European Journal of Endocrinology (2001) 144 283±290 ISSN 0804-4643

CLINICAL STUDY Circulating IGF binding protein-1 is inversely associated with leptin in non-obese men and obese postmenopausal women Stefan SoÈderberg, Bo AhreÂn1, Mats Eliasson2, Bo Dinesen4, Kerstin Brismar3 and Tommy Olsson Department of Medicine, UmeaÊ University Hospital, 1MalmoÈ University Hospital and 2LuleaÊ Hospital, Sweden, 3Department of Endocrinology and Diabetology, Karolinska University Hospital, Sweden and 4The Steno Diabetes Center, Gentofte, Denmark (Correspondence should be addressed to S SoÈderberg, Department of Medicine, UmeaÊ University Hospital, S-901 85 UmeaÊ, Sweden; Email: [email protected])

Abstract Objective: Hyperleptinaemia and hyperinsulinaemia interrelate to insulin-like growth factor binding protein 1 (IGFBP-1), and disturbances in the growth hormone±IGF-I axis are linked to obesity and cardiovascular diseases. However, whether the association between leptin and the GH±IGF-I axis is altered with increasing obesity is not known. We therefore examined the relationship between leptin, IGF-I, IGFBP-1, insulin and proinsulin in men and women with or without obesity in a population study. Design and subjects: Healthy subjects n 158; 85 men and 73 pre- and postmenopausal women) from the Northern Sweden MONICA (Monitoringˆ of Trends and Determinants in Cardiovascular Disease) population were studied with a cross-sectional design. Methods: Anthropometric measurements (body mass index (BMI) and waist circumference) and oral glucose tolerance tests were performed. Radioimmunoassays were used for the analyses of leptin, IGF-I and IGFBP-1, and ELISAs for specific insulin and proinsulin. Results: Leptin inversely correlated to IGFBP-1 in non-obese men P , 0:05 and obese postmenopausal women P , 0:05 : In contrast, leptin did not correlate to IGF-I.† IGFBP-1 was also significantly associated with† proinsulin in non-obese men P , 0:01 and non-obese premenopausal women P , 0:05 : The association between leptin and IGFBP-1† was lost after adjustment for insulin. In multivariate† analyses taking measures of adiposity into account, low proinsulin, and IGF-I in combination with old age, but not leptin, predicted high IGFBP-1 levels. Conclusions: Leptin was inversely associated with IGFBP-1 in non-obese men and obese postmenopausal women, and proinsulin was inversely associated with IGFBP-1 in non-obese men and premenopausal women. However, these associations were lost with increasing central obesity in men and premenopausal women and after control for insulin. Therefore, this study suggests (i) that leptin is of minor importance for regulation of IGFBP-1 levels and (ii) that the insulin resistance syndrome is characterised by an altered relationship between leptin, IGFBP-1 and insulin.

European Journal of Endocrinology 144 283±290

Introduction (IGFBP-1) production (10). Leptin might also be involved in this regulation, since leptin interacts with The cluster of risk factors named the insulin resistance the growth hormone (GH)±IGF axis in a bi-directional syndrome, which is associated with (central) obesity way. Besides hypothalamic effects (11), it is possible (1, 2), is characterised by hyperinsulinaemia, hyper- that leptin influences GH effects indirectly in the tension, dyslipidaemia and abnormal fibrinolysis (3). periphery through interaction with the IGFBPs (12) Increased leptin levels may mediate several of these and, conversely, levels of leptin decrease after GH manifestations through metabolic and hormonal inter- substitution (13). However, whether the association actions and effects on blood pressure (4±6). In line with between leptin and the GH±IGF-I axis is altered in this, we have reported that leptin is independently insulin resistance is not known. associated with increased risk for cardiovascular In addition, states of insulin resistance are char- disease (7, 8). In addition, the insulin resistance acterised by high circulating levels of proinsulin-like syndrome may include insufficient peripheral effects molecules (14), but the association between the of insulin-like growth factor-I (IGF-I) (9), possibly due GH±IGF-I axis and these molecules has not yet been to insulin-mediated inhibition of IGF binding protein-1 fully elucidated (15).

q 2001 Society of the European Journal of Endocrinology Online version via http://www.eje.org

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The aim of this study was to explore the association measured by ELISA without cross-reactivity to human between circulating levels of IGF-I and IGFBP-1 on one proinsulin (21). Split(32±33)- and des(31,32)-proinsu- hand and leptin and proinsulin on the other in healthy lin do not react, whereas split(65±66)- and des(64,65)- men and women representing a wide range of body proinsulin cross-react with an efficiency of 30% and composition and age, and to explore if these associa- 63% respectively, on a molar basis. The detection limit tions differ between men and women in general, and was 5 pmol/l, and the working range was 5± between pre- and postmenopausal women. 600 pmol/l. Interassay coefficients of variation (CV) were 5±6% at 80±350 pmol/l and 11% at 30 pmol/l. Proinsulin immunoreactivity was measured by ELISA Patients and methods with a detection limit of 0.25 pmol/l and a working Study population range of 0.25±100 pmol/l (22). The four major proinsulin conversion intermediates reacted 65±99% The study was performed within the framework of the on a molar basis. C-peptide and insulin did not react. Northern Sweden MONICA Project, which, in turn, is Interassay CVs were 6±7% at 5±30 pmol/l. IGF-I was part of the WHO MONICA (Monitoring of Trends and determined by RIA after separation of IGFs from IGFBPs Determinants in Cardiovascular Disease) Project (16). by acid±ethanol extraction and cryoprecipitation. To In 1994, a population was screened for cardiovascular minimise interference of remaining IGFBPs, des(1±3)- risk factors. A total of 2500 individuals in the 25±74 IGF-I was used as radioligand (23). The intra- and year range were invited by mail from a total population interassay CVs were 4% and 11% respectively. IGFBP-1 of 367 000 in this age range. Within each age group concentrations in plasma were determined according to (25±34, 35±44, 45±54, 55±64 and 65±74 years) the method of PoÂvoa et al. (24). The sensitivity of the 250 men and 250 women were randomly selected RIA was 3 mg/l, and the intra- and interassay CVs were from continuously updated population registers in 3% and 10% respectively. The leptin analysis was Norrbotten and VaÈsterbotten, the two northernmost performed using a double-antibody RIA with rabbit provinces of Sweden. In total 1921 subjects participated anti-human leptin antibodies, 125I-labelled human in the study (76.8%). Subjects were included whose leptin as tracer and human leptin as standard (Linco blood samples were taken between 0700 and 0900 h in Res., St Louis, MO, USA). Interassay CV was 1.9% at order to minimise the influence of diurnal rhythm of low levels (,5 ng/ml) and 3.2% at high levels (10± leptin (17), insulin (18) and IGFBP-1 (19). Subjects who 15 ng/ml). were pregnant, had diabetes or subclinical hypothyroid- ism, or had a history of prior myocardial infarction or stroke, or were using oestrogen replacement therapy, Statistical analysis oral contraceptives, or antihypertensive agents were All the main study variables were positively skewed excluded. Furthermore, women with irregular men- and, therefore, (ln) transformed values were used. struations were excluded. After an overnight fast, a Means (geometric for transformed values) with 95% 75 g oral glucose tolerance test was performed. Three confidence intervals (CI) are presented. Bivariate subjects were found to have previously unknown (Pearson's) and partial correlation coefficients were diabetes and were excluded from further analysis. calculated, adjusted for age and adiposity. One-way After these exclusions, 85 men and 73 women ANOVA analyses with adjustments for covariates were remained and thus formed the basis of this study. used, and significant differences between factor levels Sampling procedures have been described previously were evaluated after Bonferroni correction. Multiple (20). This study was approved by the Research Ethics linear regression analysis was performed with a Committee of UmeaÊ University. stepwise method. Introducing combination terms in the model tested interaction, and dummy variables the Anthropometric and biochemical analyses presence of group effects. The influence of potential outliers was tested by analysis of residuals and by Body mass index (BMI) was calculated as total body excluding subjects. Two-tailed tests were used and a weight in kilograms divided by the square of height in P-value ,0.05 was considered significant. All calcula- meters, and waist hip ratio (WHR) was calculated as tions were made with the statistical program SPSS the ratio of the circumference of the narrowest part of (Chicago, IL, USA) version 6.1 on a Macintosh the waist divided by the broadest part of the hip. All computer. measurements were taken with the subject standing upright and breathing lightly. Blood pressure was measured with the subjects in the sitting position Results after 5 min of rest using the random zero method. Plasma glucose was analysed by the hexokinase Baseline characteristics method (Boehringer Mannheim, Germany) on a Baseline characteristics of the study population are Hitachi 717 analyser (Tokyo, Japan). Insulin was shown in Table 1. Men had higher waist circumference, www.eje.org

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WHR and fasting glucose levels in combination with lower leptin concentrations versus both pre- and -value

P postmenopausal women. Furthermore, men had higher 0.001 ab 0.001 ab 0.001 abc 0.001 ab 0.001 abc , , , , , BMI and higher fasting proinsulin/insulin ratio com- pared with premenopausal women. IGF-I levels were highest in premenopausal women followed by men who -ratio

F had higher levels than postmenopausal women. Differences in mean IGF-I levels between groups disappeared after adjustment for age whereas differ- ences in proinsulin, glucose, IGFBP-1, and leptin concentrations remained after adjustment for both

premenopausal women±postmenopausal age and BMI (data not shown). ˆ

Relationship of IGF-I and IGFBP-1 to age,

95% CI Min±max adiposity, insulins and leptin Levels of IGF-I were decreasing, whilst levels of IGFBP-1

† were increasing, with age in men and premenopausal 28 women (data not shown). IGFBP-1 was inversely ˆ

n associated with adiposity indices (BMI, waist circum-

ference and WHR) in men and women irrespective of menstrual status, whereas IGF-I did not correlate to

men±postmenopausal women, and c adiposity once adjusted for age (data not shown). IGF-I ˆ was positively associated with insulin in non-obese men and in obese premenopausal women, and inversely with the proinsulin/insulin ratio in men and premeno- pausal women (Table 2). Leptin inversely correlated to IGFBP-1 in non-obese men and obese postmenopausal women. IGFBP-1 also 95% CI Min±max correlated inversely to insulin and proinsulin in non- obese men, but only to proinsulin in non-obese † premenopausal women and not in postmenopausal 45 men±premenopausal women, b

ˆ women. The inverse correlation between proinsulin ˆ n

and IGFBP-1 remained after further adjustment for insulin in premenopausal women r 20:33; P , 0:05 ; but not in men, and in all groups theˆ inverse association† between leptin and IGFBP-1 was lost after adjustment for insulin (data not shown). Levels of leptin did not correlate to levels of IGF-I, neither in bivariate test.

2 nor in partial correlation analysis. Finally, the inverse x association between IGF-I and IGFBP-1 in men and

Men Premenopausal women Postmenopausal womennon-obese ANOVA postmenopausal women was independent of

95% CI Min±max the effects of insulin r 20:24; P , 0:05; and r 20:64; P , 0:05 respectively). ˆ ˆ † 85

ˆ Linear regression analyses with IGFBP-1 as n dependent variable Multiple linear regression analysis with IGFBP-1 concentrations as the dependent variable was per- formed (Table 3). The model included fasting levels of insulin, proinsulin and glucose, in combination with

g/l)* 20.5 17.5±24.1IGF-I, 2.0±92.0 BMI, 25.6 waist 21.6±30.3 circumference, 4.0±69.0 34.9 27.7±44.0 age 8.0±109.0 and 6.83 gender 0.001 b (when ) 25.8 25.1±26.5 19.5±38.0 24.1 22.8±25.4 17.4±35.8 25.8 24.0±27.6 19.4±40.6 3.38 0.04 a 2 m applicable) as explanatory variables. Significant pre- Subject characteristics. g/l)* 146.7 137.6±156.4 53.0±296.0 174.4 159.5±190.9 94.0±313.0 117.8 103.2±134.5 51.0±218.0 14.28 waist to hip ratio. m dictors for high IGFBP-1 levels were low levels of ˆ proinsulin P , 0:01 and IGF-I P , 0:05 in combi- † † Fasting insulin (pmol/l)*Fasting proinsulin (pmol/l)*Fasting glucose (mmol/l)IGF-I ( 36.7 7.8 5.5 32.0±42.0 6.9±9.0 7.1±165.0 5.3±5.6 2.7±46.0 32.4 3.0±7.3 5.2 28.1±37.4 5.1 16.0±125.0 4.6±5.9 37.1 5.0±5.2 2.2±16.0 30.0±45.9 3.9±5.8 14.0±151.0 6.8 0.78 5.2 5.5±8.4 0.5 ± 5.0±5.4 3.0±31.0 7.92 4.3±6.0 0.001 a 7.20 0.001 ab WHR 0.93 0.92±0.94 0.76±1.13 0.80 0.78±0.82 0.68±1.05 0.83 0.80±0.86 0.70±1.10 64.50 Regular smokers (%) 17.9 28.9 25.0 0.3² ± IGFBP-1 ( Leptin (ng/ml)* 4.3 3.7±4.9 0.9±19.6 12.1 9.8±14.9 3.8±48.1 14.7 11.3±19.2 2.6±50.5 58.05 BMI (kg/m Waist circumference (cm) 92.2 90.3±94.2 73.0±118.0 78.7 75.4±82.1 64.5±113.5 83.4 78.5±88.3 66.0±118.0 25.56 Age (years) 48.8 45.5±52.2 25.0±74.0 37.9 35.6±40.3 25.0±53.0 60.6 58.2±62.9 50.0±73.0 29.78 Table 1 Significant differences between groups (ANOVA with Bonterom post hoc tests: a women. WHR * Geometric means. ²Difference between groups tests with nation with low waist circumference P , 0:001 and †

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Table 2 Partial correlations between determined variables. Stratified for central obesity and adjusted for age, BMI and waist circumference. Median of waist circumference used as cut-off: pre- and postmenopausal women 0.77 and 0.82 m respectively, and men 0.92 m.

Men Premenopausal women Postmenopausal women All #median .median All #median .median All #median .median

IGF-I versus: Leptin 0.19 0.23 0.15 0.23 0.09 0.39 0.21 20.06 0.23 IGFBP-1 20.32² 20.28 20.30 20.09 20.39 20.04 20.37 20.60* 20.32 Fasting insulin 0.30² 0.39* 0.26 0.29 0.27 0.51* 0.31 0.31 0.29 Fasting proinsulin 0.09 0.13 0.05 20.06 20.12 20.02 0.15 20.10 0.30 Fasting proinsulin/insulin 20.27* 20.30 20.28 20.34* 20.34 20.54* 20.14 20.30 0.03 IGFBP-1 versus: Leptin 20.25* 20.33* 20.13 20.11 20.34 0.18 20.24 0.26 20.62* Fasting insulin 20.35² 20.38* 20.33 20.33* 20.39 20.17 20.42* 0.02 20.56* Fasting proinsulin 20.32² 20.42² 20.23 20.42² 20.50* 20.44 20.22 0.22 20.38 Fasting proinsulin/insulin 0.06 20.03 0.14 20.02 0.00 20.23 0.19 0.16 0.19

P-values: * , 0:05; ² , 0:01; ³ , 0:001: old age P , 0:001 in the entire population, and these proinsulin P , 0:01 and IGF-I P , 0:01 in com- variables explained† 48% of the IGFBP-1 variation. The bination with low BMI† P , 0: 001 and† old age addition of menstrual status (dummy variables) to this P , 0:001 in men; low proinsulin †P , 0:01 ; low model revealed that premenopausal women had sig- waist circumference† P , 0:05 and old age P , 0†:001 nificantly higher IGFBP-1 levels than men P , 0:05 ; in premenopausal women; and† low IGF-I P , 0:05† and that postmenopausal women did not have sig-† and low BMI P , 0:01 in postmenopausal women.† nificantly different IGFBP-1 levels compared with men Further stratification for† waist circumference revealed and premenopausal women. BMI, waist circumference that low levels of proinsulin P , 0:01 and IGF-I or menstrual status in combination with proinsulin or P , 0:05 in combination with high waist† circum- insulin was not associated with IGFBP-1 levels. The ference P†, 0:001 were associated with high IGFBP- following variables were associated with IGFBP-1 levels 1 in non-obese premenopausal† women, whilst only after stratification for menstrual status: low levels of low waist circumference P , 0:01 predicted high †

Table 3 Linear regression model for variables associated with circulating (ln) IGFBP-1 levels. The model included (fasting) insulin, (fasting) proinsulin, IGF-I, (fasting) glucose, BMI, waist circumference, age and gender (when applicable) as explanatory variables. Analyses repeated after stratification for central obesity.

Group Factors Adjusted R2 B SE(B) b P

All subjects Age 48.2 0.020 0.004 0.421 ³ Waist circumference 20.020 0.004 20.336 ³ Fasting insulin 20.188 0.097 20.155 0.05 Fasting proinsulin 20.290 0.099 20.241 ² IGF-I 20.390 0.158 20.185 *

Men Age 47.9 0.018 0.005 0.362 ³ BMI 20.094 0.021 20.403 ³ Fasting proinsulin 20.295 0.107 20.243 ² IGF-I 20.708 0.237 20.283 ²

Premenopausal women Age 39.7 0.033 0.009 0.468 ³ Waist circumference 20.015 0.007 20.294 * Fasting proinsulin 20.529 0.182 20.395 ² #median of waist Waist circumference 67.3 0.069 0.014 0.614 ³ Fasting proinsulin 20.510 0.156 20.401 ² IGF-I 20.476 0.171 20.347 * .median of waist Waist circumference 32.4 20.042 0.013 20.598 ²

Postmenopausal women BMI 43.8 20.069 0.019 20.536 ² IGF-I 20.577 0.260 20.330 *

B regression coefficient, SE B standard error of B, and b standardised regression coefficient. P-values: *,0.05, ²,0.01, ³,0.001. ˆ †ˆ ˆ www.eje.org

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Figure 1 The relation between levels of proinsulin and IGFBP-1 in men (X) and women (A).

Figure 2 The relation between levels of insulin and IGFBP-1 in men (X) and women (A).

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IGFBP-1 levels in obese premenopausal women. The between IGFBP-1 and leptin. It is possible that these stratified analyses did not bring additional information associations reflect normal regulation by proinsulin of to the analyses of men and postmenopausal women. both IGFBP-1 and leptin in non-insulin resistant Finally, leptin was introduced in the model. Leptin subjects, whereas in obese subjects the influence by replaced BMI and IGF-I as a predictor of circulating insulin predominates. A problem with the interpret- IGFBP-1 levels P , 0:001 in postmenopausal women. ation of the data in the present study is that the cohort Leptin did not associate† to IGFBP-1 in men and was divided in subgroups with a limited number of premenopausal women. subjects in each group. Although this may limit the The relationships between levels of proinsulin and generality of the findings, the results clearly indicate insulin on one hand and IGFBP-1 levels on the other that the relation between IGFBP-1 and leptin is are further illustrated in Figs 1 and 2. In Fig. 2, it can stronger in non-obese men and in obese postmeno- be seen that there are three men with high IGFBP-1 pausal women than in other subgroups. It is not clear despite high insulin, and a premenopausal woman with why the association between IGFBP-1 and leptin is very low IGFBP-1 and high insulin levels. Exclusion of restricted to non-obese men and obese postmenopausal these subjects (all with waist above median) did not women. Therefore, the physiological relevance of these affect the association between leptin on the one hand findings remains to be established. and IGF-I and IGFBP-1 on the other. In contrast, The regulation of the IGFBPs will affect circulating insulin became inversely associated with IGFBP-1 in levels of IGFs, since IGFBPs maintain adequate levels of obese men r 20:42; P , 0:05 : Furthermore, the fol- IGFs through protection from degradation, and also lowing factors ˆ were significant predictors† for IGFBP-1 function as regulators of IGF function at the cellular in the regression analysis: insulin P , 0:01 (replaced level (10). IGFBP-1, which is mainly produced in the proinsulin) together with age, BMI and IGF-I† in men; liver, is thought to be a direct regulator of free IGF and IGF-I P , 0:01 (replaced age and waist) together levels, and thus reflects the biologically active fraction with proinsulin in premenopausal† women. of IGFs (27). Insulin regulates the hepatic production of IGFBP-1 through insulin responsive elements in the promoter region (28). Discussion We found that IGF-I did not correlate with leptin Earlier reports have shown that levels of IGFBP-1 are after adjustments in this healthy and probably weight- associated with various expressions of the insulin stable population, results consistent with earlier studies resistance syndrome (25). It was thus of interest to in both normal and GH-deficient subjects (13, 29). further clarify the role of IGFBP-1 in conjunction with Neither did IGF-I correlate to measures of adiposity leptin and proinsulin levels. Specifically, we found that once adjusted for age, suggesting that IGF-I production leptin was associated with low IGFBP-1 levels in non- is related more to lean body mass than to fat mass (30). obese men after adjustment for adiposity. This associa- In contrast, there are studies in both humans and rats tion was lost in subjects with increasing central obesity reporting a sustained decrease in leptin levels after and after adjusting for fasting insulin. This suggests an treatment with IGF-I, probably due to decreased altered relationship between leptin and IGFBP-1 in circulating levels of insulin (31, 32). Leptin can affect subjects with obesity due to the influence of insulin. In the homeostasis of IGF-I through an interaction with line with this, it was recently suggested that the the GH axis, an interaction that could be both direct reduction in leptin during fasting is associated with and indirect. For example, intracerebroventricular increased IGFBP-1 levels, and that this change is administration of leptin antiserum reduced GH secre- dependent on the concomitant reduction in insulin tion in rats (11) and GH administration to GH-deficient (26). Furthermore, we found a strong inverse associ- humans has been found to reduce leptin levels, ation between circulating levels of proinsulin and probably through reduced amount of body fat mass IGFBP-1 in non-obese men and premenopausal (13). Leptin could also influence GH effects in the women. It may be speculated that proinsulin regulates periphery through association with the GH-binding hepatic IGFBP-1 production under conditions of low protein and the IGFBPs (12, 33). insulin, to replace the influence of insulin, as in non- In conclusion, circulating leptin was inversely obese subjects. If so, this would suggest that the associated with IGFBP-1 levels in non-obese men and relation between IGFBP-1 and proinsulin is not obese postmenopausal women, whereas leptin did not primarily related to the insulin resistance syndrome, associate to IGF-I levels. Similarly, circulating levels of which is in contrast with earlier studies (15). proinsulin was inversely associated with IGFBP-1 in Interestingly, we have recently shown that circulat- non-obese men and premenopausal women. These ing levels of leptin and proinsulin are strongly associations were lost with increasing central obesity associated in non-obese men and premenopausal in men and premenopausal women and after control women, whereas the association was lost with increas- for insulin. Therefore, this study suggests (i) that ing central obesity (unpublished data). This association leptin is of minor importance for IGFBP-1 regulation between proinsulin and leptin resembles therefore that and (ii) that the insulin resistance syndrome is www.eje.org

Downloaded from Bioscientifica.com at 09/25/2021 11:48:59PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2001) 144 Leptin, IGFBP-1, proinsulin and adiposity 289 characterised by an altered relationship between leptin, 14 Rùder ME, Dinesen B, Hartling SG, Houssa P, Vestergaard H, IGFBP-1 and insulin. Sodoyez-Goffaux F et al. Intact proinsulin and beta-cell function in lean and obese subjects with and without type 2 diabetes. Diabetes Care 1999 22 609±614. 15 Mohamed-Ali V, Pinkney JH, Panahloo A, Cwyfan-Hughes S, Acknowledgements Holly JM & Yudkin JS. Insulin-like growth factor binding protein- We are indebted to the Northern Swedish MONICA 1 in NIDDM: relationship with the insulin resistance syndrome. Clinical Endocrinology 1999 50 221±228. Project and the grants supporting it. This study was 16 WHO MONICA project principal investigators (prepared by H also supported by grants from the Swedish Medical Tunstall Pedoe). 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body composition during growth hormone (GH) therapy in 33 Bjarnason R, Boguszewski M, Dahlgren J, Gelander L, Kristrom B, adults with GH deficiency. Journal of Clinical Endocrinology and Rosberg S et al. Leptin levels are strongly correlated with those of Metabolism 1997 82 223±228. GH-binding protein in prepubertal children. European Journal of 31 Bianda TL, Glatz Y, Boeni-Schnetzler M, Froesch ER & Schmid C. Endocrinology 1997 137 68±73. Effects of growth hormone (GH) and insulin-like growth factor-I on serum leptin in GH-deficient adults. Diabetologia 1997 40 363±364. 32 BoÈni-Schnetzler M, Hauri C & Zapf J. Leptin is suppressed during infusion of recombinant human insulin-like growth factor I Received 21 February 2000 (rhIGF I) in normal rats. Diabetologia 1999 42 160±166. Accepted 13 November 2000

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