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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 9 4412-4415
Copyright © 2001 by The Endocrine Society

Other Original Articles

The Acute Leptin Response to GH

Catherine A. Lissett, Peter E. Clayton and Stephen M. Shalet

Department of Endocrinology, Christie Hospital, and Academic Unit of Child Health, University of Manchester (P.E.C.), Manchester, United Kingdom M20 4BX

Address all correspondence and requests for reprints to: Prof. S. M. Shalet, Department of Endocrinology, Christie Hospital, National Health Service Trust, Wilmslow Road, Manchester, United Kingdom M20 4BX. E-mail: stephen.m.shalet{at}man.ac.uk

Abstract

The effect of an acute bolus of GH on serum leptin in normal individuals and the factors affecting this response have not previously been studied. Seventeen healthy volunteers with normal body mass index, with ages ranging from 20.5–78.2 yr were studied. Each subject received three single doses of GH in random order at least 4 wk apart. Bioimpedence analysis was performed to provide estimates of fat and lean masses. Serum samples for leptin, insulin, and IGF-I were taken 0, 18, 24, 48, 72, and 120 h after each dose of GH.

Leptin levels changed significantly after the 0.67- and 7-mg doses of GH, but not after the 0.27-mg dose. Compared with baseline, there was a significant elevation (P < 0.001) in serum leptin levels at 24 h, followed by a significant decrease (P < 0.01) at 72 h.

Baseline and peak leptin levels were significantly determined by gender, fat mass, and log10 insulin. Nadir leptin levels were significantly determined by gender and fat mass. In contrast, the increment in leptin levels was significantly determined by age, although this only accounted for 24% of the variability in the increment in leptin levels.

We have demonstrated that administration of a single bolus dose of GH significantly increases serum leptin levels, followed by a significant nadir. This occurs not only after a supraphysiological dose of GH, but also after 0.67 mg, a dose within the physiological replacement range. The increment in leptin increases with advancing age, suggesting that at the level of the adipocyte, aging increases responsiveness to GH. However, this only partially explains the changes seen, and it is likely that another factor(s) is involved in the acute impact of GH on circulating leptin levels. The presence of a significant nadir after the peak in leptin levels supports the existence of a negative feedback loop, linking circulating leptin to its own biosynthesis in adipose tissue, mediated by peripheral leptin receptors.

These data provide unequivocal evidence that GH can affect serum leptin levels in the absence of a change in body composition.

LEPTIN, THE PRODUCT of the obese (ob) gene (1), is a circulating hormone that appears to be part of a signaling pathway from adipose tissue to the brain and may play a role in appetite control and the regulation of energy expenditure. Circulating leptin levels reflect the degree of adiposity; leptin levels are elevated in obesity (2), decreased in anorexia nervosa (3), and positively correlated with the percentage of body fat (4). However, serum concentrations of leptin do not simply reflect the amount of adipose tissue within an individual. Leptin levels have a diurnal rhythm in both obese and normal subjects, with peak levels at night and a nadir in the morning (5). Leptin fluctuates with spontaneous follicular maturation (6), and E2 exposure appears to increase leptin secretion (7, 8), suggesting a direct effect of E2 on adipose tissue. In addition, leptin levels are positively associated with insulin concentrations and are increased after insulin administration.

The relationship between GH and leptin is interesting because GH has profound effects on body composition. GH deficiency in adults is associated with increased body fat and decreased lean mass, paralleled by an increase in serum leptin concentrations, and GH replacement therapy is associated with a reduction in fat mass and an increase in lean mass after both short- and long-term treatment. A further aspect of the relationship between GH and leptin is that obesity is associated with reduced spontaneous and stimulated GH secretion in humans (9). The peripheral signals that mediate this reduction are postulated to be FFA and leptin. FFA and GH maintain a classical regulatory feedback loop. GH elicits a rise in FFA by its lipolytic action and a rise in FFA inhibits spontaneous and stimulated GH secretion (10). A great deal less is known about the role of leptin, with much of the data being derived from animal models, which, in general, differ from those in humans. Assuming that leptin regulates GH secretion, to establish a classical feedback loop it is necessary to show that GH participates in the regulation of leptin secretion. No clear information exists in this respect. A number of studies in GH-deficient humans have examined the effect of chronic GH replacement and have demonstrated a decrease in serum leptin levels (11, 12). This probably reflects the changes in body composition that accompany GH therapy rather than a direct effect on leptin per se. Gill et al. (11), however, reported the effects of a supraphysiological bolus of GH and demonstrated an initial increase, followed by a decrease, in serum leptin in GH-deficient elderly patients. We examined whether this was the case in normal individuals after doses of GH that included those within the physiological replacement range used for patients with GH deficiency in adulthood.

Subjects and Methods

Subjects

Seventeen healthy subjects (nine men and eight women) with normal body mass index (mean ± SD, 25.1 ± 3.49 kg/m2) and ages ranging from 20.5–78.2 (median, 49.5) yr, were studied. All individuals were healthy and underwent an examination to exclude previously undiagnosed medical complaints. None was taking any medication known to affect the GH/IGF-I axis. Ethical committee approval and informed consent were obtained.

Study protocol

Subjects received three single sc doses of GH, 0.27, 0.67, and 7 mg (Genotropin, Pharmacia & Upjohn, Inc., Stockholm, Sweden; 1 mg = 3 IU), in random order at least 4 wk apart. Serum samples for leptin, insulin, and IGF-I determinations were taken 0, 18, 24, 48, 72, and 120 h after each dose of GH.

Body composition

Bioelectrical impedance analysis was measured with a Tanita body fat analyzer TBF-305 (Tanita Corp., Uxbridge, UK) with an alternating current of 50 kHz, 500 µA.

Assays

Serum IGF-I was measured after acid-alcohol extraction using an in-house RIA. The intraassay coefficients of variation for mean IGF-I concentrations of 45, 243, and 698 ng/ml were 9.0%, 6.5%, and 4.7%, respectively. The sensitivity of the assay was 13 ng/ml.

Serum leptin was measured by RIA (Linco Research, Inc., St. Charles, MO). The sensitivity of the assay was 0.5 ng/ml. The intraassay coefficients of variation for mean serum concentrations of 4.9, 10.4, and 25.6 ng/ml were 8.3%, 3.9%, and 3.4%, respectively.

Serum insulin was measured using an ELISA (Diagnostic Systems Laboratories, Inc., Webster, TX). The sensitivity of the assay was 0.1 ng/ml. The intraassay coefficients of variation for mean serum concentrations of 0.34 and 1.77 ng/ml were 1.3% and 2.6%, respectively.

Statistical analysis

Leptin and insulin were log10 transformed before statistical analysis, and the results are presented as the geometric mean (-1, +1 tolerance factor). All other data are presented as the mean ± 1 SD. ANOVA for repeated measures was used to examine changes in variables over time. Once a statistically significant change was detected over time, a paired t test was used to compare baseline, peak, and nadir values.

Results

The only side effect noted was mild transient ankle swelling at the 7 mg dose in one female subject. Leptin levels changed significantly after the 0.67- and 7-mg doses of GH (P < 0.0001), but not after the 0.27-mg dose (P = 0.5). Initially, there was a significant elevation in serum leptin levels at a median time of 24 h (range, 18–24 h). Leptin levels increased from 7.3 (-1, +1 tolerance factor, 2.9, 18.6) ng/ml to 9.1 (-1, +1 tolerance factor, 3.9, 21.0) ng/ml (P < 0.001) and from 8.1 (-1, +1 tolerance factor, 3.2, 21.4) ng/ml to 10.7 (-1, +1 tolerance factor, 8.9, 24.7) ng/ml (P < 0.0001) after the 0.67- and 7-mg doses of GH, respectively. Subsequently, there was a significant decrease in leptin concentrations compared with baseline at a median time of 72 h (range, 48–120 h; see Fig. 1Go). Leptin levels declined from 7.3 (-1, +1 tolerance factor, 2.9, 18.6) ng/ml to 6.12 (-1, +1 tolerance factor, 2.5, 15.2) ng/ml (P < 0.01) and from 8.1 (-1, +1 tolerance factor, 3.2, 21.6) ng/ml to 6.4 (-1, +1 tolerance factor, 2.4, 17.3) ng/ml (P < 0.0001) after the 0.67- and 7-mg doses of GH, respectively.



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  		Figure 1. The effect of a single bolus of GH on median serum leptin levels. The arrows indicate the median (range) time from baseline to peak and nadir concentrations.

 
Serum insulin levels only changed significantly after the 7-mg dose of GH (P < 0.01). There was a significant rise in insulin levels at 18 h, from 0.34 (-1, +1 tolerance factor, 0.12, 0.95) ng/ml to 1.15 (-1, +1 tolerance factor, 0.57, 2.29) ng/ml. There was no nadir, in contrast to the leptin response to GH.

IGF-I levels increased significantly after all three doses of GH (P < 0.01, P < 0.0001, and P < 0.0001 for 0.27, 0.67, and 7 mg GH, respectively). Levels increased from 208.2 (74.6) ng/ml to 260.0 (99.7) ng/ml (P < 0.001), from 206.8 (92.4) ng/ml to 299.4 (86.2) ng/ml (P < 0.0001), and from 206.8 (79.1) ng/ml to 431.8 (126.6) ng/ml (P < 0.0001) after 0.27, 0.67, and 7 mg GH, respectively.

Stepwise multiple linear regression analysis was used to examine determinants of leptin concentrations at baseline, peak, and nadir as well as the increment in serum leptin. Log10 leptin was entered as the dependent variable, and gender, age, fat mass, lean mass, the matched log10 insulin and IGF-I values, and GH dose were entered as the dependent variables, except when baseline leptin was examined when GH dose was omitted (Fig. 2Go).



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  		Figure 2. Correlation between log10 increment in leptin and age.

 
Gender contributed 64% toward the variability in baseline leptin levels (positive in women, negative in men; P < 0.001; r2 = 0.64); in addition, fat mass contributed a further 14% (positive; P < 0.001; r2 = 0.78), and log10 (insulin) contributed an additional 3% (positive; P < 0.01; r2 = 0.81). Similarly, peak leptin levels were significantly determined by gender, fat mass, and log10 insulin (see Table 1Go). Nadir leptin levels were significantly determined by gender and fat mass (see Table 1Go).


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  		Table 1. Stepwise multiple linear regression analysis of variables affecting serum leptin levels

 
In contrast, the increments in leptin levels were only significantly determined by age (positive), although this only accounted for 24% of the variability in the increment in leptin levels (P < 0.01; r2 = 0.24). The increment in IGF-I was significantly associated with the increment in leptin (P < 0.01; r2 = 0.22); however, this association disappeared when the effect of age was taken into consideration. There was no association between the increment in leptin and that in insulin (P = 0.09), gender, or fat mass (P = 0.1).

Discussion

We have examined the leptin response to three different bolus doses of GH in healthy individuals, aged 20–78 yr. Leptin levels initially increased and then decreased below baseline after the two higher doses of GH (0.67 and 7 mg), but did not significantly change after the 0.27-mg dose of GH. The peak response occurred at a median time of 24 h; leptin levels then declined significantly to a nadir at a median time of 72 h compared with baseline values, although the timing of the peak and nadir leptin levels can only be approximately estimated in view of the time between each sample in this study. GH levels would be expected to peak between 3–4.5 h after administration of sc GH.

The mechanism behind this effect remains unknown. Mature adipocytes possess large numbers of GH receptors, but no functional IGF-I receptors (13); hence, one potential mechanism is that GH directly regulates ob gene expression.

Some animal data are available to shed light on the situation, although an important caveat is that the relationship between GH and leptin in rodents appears to be a mirror image of that in the human. In rodents, GH can stimulate leptin release in the presence of the appropriate hormonal milieu; Boni Schnetzler et al. (14) in 1996 determined ob mRNA levels in epididymal fat pads of hypophysectomized rats treated with GH and normal, weight-matched controls and found that ob mRNA was markedly suppressed after hypophysectomy, but that GH infusion had no effect on ob mRNA. Fain and Bahouth (15) examined the effects of GH on leptin release by cultured rat adipose tissue incubated for 24 h in primary culture. Stimulation of leptin release by GH was found in the presence of 25 nmol/liter dexamethasone, and this was accompanied by a 28% increase in leptin mRNA content.

A negative feedback loop linking circulating leptin to its own biosynthesis would potentially explain the nadir in leptin levels that we have demonstrated at 72 h. Wang et al. (16), examining the effects of both intracerebroventricular and systemic infusions of leptin in rats, demonstrated that moderate increases in circulating leptin levels considerably decreased ob gene expression in adipose tissue. The researchers hypothesized the existence of such a loop, mediated by peripheral leptin receptors.

Human data in this field are limited. The extent and pattern of the leptin response to GH that we have demonstrated is similar to those reported by Gill et al. (11), although the latter group used a single dose of 7 mg/kg in GH-deficient elderly patients. The finding of a nadir in leptin levels is also a common feature. The time course of this decrease is such that it is unlikely to be secondary to negative feedback via the hypothalamus. Our observations suggest that a feedback loop similar to that postulated by Wang et al. (16) exists in humans, as described above. The demonstration by Bornstein et al. (17) that the leptin receptor (Ob-r) in human white adipose tissue is not restricted to adipocytes, but is also present in resident endothelial and immune cells, suggesting important autocrine and paracrine roles for leptin in human adipose tissue, further supports this hypothesis.

It is interesting to observe that although insulin significantly determined leptin levels at baseline and peak, contributing 3% and 4%, respectively, to the variability in serum leptin, it did not contribute significantly to the increase in serum leptin. This fact suggests that the elevation in circulating insulin in response to GH is not responsible for the increase in serum leptin. Also in support of this suggestion is that insulin only significantly increased after the 7-mg dose of GH.

When interpreting the results of the linear regression, an important caveat is that bioelectrical impedance, used to calculate lean mass and fat mass, is not a gold standard method under all circumstances and hence could introduce errors into the linear regression calculation. These errors should be small, however, as values calculated by bioelectrical impedance should be close to actual values in adults of normal body composition, such as those used in this study.

The only significant determinant of the increase in serum leptin was age, with a positive relationship seen between advancing age and the increment in serum leptin. This suggests that increasing age is associated with increasing responsiveness to GH at the level of the adipocyte. Studies of the effect of age on unstimulated serum leptin levels are contradictory, showing both an increase and a decrease in leptin levels with age (18, 19, 20). The changes in body composition and sex steroid levels that accompany aging are confounding variables that complicate interpretation of these data.

In summary, we have demonstrated that a single bolus of GH, including a dose within the physiological replacement range, increases serum leptin levels. Further studies are required to determine the mechanism mediating this effect; however, these data provide unequivocal evidence that GH can affect serum leptin levels in the absence of a change in body composition.

Acknowledgments

Received January 5, 2001.

Accepted May 16, 2001.

References

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  7. Lavoie HB, Taylor AE, Sharpless JL, Anderson EJ, Strauss CC, Hall JE 1999 Effects of short-term hormone replacement on serum leptin levels in postmenopausal women. Clin Endocrinol (Oxf) 51:415–422[CrossRef][Medline]
  8. Shimizu H, Shimomura Y, Nakanishi Y, et al. 1997 Estrogen increases in vivo leptin production in rats and human subjects. J Endocrinol 154:285–292[Abstract/Free Full Text]
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