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Division of Medical Sciences (J.W.T., A.A.T., P.M.S.) and Department of Nuclear Medicine (N.C.), Queen Elizabeth Hospital, University of Birmingham, Birmingham, United Kingdom B15 2TH; Regional Endocrine Laboratory (P.M.S.C., G.H.), Department of Clinical Biochemistry, University Hospital Birmingham NHS Trust, Birmingham, United Kingdom B29 6JD; and Children’s Hospital (C.H.L.S.), Oakland Research Institute, Oakland, California 94609-1809
Address all correspondence and requests for reprints to: Prof. P. M. Stewart, M.D., F.R.C.P., F.Med.Sci., Division of Medical Sciences, University of Birmingham, Queen Elizabeth Hospital, Birmingham, United Kingdom B15 2TH. E-mail: p.m.stewart{at}bham.ac.uk.
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Clinical studies treating hypopituitary, GHD patients with recombinant GH have generally shown a decrease in adipose tissue mass (7), and this has prompted GH treatment studies in simple obesity (10, 11, 12, 13, 14). Results have been variable with some studies (13, 14) but not others (10, 11, 12), showing a reduction in fat mass in comparison with controls. However, without exception, when GH treatment has been prolonged in simple obesity, the doses of GH used have been large (0.7–3 mg/d) with a high incidence of side effects.
Clinical observations in patients with Cushing’s syndrome have highlighted the link between glucocorticoids and obesity. However, Cushing’s syndrome is rare and simple obesity is not a state of circulating cortisol excess (15). We have argued, however that glucocorticoids might still be involved in the pathogenesis of obesity through the autocrine generation of cortisol within adipose tissue itself via the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). This enzyme converts the biologically inactive glucocorticoid, cortisone (E) to active cortisol (F) at a prereceptor level. 11ß-HSD1 is highly expressed in adipose tissue (16) and more highly expressed in omental compared with sc preadipocytes (17). Overexpression of 11ß-HSD1 in a rodent model leads to a centrally obese phenotype as a consequence of elevated adipose tissue corticosterone levels (18). On this background, we have postulated that reduction in the local generation of F in adipose tissue through inhibition of 11ß-HSD1 may regulate fat mass (17), possibly resulting in weight reduction.
GH, probably acting via IGF-I, limits F availability to the glucocorticoid receptor (GR) by inhibiting 11ß-HSD1 (19, 20). We have, therefore, used low-dose GH replacement as a physiological inhibitor of 11ß-HSD1 and performed a double-blind, placebo-controlled study of GH therapy in obesity over an 8-month period with the aim of assessing alterations in body composition in the context of global measurements of 11ß-HSD1 activity.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Screening fasting bloods were drawn at 0900 h, and all subjects had normal blood counts and normal glucose, liver, renal, and thyroid function. At baseline, subjects had measurements of BMI, waist (measured supine at the level of the umbilicus) and hip (at the level of the greater trochanter) circumference, and blood pressure (average of three readings, measured supine after 10-min rest using Dynamap, Critikon, Tampa, FL). Further fasting 0900-h bloods were drawn for measurement of total cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides, IGF-I, F, E, glucose, insulin, and ACTH. In addition, all patients performed a 24-h urine collection for corticosteroid metabolite analysis using gas chromatography/mass spectrometry as previously described (21). Measurement of the ratio of total F (Fm = F + THF + 5
THF + -cortol + ß-cortol + 20DHF + 20ßDHF + 6ßOH-F) to total E metabolites (Em = E + THE + -cortolone + ß-cortolone + 20DHE + 20ßDHE) provides an assessment of the global set point of cortisol to cortisone conversion. More specifically, the ratio of tetrahydrometabolites of F (THF + 5THF) to those of cortisone (THE) provides a reflection of 11ß-HSD1 activity. The ratio of urinary free F (UFF) to E (UFE) reflects renal 11ß-HSD2 activity. The activities of 5- and 5ß-reductases can be inferred from measurement of the ratio THF/5THF.
Subjects were randomly assigned to treatment with GH 0.4 mg/d (1.2 IU/d; Genotropin, Pharmacia, Stockholm, Sweden) or placebo (Pharmacia) for 8 months. Patients were taught to self-administer treatment and advised to give injections in the late evening. Patients were followed up monthly and fasting bloods drawn at 0900 h for measurement of serum lipids (total cholesterol, HDL cholesterol, and triglycerides), IGF-I, F, E, plasma ACTH and urea, creatinine, and electrolytes. Additionally, anthropometric measurements were taken monthly. At months 4 and 8, blood pressure was measured as described above and a 24-h urine collection performed. Dual-energy x-ray absorptiometry (DEXA) scanning was performed at baseline and at 4 and 8 months to assess change in body composition. Whole-body DEXA measurements were performed with a total body scanner (DPX-L, Lunar Corp., Madison, WI). For total fat and lean mass measurements, coefficients of variation were less than 3%. Regional fat mass was analyzed as previously described (22).
Biochemical assays
F was assayed using a chemiluminescent immunoassay (Advia Centaur, Bayer Corp. Diagnostics, Newbury, UK) with interassay coefficients of variation of 10.2% at 76 nmol/liter, 7.7% at 528 nmol/liter and 7.4% at 882 nmol/liter. E was assayed after extraction from serum and then RIA of the extract with 125I-cortisone and Sac-Cel (IDS Ltd., Tyne and Weir, UK) second antibody separation. The interassay coefficient of variation for 10 consecutive assays was less than 15% for values between 50 and 100 nmol/liter and less than 10% for values over 100 nmol/liter. ACTH was assayed using an immunoradiometric assay (Nichols Allegro, Nichols Diagnostic, San Juan Capistrano, CA) calibrated against synthetic human ACTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) (National Pituitary Agency, University of Maryland School of Medicine, Baltimore, MD). Interassay coefficients of variation were 15.5% at 8.5 ng/liter and less than 10% over the range 67.8–1333 ng/liter. IGF-I was measured using an in-house acid/ethanol extraction RIA calibrated against IS 87/518. Interassay coefficients of variation were less than 10% over the range 5–33 nmol/liter. Serum insulin was measured using an immunoenzymometric assay with no significant cross-reactivity with pro-insulin (s), calibrated against IRP 66/304 (Medgenix Insulin-EASIA, Biosource Technologies, Inc., Camarillo, CA). Interassay coefficients of variation were less than 10% over the range 95–1038 pmol/liter.
Urea, creatinine and electrolytes, cholesterol, HDL cholesterol, and triglycerides were measured using standard laboratory methods (Instrumentation Laboratory, Warrington, UK). Glucose was assayed using the hexokinase method (Instrumentation Laboratory) with interassay coefficients of variation of 2.0% at 4.7 mmol/liter and 1.7% at 33.4 mmol/liter.
Measurements of insulin resistance were derived from fasting glucose and insulin data, using the homeostasis model assessment (HOMA) mathematical model as previously described (23), values greater than 1 suggesting insulin resistance.
Statistical analysis
The data are presented as mean ± SE unless otherwise stated. Statistical analysis was performed using repeated-measures ANOVA. For comparisons of single variables measured at one time point, a t test was used provided, the data were normally distributed. For data that were not normally distributed, the Mann-Whitney U test was used.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Baseline characteristics of both placebo- and GH-treated groups are presented in Table 1
. There were no significant differences at baseline in anthropometric measurements, body composition as measured by DEXA, fasting lipid profile, insulin sensitivity, or indices of cortisol secretion or metabolism between GH- and placebo-treated groups. Absolute IGF-I concentrations were higher at baseline in the GH-treated group (31.6 ± 3.7 vs. 17.9 ± 1.8 nmol/liter, GH vs. placebo, P < 0.05). However, when corrected for age (IGF-I concentrations expressed as a percentage of the upper limit of the age related reference range), there was no difference at baseline between GH- and placebo-treated groups (68.2 ± 4.8% vs. 54.9 ± 5.6%, GH vs. placebo, P = 0.09).
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). This elevation persisted throughout the duration of the study, although the peak IGF-I was observed at 4 months (119.1 ± 8.7% vs. 53.0 ± 5.3%, P < 0.005, GH vs. placebo) (Fig. 1).
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BMI did not change in either group during the duration of the study. Treatment with GH failed to alter total fat mass. There was no change in total fat mass on DEXA scanning over time or between GH and placebo groups. In addition, there was no change in regional fat mass (trunk, arms, legs) and no alteration in fat distribution (trunk to leg ratio) (Table 2). These findings were also reflected in measurements of waist circumference and waist to hip ratio.
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Fasting lipids and insulin sensitivity
Fasting total cholesterol, HDL cholesterol, and triglycerides did not change significantly in either treatment group throughout the duration of the study. Insulin sensitivity as measured by fasting glucose to insulin ratio and HOMA analysis also did not change significantly in either treatment group during the study (data not shown).
Blood pressure
Systolic blood pressure increased in the GH-treated group [119 ± 3 (baseline); 125 ± 3 mm Hg, P = ns (4 months]); 130 ± 4, P < 0.05 (8 months)]. There was no significant alteration in diastolic blood pressure [70 ± 3 vs. 73 ± 3 mm Hg, P = ns (baseline vs. 8 months)]. In the placebo-treated group, neither systolic nor diastolic blood pressure altered significantly across the 8-month period.
F secretion and metabolism
The secretion of total F metabolites did not change throughout the study, and there were no significant differences between GH- and placebo-treated groups at any time point (Table 3). In addition, ACTH concentrations did not differ significantly between GH- and placebo-treated groups (Table 3).
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THF/THE ratio at 8 months (mean ratio change, -0.13 ± 0.05 vs. +0.09 ± 0.09, GH vs. placebo, P = 0.07). There was no significant change in either the UFF/UFE ratio (mean ratio change, -0.04 ± 0.04 vs. -0.04 ± 0.04, GH vs. placebo, P = 1.0) or the THF/5THF ratio (mean ratio change, +0.08 ± 0.09 vs. -0.17 ± 0.09, GH vs. placebo, P = 0.11) (Fig. 2). Complete urinary steroid metabolite data are presented in Table 3
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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GH has complex actions on adipose tissue biology and has been postulated as an important physiological regulator of fat mass. Accumulation of adipose tissue mass occurs in several ways: adipocyte differentiation, increased lipid accumulation within already differentiated cells, preadipocyte proliferation, increasing the pool of cells available to undergo differentiation, and recruitment of pluripotent stem cells to become preadipocytes. The lipolytic action of GH in vivo and in vitro is well documented (24, 25). At high doses, it is this effect that appears to predominate and explains the decrease in fat mass observed in patients with GHD treated with GH. However, in vitro, GH acting through IGF-I also promotes preadipocyte proliferation and adipocyte differentiation (26, 27, 28). This work has been largely performed in animal models, and confirmation in humans is awaited. Nevertheless, the actions of GH on adipose tissue are a balance among lipolysis, proliferation, and differentiation.
In vitro, IGF-I at physiological doses, inhibits 11ß-HSD1 in both sc and omental preadipocytes, thereby decreasing local F generation (20). The degree of inhibition is modest, and the exact molecular mechanism underpinning this regulation is as yet undefined. Studies using rodent models have also shown the close relationship between GH and 11ß-HSD1 activity and expression. GHD dwarf male rats given a female pattern of GH replacement develop a female pattern of hepatic 11ß-HSD1 expression (29). These experimental data are borne out in clinical studies. Patients with GHD have increased 11ß-HSD1 activity as measured by urinary corticosteroid metabolites, and this decreases with GH replacement therapy (30, 31). In addition, acromegalic patients with uncontrolled GH excess have decreased activity that increases with effective control of GH secretion (19). Furthermore, 11ß-HSD1 activity increases in acromegalic patients treated with the novel GH receptor antagonist, Pegvisomant, an effect that is clearly mediated through a reduction in circulating IGF-I (32). In this study we have shown that exogenous low-dose GH is able to inhibit 11ß-HSD1 activity and alter the set point of E to F conversion in patients with simple obesity. These effects are likely to be IGF-I dependent. Prolonged exposure to elevated IGF-I levels may be critical and also explain why the most marked alterations in F metabolism were observed at the 8-month time point.
The role of 11ß-HSD1 in the pathogenesis of human obesity and the regulation of adipose tissue mass have not been fully characterized. 11ß-HSD1 is highly expressed in human adipose tissue (16, 17), and overexpression in adipocytes in a rodent model leads to a centrally obese phenotype (18). However, human obesity is unlikely to be caused by simple overexpression of 11ß-HSD1 (33), although reports of sc adipose tissue overexpression are cited (34, 35, 36). Indeed, global measures of 11ß-HSD1 activity suggest decreased rather than increased activity in obese patients in comparison with lean controls (22, 34, 35). It is plausible that decreased 11ß-HSD1 activity represents a compensatory mechanism to the adverse metabolic profile in obesity. Although the effects of F excess on adipose tissue mass are well recognized, as exemplified by patients with Cushing’s syndrome, the impact of the autocrine generation of F on fat mass regulation is less clear. F promotes adipocyte differentiation and inhibits preadipocyte proliferation, and we have previously shown that limiting cortisol availability to the GR through inhibition of 11ß-HSD1 inhibits adipocyte differentiation (37) and promotes preadipocyte proliferation (33). Therefore, it is possible that inhibition of 11ß-HSD1 by GH will decrease the autocrine generation of F within adipose tissue and reduce adipocyte differentiation and enhance preadipocyte proliferation. Once again there is a delicate balance of actions between increasing and decreasing adipose tissue mass, and this may explain why GH did not alter absolute fat mass despite inhibiting 11ß-HSD1. Furthermore, the degree of inhibition mediated by IGF-I may not be sufficient to truly limit the availability of F to the GR. Other factors that may explain the lack of effect of GH on fat mass in this study also need to be considered. The impact of IGF-binding proteins was not assessed, and increased appetite and calorie intake in the GH-treated group could explain the lack of reduction in fat mass.
Inhibition of hepatic 11ß-HSD1 improves insulin sensitivity (38). In this study, we were unable to detect alterations in insulin resistance as measured by HOMA. Although this is a relatively crude test, the lack of effect may be due to the multiple actions of GH. Recently a novel class of compounds that inhibit 11ß-HSD1 with a greater than 200-fold selectivity over 11ß-HSD2 has been identified (39). These compounds improve insulin sensitivity in rodents. Selective 11ß-HSD1 inhibitors may, therefore, not only help to unravel the complexities and role of the autocrine generation of F in the regulation of fat mass but also have potential as a novel adjunctive therapy in the treatment of diabetes mellitus.
The failure of GH to alter fat mass in this study, despite inhibition of 11ß-HSD1, may be a reflection of its complex direct and indirect actions on adipose tissue. Clearly the dose of GH treatment is likely to be clinically relevant. To date, there has been no accurate characterization of dose-specific responses of GH treatment on adipocyte biology. However, in elderly hypopituitary patients, modulation of 11ß-HSD1 activity occurs at doses lower than those needed to cause a reduction in fat mass (40). Previous studies have focused on global measures of 11ß-HSD1 activity; further clinical studies are needed in larger cohorts of patients to examine the effects of GH on 11ß-HSD1 expression specifically within adipose tissue. Although GH treatment may not represent an effective therapy in simple obesity because of its many complex actions and perhaps because of its lack of potency as an inhibitor of 11ß-HSD1, selective, potent inhibition of 11ß-HSD1 may represent a novel therapeutic strategy for insulin sensitization.
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Acknowledgments |
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Footnotes |
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Abbreviations: 11ß-HSD1, 11ß-Hydroxysteroid dehydrogenase type 1; BMI, body mass index; DEXA, dual-energy x-ray absorptiometry; E, cortisone; F, cortisol; GHD, GH deficiency; GR, glucocorticoid receptor; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; THE, tetrahydrometabolites of E; THF + 5THF, tetrahydrometabolites of F; UFE, urinary free E; UFF, urinary free F.
Received December 5, 2002.
Accepted February 3, 2003.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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, P < 0.01; 
, P < 0.005).