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Age Dimorphism in the Association between Growth-Hormone Status and the Respiratory Quotient Jens J. Christiansen, Nina Vahl, Sanne Fisker, Niels Møller, Jens S. Christiansen, and Jens O.L. Jørgensen

Abstract CHRISTIANSEN, JENS J., NINA VAHL, SANNE FISKER, NIELS MØLLER, JENS. S. CHRISTIANSEN, AND JENS O.L. JØRGENSEN. Age dimorphism in the association between growth-hormone status and the respiratory quotient. Obes Res. 2002;10:284 –290. Objective: To investigate the impact of age on the association between the respiratory quotient (RQ) and growthhormone (GH) secretion and to investigate the acute lipolytic response to an exogenous GH bolus. Research Methods and Procedures: A cross-sectional study of 36 non-obese healthy subjects (18 women and 18 men) from two age groups was used: “younger” (mean age, 29.5 years; range, 27 to 34 years) and “older” (mean age, 50.8 years; range, 47 to 59 years). Endogenous GH secretion by means of deconvolution analysis of 24-hour serum GH concentrations was measured every 20 minutes. Resting RQ was measured after a 12-hour overnight fast. The lipolytic response to an intravenous exogenous GH bolus (200 ␮g) was assessed by measuring serum levels of free fatty acids as well as changes in RQ. Additional measurements included body composition (regional computed tomography scan and DXA) and physical fitness (VO2max). Results: Resting RQ did not differ between the two age groups: 0.81 ⫾ 0.01 (young) vs. 0.82 ⫾ 0.01 (older; not significant). Several estimates of GH release correlated positively with RQ in the younger group, whereas a negative correlation was detected in the older subjects [GH production rate (␮g/liter ⫻ kg) vs. RQ: r ⫽ 0.62, p ⬍ 0.01 (younger); r ⫽ ⫺0.53; p ⫽ 0.02 (older)]. By regression analysis, 52% to 58% of the variation in RQ could be

Submitted for publication May 1, 2001. Accepted for publication in final form January 2, 2002. Medical Department M (Endocrinology and Diabetes), Aarhus University Hospital and Institute of Experimental Clinical Research, Aarhus University, Aarhus, Denmark. Address correspondence to Jens O.L. Jørgensen, Medical Department M, Aarhus Kommunehospital, DK 8000 C Aarhus, Denmark. E-mail: [email protected] Copyright © 2002 NAASO

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explained by GH status. After an exogenous GH bolus, the incremental response in nonesterified fatty acid was slightly higher in younger individuals (p ⫽ 0.09). Discussion: Resting RQ is significantly correlated with GH status. This association is positive in younger individuals and negative in older individuals. The lipolytic response to exogenous GH is moderately higher in younger compared with older individuals. GH status should be taken into account when investigating the residual variation in RQ. Key words: body composition, lipolysis, deconvolution analysis, cross-sectional study

Introduction Obesity is a prevalent condition associated with excess morbidity and mortality (1). It is therefore important to gain further insight into the physiological regulation of body weight. A high respiratory quotient (RQ), i.e., a low fat-to-carbohydrate oxidation rate, has been shown to be a predictor of subsequent weight gain (2,3). In subjects on a balanced weight-maintaining diet, the RQ after an overnight fast was ⬃0.80, reflecting predominant oxidation of fat. There is, however, an interindividual variation in RQ, not accounted for by diet (4), that is not fully understood. Putative predictors include familial traits, sex, age (2,5), adiposity (2,6,7), and muscle sympathetic nerve activity (8). Growth hormone (GH) regulates lipid and glucose metabolism (9 –11). Active acromegaly is associated with enhanced lipid oxidation and reduced glucose oxidation (12), and even a physiological GH bolus given to healthy adults is accompanied by a significant stimulation of lipolysis (11). Moreover, GH secretion declines with age and is higher in women than in men. It has, however, recently been reported that these associations are secondary to a strong inverse correlation between abdominal fat mass (13). Overall, these data indicate that GH status is a significant regulator of fat metabolism in adults. It

Growth Hormone and the Respiratory Quotient, Christiansen et al.

Table 1. Data of the study population: body composition, GH status, physical fitness, and RQ: differences between the two age groups Variable Age (yr) BMI (kg/m2) WH Intra-abdominal fat (cm2) LBM (%) IGF-I (␮g/l) VO2MAX (mL/min ⫻ kg) RQ GH production rate (␮g/liter ⫻ kg) GH burst (no.) GH mass (␮g/liter) Mean 24-hour GH (␮g/liter)

Younger

Older

p

29.6 ⫾ 0.4 22.8 ⫾ 0.6 0.82 ⫾ 0.02 53.5 ⫾ 6.5 0.79 ⫾ 0.01 169.1 ⫾ 11.0 50.1 ⫾ 2.3 0.81 ⫾ 0.01 31.2 ⫾ 5.4 9.6 ⫾ 0.8 3.5 ⫾ 0.6 0.69 ⫾ 0.1

51.0 ⫾ 0.8 25.7 ⫾ 0.8 0.90 ⫾ 0.02 142.0 ⫾ 17.4 0.74 ⫾ 0.01 145.8 ⫾ 9.3 34.0 ⫾ 2.3 0.82 ⫾ 0.01 18.7 ⫾ 3.2 10.7 ⫾ 0.7 1.7 ⫾ 0.3 0.41 ⫾ 0.1

0.00 0.01 0.01 0.00 0.03 NS 0.00 NS 0.05 NS 0.01 NS

GH, growth hormone; RQ, respiratory quotient; BMI, body mass index; WH, waist/hip ratio; LBM, lean body mass; IGF-I, insulin-like growth factor; NS, not significant.

remains to be assessed if an association exists in different age groups between GH secretion and RQ and also whether the lipolytic response to GH administration changes with age. We therefore evaluated the relationship between resting RQ and spontaneous 24-hour GH secretion in healthy adults of two separate age groups. Our study also included detailed measures of body composition, physical fitness, and the acute metabolic response to a GH bolus.

Research Methods and Procedures Subjects Thirty-six healthy adults (18 women and 18 men) participated in the study (Table 1). They comprised two distinctly separate age groups: “younger” (n ⫽ 18; mean age ⫾ SE, 29.6 ⫾ 0.4 years; range, 27 to 34 years) and “older” (n ⫽ 18; mean age ⫾ SE, 51.0 ⫾ 0.8 years; range, 47 to 59 years). Premenopausal women were studied during the early follicular phase of the menstrual cycle and

Table 2. Pearson’s product-moment correlation: r and p values in the two age groups Respiratory quotient All Variable Intra-abdominal fat IGF-I GH production rate GH mass GH burst Mean 24-hour GH ⌬ NEFAAUC VO2max

Younger

Older

r

p

r

p

r

p

0.09 0.26 0.17 0.17 0.13 0.28 ⫺0.11 ⫺0.02

0.61 0.14 0.33 0.33 0.44 0.10 0.51 0.67

⫺0.45 0.12 0.62 0.49 0.34 0.7 ⫺0.11 0.48

0.07 0.65 ⬍0.01 0.04 0.16 ⬍0.01 0.66 0.05

0.22 0.42 ⫺0.53 ⫺0.59 0.05 ⫺0.33 ⫺0.30 ⫺0.51

0.43 0.10 0.02 0.01 0.86 0.18 0.24 0.07

IGF-I, insulin-like growth factor I; GH, growth hormone; NEFA, nonesterified fatty acids.


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none received estrogen replacement. None of the subjects received any medications. The study was approved by the regional ethics committee, and all subjects gave their written informed consent. Data from this population on the association between body composition and GH status have been published previously (14,15). GH Status Starting at 9:00 AM after a 12-hour overnight fast, serum was sampled for GH measurements. Three meals a day were served, and subjects were allowed normal ambulatory activity until 11:00 PM, after which they remained supine. Deconvolution analyses on spontaneous GH concentrations, measured every 20 minutes for 24 hours, were performed by Veldhuis et al. (16) as previously described. Deconvolution analysis estimates used in this study were the daily production rate (micrograms per liter distribution volume per 24 hours), secretory burst amplitude (micrograms per liter per minute), mass secreted per burst (micrograms per liter), and mean 24-hour GH levels (micrograms per liter). Body Composition The amount of intra-abdominal (visceral) fat was evaluated by computed tomography with a Somatom Plus-S scanner (Siemens, Erlangen, Holland). All scans were performed by the same technician, and the scans were analyzed by the same radiologist in a blinded fashion. The percentages of body fat and lean body mass (LBM) were measured with DXA with a QDR-2000 densitometer (Hologic, Waltham, MA). Additional anthropometric measurements included body mass index (BMI) and waist-to-hip ratio. Physical Fitness Maximal oxygen consumption (VO2max) was estimated by means of cycle ergometry, according to the Aastrand protocol. RQ RQ and energy expenditure (EE) were assessed by indirect calorimetry. A computerized open-circuit system was used to measure gas exchanges across a 25-liter canopy (Deltatrac; Datex Instrumentarium, Helsinki, Finland). The monitor determines carbon dioxide production and oxygen consumption by multiplying dry air flow through the canopy with the alterations in gas concentrations over the canopy. The procedure was conducted on two occasions with the subject in the resting state after an overnight fast. The initial 5 minutes of calorimetry were used for acclimatization, and the calculations represent mean values of 2 ⫻ 25 measurements performed at 1-minute intervals. Lipolytic Response to GH Bolus After a 12-hour overnight fast, an intravenous bolus of 200 ␮g of biosynthetic human GH (Norditropin; Novo 286


Figure 1: Respiratory quotient in relation to indices of growth hormone secretion. Younger group.

Nordisk, Gentofte, Denmark) or saline was administered, in an exponentially declining fashion, over the course of 8 minutes. Nonesterified fatty acids (NEFAs) were measured at ⫺120, 0, 20, 60, 100, 140, 180, 220, 260, and 300 minute(s). The RQ was calculated from indirect calorimetry performed before and 120 minutes after the GH bolus.


Hormone and Lipid Analyses GH measurements were performed with a DELFIA assay (Wallac). Intra- and interassay coefficients of variation ranged from 1.8% to 3.0% and 1.6% to 2.3% for GH concentrations of 0.71 to 31.4 ␮g/liter, and the lower detection limit was, at most, 0.01 ␮g/liter. Insulin-like growth factor I (IGF-I) analyses were performed with an in-house, time-resolved immunofluorometric assay after extraction of serum to remove binding proteins, as previously described (17). NEFAs were determined with a commercial kit by a colorimetric method (Wako Chemicals, Neuss, Germany). Plasma triglycerides (TGs) were measured with a standard enzymatic kit. Statistics Comparisons of the variables between age groups were done with Student’s t-test. The strength of association between variables was estimated with Pearson’s product–moment correlation. Multiple linear regression and forward stepwise regression analyses were used to determine the strongest predictors of RQ. The z-score at any time-point was calculated with baseline values as references; i.e., (concentration at the actual time ⫺ concentration at time 0)/SD at time 0. Delta values expressed in z-scores were the differences in z-scores between the GH and saline infusions. Data not normally distributed were log-transformed before analysis. Data are given as means ⫾ SEM. Statistical significance was assumed when p ⬍ 0.05.

Results Physical characteristics, fitness, and hormone levels according to age are presented in Table 1. The older age group had a higher BMI and fat mass and a lower LBM, and the older group showed a higher proportion of central adiposity with an increased waist-to-hip ratio and intra-abdominal fat mass. Physical fitness, as estimated by VO2max, was higher in the younger group. Plasma TGs were significantly elevated in the older age group (p ⫽ 0.03). Spontaneous GH release was significantly increased in younger compared with older subjects. The RQ after an overnight fast did not differ between the two age groups: 0.81 ⫾ 0.01 (younger) vs. 0.82 ⫾ 0.01 (older; not significant). Simple correlation analysis (Table 2) revealed several indices of 24-hour spontaneous GH secretion (production rate, mass, and mean 24-hour GH level) that correlated positively to the RQ in the younger group (Figure 1), whereas significant negative correlations between GH variables and RQ were recorded in the older group (Figure 2). Subdividing age groups into men and women did not change the age dichotomy pattern (data not shown). Stepwise regression analysis showed that 58% of the variability in RQ (p ⬍ 0.01) was explained by the mean 24-hour GH level in the younger group, whereas in the older age group, 52% of the variability in RQ could be

Figure 2: Respiratory quotient in relation to indices of growth hormone secretion. Older group.

explained by production rate and mean 24-hour GH level (p ⫽ 0.02). Plasma TG levels were inversely correlated with several indices of GH secretion when the two age groups were combined, but no significant association was recorded between TGs and RQ when each age group was evaluated separately. The incremental response in NEFAs after the exogenous GH bolus was slightly higher in the younger group [⌬AUCNEFA (mM): 65.3 ⫾ 10.4 (younger) vs. 40.3 ⫾ 9.9 OBESITY RESEARCH Vol. 10 No. 4 April 2002

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Figure 3: Differences between age groups in the lipolytic response to a growth hormone bolus. F, younger; E, older.

(older); p ⫽ 0.09; Figure 3]. The GH-induced reduction in RQ did not, however, differ significantly between the two groups (Figure 4). Resting EE (REE) did not differ between the two age groups [REE/LBM (kcal/24 hours ⫻ kg): 31.0 ⫾ 07 (younger) vs. 32 ⫾ 0.9 (older)]. Total body fat was the single most important predictor of REE/LBM in multiple regression analysis including the entire population. No significant correlation existed between REE/LBM and RQ (R ⫽ 0.089; p ⫽ 0.605).

Discussion In this study we investigated the association between RQ after an overnight fast and spontaneous 24-hour GH secretion in healthy adults of both sexes. An age dimorphism occurred such that the correlation between RQ and GH secretion was positive in younger subjects but negative in older subjects. After an exogenous GH bolus, the increase in serum NEFA levels was slightly more pronounced in the younger group compared with the older group, whereas the accompanying decline in RQ did not differ between the two age groups. It is well-established that GH administration significantly reduces RQ in healthy adults (18,19), obese women (20), and GH-deficient patients (21). Moreover, a GH dose-dependent reduction in RQ has been observed in healthy adults (22), and active acromegaly is associated with a reduced RQ, which normalizes after successful adenomectomy (12). It is also accepted that GH secretion, in turn, is regulated by fat mass and circulating free fatty acids. In this study, intra-abdominal fat mass in these clinically nonobese subjects was the most important and negative determinant of both spontaneous and stimulated GH release (13,14). In experimental studies, circulating NEFAs suppressed GH secretion (23), but it remains unknown whether 288


Figure 4: Respiratory quotient before and after growth hormone infusion in the two age groups.

adiposity is the cause or the effect of reduced GH. The present report is, however, the first to evaluate the association between endogenous GH levels and fat oxidation. Based on our observations, we speculate that, in older subjects, endogenous GH was a primary stimulator of lipolysis and subsequent fat oxidation in the postabsorptive state. In contrast, in younger subjects, spontaneous GH secretion was positively correlated to RQ, and multiple regression analysis revealed GH secretion was a significant negative predictor of lipid oxidation in this age group. At the same time the ability of exogenous GH to acutely stimulate both lipolysis and lipid oxidation was fully preserved in the younger age group. Based on all available data, it is unlikely that GH directly increases RQ, i.e., stimulates postabsorptive carbohydrate oxidation. The observed correlation therefore seems to indicate that lipid oxidation in the younger age group is predominantly driven by non-GH-related mechanisms. The increase in NEFA levels would subsequently suppress GH release. GH also stimulates the production of IGF-I, which exerts insulin-like effects on glucose metabolism (i.e., increases RQ). Serum total IGF-I levels, however, did not differ significantly between the two age groups, and no correlation was detected between this parameter and RQ. Ghrelin, the endogenous ligand for the GH secretagogue receptor, has recently been identified in specific cells within the stomach (24). Ghrelin stimulates GH secretion at both the hypothalamic and pituitary levels, but ghrelin also exhibits orexigenic properties through GH-independent mechanisms. Theoretically, the net result of ghrelin-stimulated GH release on substrate metabolism could, therefore, be an increase in RQ. A possible association between age and ghrelin secretion remains to be investigated. There are certain limitations to this study that merit attention. First, prior changes in body weight and acute energy balance are major determinants of RQ (2) and were not accounted for in this study. If these features differed


between the two age groups, this could also influence the association between GH and RQ. Second, we only measured RQ in the postabsorptive state, which does not allow an extrapolation to 24-hour RQ. As expected, RQ was close to 0.80 in both groups, which reflects predominant oxidation of fat. Twenty-four-hour RQ during a balanced diet is subject to a wide, and largely unexplained, interindividual variation, which implies differences in the partitioning of fat and carbohydrate substrates for oxidation (2). The role of GH in this context would be a relevant factor to evaluate. Third, integrated estimates of 24-hour GH secretion are not necessarily the optimal parameters for evaluating the impact of GH on RQ after an overnight fast. Fourth, a crosssectional design cannot be used to predict longitudinal (prospective) intraindividual changes. It would therefore be of interest to make longitudinal observations in our study population to assess whether the association between GH status and RQ changes with age in a given individual. It is also noteworthy that GH may influence lipid metabolism through other mechanisms than stimulation of lipolysis (25– 27). Moreover, GH has been shown to facilitate hepatic uptake and degradation of lipoproteins (28). To what extent these effects of GH are age-dependent and ultimately influence RQ is presently unclear. Conflicting data exist regarding the association between sex and RQ (2,5,6), and we were unable to detect a sex difference in RQ. We observed no significant association between RQ and fat mass by either simple correlation or multiple regression analysis, even though intra-abdominal fat was weakly and negatively correlated with RQ in the younger subjects. This contrasts with previous reports (2,6,7) but may be explained by the fact that we only studied relatively lean subjects. In conclusion, the present data suggest a hitherto unrecognized age-dependent association between GH status and postabsorptive lipid oxidation in clinically non-obese subjects. The physiological significance remains to be further scrutinized, but we suggest that GH status should be taken into account when investigating the residual variation in RQ.

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