ 0.7 0.2 ⫾ 0.0 0.40 ⫾ 0.1 0.78 ⫾ 0.16* 1.3 ⫾ 0.2*

12.8 ⫾ 1.7† 1.9 ⫾ 0.2 177.1 ⫾ 17.4*† 11.5 ⫾ 1.4 62.2 ⫾ 6.3*† 232.8 ⫾ 22.7*† 15.7 ⫾ 1.5*† 431.4 ⫾ 53.6* 1.6 ⫾ 0.1*† 47.7 ⫾ 7.0*† 1.5 ⫾ 0.2*† 1.5 ⫾ 0.2† 1.3 ⫾ 0.2† 1.7 ⫾ 0.2† 5.2 ⫾ 0.9† 0.5 ⫾ 0.1† 0.6 ⫾ 0.1† 1.9 ⫾ 0.2† 2.5 ⫾ 0.3†

10.0 ⫾ 1.5*† 1.6 ⫾ 0.2 217.2 ⫾ 25.9*† 8.4 ⫾ 1.2* 82.6 ⫾ 10.9*† 297.5 ⫾ 39.6*† 20.8 ⫾ 2.7*† 747.9 ⫾ 129.5*† 2.0 ⫾ 0.3*† 84.0 ⫾ 17.1*† 2.5 ⫾ 0.5*† 1.9 ⫾ 0.2† 1.7 ⫾ 0.2*† 2.4 ⫾ 0.6 6.0 ⫾ 0.9† 0.6 ⫾ 0.1† 0.6 ⫾ 0.1† 2.2 ⫾ 0.2† 2.9 ⫾ 0.3†

10.6 ⫾ 1.9* 2.0 ⫾ 0.3 323.2 ⫾ 45.9*† 8.8 ⫾ 1.0* 122.8 ⫾ 17.3*† 459.5 ⫾ 74.4*† 32.5 ⫾ 5.0*† 1338.9 ⫾ 264.0* 3.0 ⫾ 0.5*† 156.0 ⫾ 34.4*† 4.4 ⫾ 0.9*† 2.9 ⫾ 0.4*† 2.7 ⫾ 0.5*† 3.9 ⫾ 1.4 7.5 ⫾ 1.1† 1.0 ⫾ 0.2† 0.8 ⫾ 0.1† 2.8 ⫾ 0.4*† 3.7 ⫾ 0.5*†

14.7 ⫾ 1.8 2.0 ⫾ 0.3 111.5 ⫾ 16.6 15.1 ⫾ 3.7 50.3 ⫾ 8.8 158.4 ⫾ 26.1 9.1 ⫾ 1.9 79.5 ⫾ 10.4 0.5 ⫾ 0.1 4.0 ⫾ 0.6 0.3 ⫾ 0.0 1.2 ⫾ 0.2 1.0 ⫾ 0.2 1.2 ⫾ 0.1 5.0 ⫾ 0.9 0.6 ⫾ 0.1 0.6 ⫾ 0.1 1.6 ⫾ 0.2 2.3 ⫾ 0.3

13.3 ⫾ 2.0 1.8 ⫾ 0.3 108.6 ⫾ 10.6 14.3 ⫾ 3.5 43.7 ⫾ 5.8 156.0 ⫾ 19.6 9.0 ⫾ 1.3 83.9 ⫾ 6.7 0.7 ⫾ 0.1 11.2 ⫾ 6.6 0.4 ⫾ 0.1 1.2 ⫾ 0.2 1.0 ⫾ 0.1 1.3 ⫾ 0.1 6.2 ⫾ 1.3 0.8 ⫾ 0.2 0.5 ⫾ 0.2 1.7 ⫾ 0.3 2.5 ⫾ 0.3

16.0 ⫾ 1.5 2.2 ⫾ 0.2 116.1 ⫾ 10.1 19.0 ⫾ 2.9 43.5 ⫾ 4.2 174.8 ⫾ 17.4 9.6 ⫾ 1.3 84.0 ⫾ 6.9 0.5 ⫾ 0.1 4.4 ⫾ 0.6 0.3 ⫾ 0.0 1.3 ⫾ 0.1 0.9 ⫾ 0.1 1.1 ⫾ 0.1 6.3 ⫾ 0.9 0.6 ⫾ 0.2 0.5 ⫾ 0.1 1.7 ⫾ 0.2 2.2 ⫾ 0.2

16.1 ⫾ 0.8 2.2 ⫾ 0.1 117.0 ⫾ 10.4 19.1 ⫾ 2.4 39.0 ⫾ 5.4 170.2 ⫾ 16.4 9.5 ⫾ 1.5 85.7 ⫾ 7.6 0.4 ⫾ 0.1 4.5 ⫾ 0.4 0.3 ⫾ 0.0 1.2 ⫾ 0.2 0.8 ⫾ 0.2 1.0 ⫾ 0.1 5.0 ⫾ 1.4 0.5 ⫾ 0.2 0.5 ⫾ 0.1 1.5 ⫾ 0.2 2.0 ⫾ 0.3

Free fatty acids during infusion of lipid/heparin (n ⫽ 6) and glycerol (n ⫽ 5). Data are given in ␮M (means ⫾ SEM). * p ⬍ 0.05 vs. glycerol. † p ⬍ 0.05 vs. 0 minute.

decline could be related to (1) increased glucagon release, (2) increased insulin secretion during arginine stimulation of ␤-cells in healthy and type 2 diabetics, (3) the simultaneous decrease of plasma FFA, and finally, (4) an effect of arginine per se on leptin production and/or secretion. Plasma glucagon concentrations identically increased by ⬃85% in all subjects during arginine stimulation. Although fasting plasma leptin correlates with both glucagon and insulin secretion in healthy women (26,34), glucagon infusion does not affect plasma leptin (35), arguing against a major role of glucagon in the regulation of leptin secretion. In this study, plasma insulin concentrations were closely related to total plasma leptin, BMI-adjusted leptin, and the decrease in plasma leptin during arginine infusion. Such correlation between plasma insulin and leptin was observed in both lean and obese healthy and insulin-resistant subjects (11,36) and hyperinsulinemic hypothyroidism (37), but not in other populations (14 –16,38) The interaction between insulin and plasma leptin concentration is not yet clear, but evidence has been provided that insulin can stimulate leptin secretion. In adipocytes, insulin increases ob gene expression and leptin secretion (39,40). Likewise, insulin causes a transient and delayed increase of leptin under in vivo conditions (10,39). From these data, it is rather surprising that 1116


the arginine-dependent rise in insulin secretion did not increase plasma leptin in this study. As the studies were stopped at 120 minutes, we cannot rule out a possible increase of plasma leptin during the subsequent few hours. Of note, prolonged excessive hyperinsulinemia for 72 hours (23), but not short-term insulin exposure (35,41), was found to increase serum leptin concentrations. In moderately obese men, insulin infusion increased plasma leptin and lowered plasma FFA in concentrationdependent manner (8). The half-maximally effective insulin concentrations for both effects were similar so a direct interaction between plasma leptin and FFA could not be ruled out. In the present study, plasma FFA concentrations decreased during arginine infusion in all subjects. However, this decrease in plasma FFA did not correlate with that of plasma leptin, which is at odds with a previous study, where a positive correlation between fasting leptin and plasma FFA concentration was found in patients and healthy subjects (42). On the other hand, plasma FFA elevation induced by a lipid infusion caused a rise in plasma leptin in nondiabetic subjects during hyperinsulinemic-hyperglycemic clamps (25). When we employed a pancreatic clamp protocol and matched glycerol infusion in a control study, we did not detect any influence of FFA on plasma leptin concen-


Figure 4: Plasma concentrations (means ⫾ SEM) of leptin during infusion of lipid/heparin (n ⫽ 7, F) and glycerol (n ⫽ 7, 䡺) (A) at basal conditions and (B) in the presence of somatostatin-insulinglucagon infusions. *p ⬍ 0.05 vs. basal.

trations. This in line with two in vivo studies, where changes of plasma FFA induced by acipimox or lipid infusion did not correlate with changes in plasma leptin in normal and abdominally obese subjects (21,22), but contrasts with a recent in vitro study reporting down-regulation of both leptin protein and mRNA by FFA (24). Different composition of plasma FFAs could help to explain the divergent results of the above discussed studies. In this study, C16:0, C18:0, C18:1(n-9), C18:2(n-6), and C18: 3(n-3) fatty acids contributed mostly to the rise in total plasma FFA (Table 3). The C18:0 fatty acid is also increased in type 2 diabetes (43). Likewise, the percentage of saturated and monounsaturated fatty acids is higher in hyperlipidemic type 2 diabetic patients (44). At present, no other data on the contribution of plasma FFA species during experimental conditions have been reported. Finally, infusion of arginine per se could account for the decreased plasma leptin and FFA concentrations. Arginine was particularly chosen for the evaluation of ␤-cell function because of its superior ability over glucose to stimulate endogenous insulin release in type 2 diabetes (45,46) and because of the excellent reproducibility of this test (47). Arginine stimulates insulin secretion mainly by directly entering the cell through cationic amino acid transporters inducing depolarization of the ␤-cell membrane (48,49). Few data are available at present on the impact of arginine

or other amino acids on production and/or secretion of leptin. Our results are in line with a study reporting a decrease in plasma leptin in postmenopausal women (26), but in contrast to findings in healthy children, in whom arginine infusion did not modulate leptin secretion (27). These differences could be explained by differences in age or data analysis that could not detect the rather small decrease observed in our study. Interestingly, patients using peritoneal dialysis, an amino acid– based dialysis solution, had a transient decrease of plasma leptin concentrations during 3 months (50). In the lipid/heparin and glycerol infusion protocols, plasma leptin concentrations continuously declined both under basal and under somatostatininsulin-glucagon clamp conditions, which is in line with other studies without any manipulations (7,8). This could suggest a similar decline during the arginine infusion protocols, indicating that arginine counteracted a further decline of leptin. On the other hand, plasma leptin decreased by ⬃11% within the first 180 minutes of the lipid infusion study, whereas it declined to similar extent (by ⬃12%) within only 30 minutes of the arginine infusion study. It is therefore tempting to assume that arginine per se exerts a direct effect on plasma leptin concentrations. After the arginine infusion, leptin concentrations increased again during the last 90 minutes of the study. It is of note that subcutaneous injection of a physiological dose of growth hormone increases plasma leptin by ⬃25% within 24 hours (51). Thus, we cannot completely rule out that a rise in plasma growth hormone during arginine infusion could have contributed to the re-increase of leptin. In conclusion, arginine infusion transiently decreased plasma leptin concentrations both in insulin-deficient and hyperinsulinemic diabetic patients, which suggests a direct inhibitory effect of the amino acid rather than short-term effects of insulin or FFAs.

Acknowledgments These studies were supported by grants from the Austrian Science Foundation (FWF, P13213-MOB) to M.R. and from ¨ NB, 8196, 5033) to W.W. and the Austrian National Bank (O M. R. We thank C. Ludwig, the staff of the Metabolic Unit (A. Hofer, O.H. Lentner), and the Laboratory of the Division of Clinical Endocrinology and Metabolism, Department of Internal Medicine III for excellent assistance. References 1. Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 1996;334:292–5. 2. Kennedy A, Gettys TW, Watson P, et al. The metabolic significance of leptin in humans: gender-based differences in relationship to adiposity, insulin sensitivity, and energy expenditure. J Clin Endocrinol Metab. 1997;82:1293–300. 3. Reaven GM. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes. 1988;37:1595– 607. OBESITY RESEARCH Vol. 10 No. 11 November 2002

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