0021-972X/91/7201-0051$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 72, No. 1 Printed in U.S.A.
Dual Defects in Pulsatile Growth Hormone Secretion and Clearance Subserve the Hyposomatotropism of Obesity in Man* JOHANNES D. VELDHUIS, ALI IRANMANESH, KEN K. Y. HO, MICHAEL J. WATERS, MICHAEL L. JOHNSON, AND GERMAN LIZARRALDE Division of Endocrinology and Metabolism and the Interdisciplinary Graduate Biophysics Program, Departments of Internal Medicine and Pharmacology, University of Virginia (J.D. V., M.L.J.), Charlottesville, Virginia 22908; Salem Veterans Administration Hospital (A.I., G.L.), Salem, Virginia 24153; and Garvan Institute of Medical Research, St. Vincent's Hospital (K.K.Y.H.), Darlinghurst, New South Wales, and the Department of Physiology and Pharmacology, University of Queensland (M.J. W.), Queensland 4067, Australia
significantly prolonged mean intersecretory burst intervals (282 ± 65 vs. 131 ± 11 min; P < 0.05). The resultant daily GH production rate in obese men was reduced to one fourth that in normal weight individuals. Both GH secretion rate and burst frequency were negatively correlated with the degree of obesity (ponderal index). The decreases in GH burst frequency and halflife were specific, since GH secretory pulse amplitude (maximal rate of GH release), the mass of GH released per burst, and the duration of computer-resolved GH secretory bursts were not different in obese and normal weight men. We conclude that obese men harbor a double defect in GH dynamics involving both GH secretion and clearance, and that the severity of the GH secretory deficiency is proportionate to the degree of obesity. (J Clin Endocrinol Metab 72: 51-59, 1991)
ABSTRACT. We have examined the mechanisms underlying reduced circulating GH concentrations in the obese human. Computer-assisted (deconvolution) analysis was used to determine endogenous GH secretory and clearance rates quantitatively from entire 24-h plasma GH concentration profiles. These analyses revealed that the half-life (ti/2) of endogenous GH was significantly shorter in obese (11.7 ± 1.6 min) than in normal weight subjects (15.5 ± 0.81 min; P < 0.01). The accelerated blood disposal rate of GH was not due to decreased circulating concentrations of GH-binding protein, since the latter were similar in obese (25 ± 1.0%) and normal weight (24 ± 2.3%) men. However, obese men had significantly fewer GH secretory bursts (3.2 ± 0.53 us. 9.7 ± 0.67/day; P < 0.01). Among the rare GH secretory bursts that occurred in obese subjects, there were
U
in man are associated with profound increases in the frequency and amplitude of intermittent GH release (9, 10). In rats, major changes in GH pulse properties (albeit directionally opposite those in man) also occur when glucose concentrations are altered (11, 12). On the other hand, suppressed mean circulating GH concentrations have been recognized in obesity, with diminished acute secretory responses to dynamic stimuli such as insulin, arginine, opiates, glucagon, levo-dopa, sleep, and GHreleasing hormones (GHRH) (13-33). Such observations have suggested the hypothesis that one or more neuroregulatory defects in pulsatile GH secretion exist in obese individuals and result in hyposomatotropism. However, the explicit nature of such regulatory defects is not known. In addition, studies in nonhuman species have demonstrated accelerated metabolic clearance of exogenously administered GH in some obese animals (34), but not in others (13), suggesting that increased metabolic disposal of GH may contribute to hyposomatotropism. The precise and valid assessment of alterations in the
NDER a variety of experimental and physiological conditions, the pulsatile mode of GH, glucagon, insulin, or ACTH secretion conveys important signaling information to target tissues (1-4). Moreover, patterns of episodic GH release are susceptible to physiological regulation and specific pathophysiological alterations (58). For example, sex steroids, age, gender, puberty, and nutritional status, including feeding and short term fasting, all modulate the pulsatile mode of GH release (510). Short term caloric deprivation and diabetes mellitus Received May 23,1990. Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Division of Endocrinology and Metabolism, Box 202, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908. * This work was supported in part by NIH Grant RR-00847 (to the Clinical Research Center of the University of Virginia), Research Career Development Award 1-KO4-HD-00634 (to J.D.V.), NIH Grant GM-28928 (to M.L.J.), Diabetes and Endocrinology Research Center Grant NIH DK-38942, NIH-supported Clinfo Data Reduction Systems, V.A. Medical Research Funds (to A.I. and G.L.), and support by the Pratt Foundation. 51
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VELDHUIS ET AL.
52
amplitude or frequency of pulsatile hormone release requires intensive and extended venous sampling procedures to capture the majority of spontaneous hormone release episodes that occur in vivo (35, 36). Moreover, to calculate endogenous hormone secretory rates, quantitative estimates of subject-specific MCRs are required (36, 37). Here, we have applied an intensive venous sampling paradigm and recent developments in deconvolution technology to evaluate quantitatively subjectspecific GH secretory events and endogenous GH MCRs in individual subjects in vivo (36, 37). In particular, we tested the following three null hypotheses: 1) the numbers of spontaneous GH secretory bursts per 24 h are not different in obese and normal weight men; 2) the amplitude, duration, and mass of GH secreted per spontaneous pulse are not different in obese and control individuals; and 3) the subject-specific half-life of disappearance of endogenous GH is no different in obese and control subjects. The resultant analyses have allowed us to reject certain of these null hypotheses with high probability, thus implicating specific mechanisms that subserve the hyposomatotropism of obesity in man.
JCE & M • 1991 Vol 72 • No 1
were extrapolated from and expressed in relation to GH standards diluted in human serum (38, 39). The assay sensitivity was 0.08 ng/dL. The serum somatomedin-C content was determined by Nichols Laboratories. GH-binding protein was assayed in each subject in a pool of 24-h sera using the methods described previously (40). In brief, [125I]GH (-50,000 cpm) was incubated overnight with 50 nL patient sera with or without 5 ng human recombinant GH. The reaction mixture was added to an Ultrogel AcA 44 minicolumn (0.9 X 30 cm) and eluted with phosphate-buffered saline (pH 7.4) containing 0.5% BSA at a rate of 300 /uL/min. The same column was always used for paired elution of any individual's sample. [125I]GH eluted from the column in two peaks, the first representing [125I]GH bound to GH-binding protein. The specific binding was defined as tracer bound in the first peak minus that bound in the presence of excess GH, expressed as a percentage of the total counts recovered. The inter- and intracolumn coefficients of variation in measurement were 3.9% and 2.8%, respectively. The level of specific binding of [125I]GH may be reduced by the level of endogenous GH in serum. Our studies have shown that endogenous GH concentrations below 40 mlU/L (20 ^g/L) d° not have a significant effect in reducing specific binding of tracer (unpublished data). In the present study all serum samples contained GH concentrations below this value.
Materials and Methods Clinical protocol Pulsatile GH secretion and endogenous GH clearance were studied in 7 control and 10 healthy obese men (see body mass index below). The mean ages (±SEM) of the normal and obese subjects were, respectively, 48 ± 4.7 and 40 ± 3.1 yr (P = NS). All subjects were studied after they provided written informed consent for the study, which was approved by the Human Investigation Committee of the University of Virginia School of Medicine. Each subject had an unremarkable clinical history and physical examination, with normal fasting plasma glucose and biochemical tests of renal, hepatic, and hematological function, and normal morning serum concentrations of T4 and/ or TSH, PRL, estradiol, testosterone, immunoactive LH and FSH, and somatomedin-C. Participants were admitted to the Clinical Research Center the evening or morning before study, and then underwent blood sampling at 10-min intervals for 24 h beginning at 0800 h, at least 1 h after venipuncture. Blood was withdrawn through an iv catheter placed in a forearm vein. Subjects were permitted to ambulate, were given 3 meals/day (0800,1200, and 1800 h) with ad libitum calories, and were not permitted to nap or sleep until 2200 h. The blood was permitted to clot at room temperature, and the subsequent sera were frozen for later immunoradiometric assay (IRMA) of GH. Assays Serum GH concentrations were determined in replicate (duplicate) by IRMA (Nichols Laboratories, San Juan Capistrano, CA), in which the median intraassay coefficient of variation determined from all 145 replicated samples in each subject averaged 5.7% (range, 2.3-10.7%) in the 7 normal subjects and 5.1% (2.7-10.1%) in the 10 obese subjects (P = NS). All data
Index of obesity The body mass index, defined as the ratio of the subject's weight in kilograms to the square of the subject's height in meters, averaged 28 ± 1.3 (range, 23-33) in the normal weight subjects and 46 ± 1.6 (range, 41-58) in the obese (P < 0.001). Absolute weights were 89 ± 5.0 kg (range, 68-107) in the control men and 145 ± 8.6 kg (range, 113-199) in the obese volunteers (P < 0.001). Deconuolution modeling Deconvolution provides quantitative estimates of individual secretory parameters and the calculation of subject-specific endogenous MCRs by using all serum hormone concentrations and their variances considered simultaneously (36, 37). In this biophysical model of hormone secretion and clearance, we assume that the plasma concentration of GH at any given instant reflects the simultaneous operation of four distinct, finite, and determinable secretory and clearance parameters of interest: 1) the locations, 2) the amplitudes, and 3) the durations of all GH secretory bursts, acted upon by 4) endogenous subject-specific clearance kinetics, as described previously (37). The secretory function, which denotes the secretory rate as a function of time, was constructed as a finite series of discrete secretory bursts, with determinable, statistically bounded amplitudes, half-durations (duration at half-maximal amplitude), and temporal positions (times) distributed randomly throughout the sampling interval. No tonic secretion function was required to model the present data. A distinct secretory burst was defined algebraically as a random (Gaussian) distribution of instantaneous molecular secretory rates (37), whose fitted amplitude could be distinguished from zero (pure noise) at P
Vol. 72, No. 1 Printed in U.S.A.
Dual Defects in Pulsatile Growth Hormone Secretion and Clearance Subserve the Hyposomatotropism of Obesity in Man* JOHANNES D. VELDHUIS, ALI IRANMANESH, KEN K. Y. HO, MICHAEL J. WATERS, MICHAEL L. JOHNSON, AND GERMAN LIZARRALDE Division of Endocrinology and Metabolism and the Interdisciplinary Graduate Biophysics Program, Departments of Internal Medicine and Pharmacology, University of Virginia (J.D. V., M.L.J.), Charlottesville, Virginia 22908; Salem Veterans Administration Hospital (A.I., G.L.), Salem, Virginia 24153; and Garvan Institute of Medical Research, St. Vincent's Hospital (K.K.Y.H.), Darlinghurst, New South Wales, and the Department of Physiology and Pharmacology, University of Queensland (M.J. W.), Queensland 4067, Australia
significantly prolonged mean intersecretory burst intervals (282 ± 65 vs. 131 ± 11 min; P < 0.05). The resultant daily GH production rate in obese men was reduced to one fourth that in normal weight individuals. Both GH secretion rate and burst frequency were negatively correlated with the degree of obesity (ponderal index). The decreases in GH burst frequency and halflife were specific, since GH secretory pulse amplitude (maximal rate of GH release), the mass of GH released per burst, and the duration of computer-resolved GH secretory bursts were not different in obese and normal weight men. We conclude that obese men harbor a double defect in GH dynamics involving both GH secretion and clearance, and that the severity of the GH secretory deficiency is proportionate to the degree of obesity. (J Clin Endocrinol Metab 72: 51-59, 1991)
ABSTRACT. We have examined the mechanisms underlying reduced circulating GH concentrations in the obese human. Computer-assisted (deconvolution) analysis was used to determine endogenous GH secretory and clearance rates quantitatively from entire 24-h plasma GH concentration profiles. These analyses revealed that the half-life (ti/2) of endogenous GH was significantly shorter in obese (11.7 ± 1.6 min) than in normal weight subjects (15.5 ± 0.81 min; P < 0.01). The accelerated blood disposal rate of GH was not due to decreased circulating concentrations of GH-binding protein, since the latter were similar in obese (25 ± 1.0%) and normal weight (24 ± 2.3%) men. However, obese men had significantly fewer GH secretory bursts (3.2 ± 0.53 us. 9.7 ± 0.67/day; P < 0.01). Among the rare GH secretory bursts that occurred in obese subjects, there were
U
in man are associated with profound increases in the frequency and amplitude of intermittent GH release (9, 10). In rats, major changes in GH pulse properties (albeit directionally opposite those in man) also occur when glucose concentrations are altered (11, 12). On the other hand, suppressed mean circulating GH concentrations have been recognized in obesity, with diminished acute secretory responses to dynamic stimuli such as insulin, arginine, opiates, glucagon, levo-dopa, sleep, and GHreleasing hormones (GHRH) (13-33). Such observations have suggested the hypothesis that one or more neuroregulatory defects in pulsatile GH secretion exist in obese individuals and result in hyposomatotropism. However, the explicit nature of such regulatory defects is not known. In addition, studies in nonhuman species have demonstrated accelerated metabolic clearance of exogenously administered GH in some obese animals (34), but not in others (13), suggesting that increased metabolic disposal of GH may contribute to hyposomatotropism. The precise and valid assessment of alterations in the
NDER a variety of experimental and physiological conditions, the pulsatile mode of GH, glucagon, insulin, or ACTH secretion conveys important signaling information to target tissues (1-4). Moreover, patterns of episodic GH release are susceptible to physiological regulation and specific pathophysiological alterations (58). For example, sex steroids, age, gender, puberty, and nutritional status, including feeding and short term fasting, all modulate the pulsatile mode of GH release (510). Short term caloric deprivation and diabetes mellitus Received May 23,1990. Address all correspondence and requests for reprints to: Dr. Johannes D. Veldhuis, Division of Endocrinology and Metabolism, Box 202, Department of Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908. * This work was supported in part by NIH Grant RR-00847 (to the Clinical Research Center of the University of Virginia), Research Career Development Award 1-KO4-HD-00634 (to J.D.V.), NIH Grant GM-28928 (to M.L.J.), Diabetes and Endocrinology Research Center Grant NIH DK-38942, NIH-supported Clinfo Data Reduction Systems, V.A. Medical Research Funds (to A.I. and G.L.), and support by the Pratt Foundation. 51
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 14:38 For personal use only. No other uses without permission. . All rights reserved.
VELDHUIS ET AL.
52
amplitude or frequency of pulsatile hormone release requires intensive and extended venous sampling procedures to capture the majority of spontaneous hormone release episodes that occur in vivo (35, 36). Moreover, to calculate endogenous hormone secretory rates, quantitative estimates of subject-specific MCRs are required (36, 37). Here, we have applied an intensive venous sampling paradigm and recent developments in deconvolution technology to evaluate quantitatively subjectspecific GH secretory events and endogenous GH MCRs in individual subjects in vivo (36, 37). In particular, we tested the following three null hypotheses: 1) the numbers of spontaneous GH secretory bursts per 24 h are not different in obese and normal weight men; 2) the amplitude, duration, and mass of GH secreted per spontaneous pulse are not different in obese and control individuals; and 3) the subject-specific half-life of disappearance of endogenous GH is no different in obese and control subjects. The resultant analyses have allowed us to reject certain of these null hypotheses with high probability, thus implicating specific mechanisms that subserve the hyposomatotropism of obesity in man.
JCE & M • 1991 Vol 72 • No 1
were extrapolated from and expressed in relation to GH standards diluted in human serum (38, 39). The assay sensitivity was 0.08 ng/dL. The serum somatomedin-C content was determined by Nichols Laboratories. GH-binding protein was assayed in each subject in a pool of 24-h sera using the methods described previously (40). In brief, [125I]GH (-50,000 cpm) was incubated overnight with 50 nL patient sera with or without 5 ng human recombinant GH. The reaction mixture was added to an Ultrogel AcA 44 minicolumn (0.9 X 30 cm) and eluted with phosphate-buffered saline (pH 7.4) containing 0.5% BSA at a rate of 300 /uL/min. The same column was always used for paired elution of any individual's sample. [125I]GH eluted from the column in two peaks, the first representing [125I]GH bound to GH-binding protein. The specific binding was defined as tracer bound in the first peak minus that bound in the presence of excess GH, expressed as a percentage of the total counts recovered. The inter- and intracolumn coefficients of variation in measurement were 3.9% and 2.8%, respectively. The level of specific binding of [125I]GH may be reduced by the level of endogenous GH in serum. Our studies have shown that endogenous GH concentrations below 40 mlU/L (20 ^g/L) d° not have a significant effect in reducing specific binding of tracer (unpublished data). In the present study all serum samples contained GH concentrations below this value.
Materials and Methods Clinical protocol Pulsatile GH secretion and endogenous GH clearance were studied in 7 control and 10 healthy obese men (see body mass index below). The mean ages (±SEM) of the normal and obese subjects were, respectively, 48 ± 4.7 and 40 ± 3.1 yr (P = NS). All subjects were studied after they provided written informed consent for the study, which was approved by the Human Investigation Committee of the University of Virginia School of Medicine. Each subject had an unremarkable clinical history and physical examination, with normal fasting plasma glucose and biochemical tests of renal, hepatic, and hematological function, and normal morning serum concentrations of T4 and/ or TSH, PRL, estradiol, testosterone, immunoactive LH and FSH, and somatomedin-C. Participants were admitted to the Clinical Research Center the evening or morning before study, and then underwent blood sampling at 10-min intervals for 24 h beginning at 0800 h, at least 1 h after venipuncture. Blood was withdrawn through an iv catheter placed in a forearm vein. Subjects were permitted to ambulate, were given 3 meals/day (0800,1200, and 1800 h) with ad libitum calories, and were not permitted to nap or sleep until 2200 h. The blood was permitted to clot at room temperature, and the subsequent sera were frozen for later immunoradiometric assay (IRMA) of GH. Assays Serum GH concentrations were determined in replicate (duplicate) by IRMA (Nichols Laboratories, San Juan Capistrano, CA), in which the median intraassay coefficient of variation determined from all 145 replicated samples in each subject averaged 5.7% (range, 2.3-10.7%) in the 7 normal subjects and 5.1% (2.7-10.1%) in the 10 obese subjects (P = NS). All data
Index of obesity The body mass index, defined as the ratio of the subject's weight in kilograms to the square of the subject's height in meters, averaged 28 ± 1.3 (range, 23-33) in the normal weight subjects and 46 ± 1.6 (range, 41-58) in the obese (P < 0.001). Absolute weights were 89 ± 5.0 kg (range, 68-107) in the control men and 145 ± 8.6 kg (range, 113-199) in the obese volunteers (P < 0.001). Deconuolution modeling Deconvolution provides quantitative estimates of individual secretory parameters and the calculation of subject-specific endogenous MCRs by using all serum hormone concentrations and their variances considered simultaneously (36, 37). In this biophysical model of hormone secretion and clearance, we assume that the plasma concentration of GH at any given instant reflects the simultaneous operation of four distinct, finite, and determinable secretory and clearance parameters of interest: 1) the locations, 2) the amplitudes, and 3) the durations of all GH secretory bursts, acted upon by 4) endogenous subject-specific clearance kinetics, as described previously (37). The secretory function, which denotes the secretory rate as a function of time, was constructed as a finite series of discrete secretory bursts, with determinable, statistically bounded amplitudes, half-durations (duration at half-maximal amplitude), and temporal positions (times) distributed randomly throughout the sampling interval. No tonic secretion function was required to model the present data. A distinct secretory burst was defined algebraically as a random (Gaussian) distribution of instantaneous molecular secretory rates (37), whose fitted amplitude could be distinguished from zero (pure noise) at P
