Growth Hormone (GH) Responses to GH-Releasing Hormone-Arginine Testing in Human Immunodeficiency Virus Lipodystrophy
http://www.100md.com
《临床内分泌与代谢杂志》
Abstract
Prior studies suggest reduced overnight GH secretion in association with excess visceral adiposity among patients with HIV lipodystrophy (LIPO, i.e. with fat redistribution). We now investigate GH responses to standardized GHRH-arginine in LIPO patients (n = 39) in comparison with body mass index- and age-matched control groups [HIV patients without fat distribution (NONLIPO, n = 17)] and healthy subjects (C, n = 16). IGF-I [242 ± 17; 345 ± 38; 291 ± 27 ng/ml (P < 0.05 vs. NONLIPO)] was lowest in the LIPO group. Our data demonstrate failure rates of 18% for the LIPO group vs. 5.9% for the NONLIPO group and 0% for the C group, using a stringent criterion of 3.3 ng/ml for peak GH response to GHRH-arginine (P < 0.05 LIPO vs. C). Using less stringent cutoffs, the failure rate in the LIPO group rises to 38.5% at 7.5 ng/ml. Among the LIPO patients, the peak GH response to GHRH-arginine was significantly predicted by visceral adipose tissue (P = 0.008), free fatty acid (P = 0.04), and insulin level (P = 0.007) in regression modeling controlling for age, body mass index, sc fat area, and triglyceride level. These data demonstrate increased failure rates to standardized stimulation testing with GHRH-arginine in LIPO patients, in association with increased visceral adiposity. The effects of low-dose GH should be assessed in the large subset of LIPO patients with abnormal GH stimulation testing.
Introduction
HIV LIPODYSTROPHY IS characterized by changes in fat distribution and insulin resistance (1, 2, 3). Fat distribution changes are heterogeneous and can include reduced sc fat as well as increased visceral fat. We have previously shown decreased overnight GH secretion and pulse amplitude in patients with HIV lipodystrophy (LIPO, i.e. with fat redistribution) (4), but rates of response to standardized stimulation testing remain unknown.
The diagnosis of GH deficiency (GHD) in adults can be difficult because of the lack of a specific cutoff to establish the condition. Furthermore, GHD may be partial. Most often, the diagnosis of true GHD in the adult is made using the results from standardized stimulation testing in the context of a pituitary mass or prior insult, such as radiation therapy. However, increasingly, it is becoming recognized that nutritional status can affect GH secretion, and reduced GH secretion has been shown in obesity (5) and in relationship to excess visceral fat in HIV-infected patients (4). Whether such patients have true GHD or functional reduction in GH secretion and whether they might benefit from GH replacement remain controversial.
In this study, we investigated GH response rates to standardized GHRH-arginine stimulation testing among HIV patients with (LIPO) and without (NONLIPO)lipodystrophy, in comparison with C subjects of similar age and body mass index (BMI).
Subjects and Methods
Clinical research protocol
Inclusion and exclusion criteria. Seventy-two male subjects [HIV-infected (n = 56) and healthy C subjects (n = 16) similar in age and BMI] were enrolled in the study between October 2001 and December 2003. HIV-infected subjects were characterized at study entry as LIPO (n = 39) and NONLIPO (n = 17), based on evidence of fat redistribution as previously described. All HIV-infected subjects were required to be on a stable antiretroviral regimen for at least 6 wk before entrance into the study. Subjects were categorized as LIPO based on a waist to hip ratio (WHR) of at least 0.90 and evidence of fat redistribution, including increased fat under the chin, at the back of the neck, or in the abdominal, chest, or breast areas or decreased fat in the arms, legs, or face. A score for fat change (between 0 and 2) was graded for each site, and subjects with an increased WHR and lipodystrophy score of at least 2.0 were characterized as LIPO. HIV-infected subjects with a lipodystrophy score of less than 2.0 (without significant fat redistribution) were characterized as NONLIPO patients. Similar criteria were used in prior studies from our group to identify LIPO patients (4). Subjects with diabetes mellitus, BMI less than 20 kg/m2, hemoglobin less than 9 g/dl, and use of GH, GHRH, oral, or parenteral glucocorticoids, megesterol acetate, or antidiabetic agents within the 3 months before study initiation were excluded. Subjects with symptoms of pituitary disease, known history of pituitary disease, or history of radiation were excluded.
Written informed consent was obtained from each subject before testing, in accordance with the Committee on the Use of Humans as Experimental Subjects of the Massachusetts Institute of Technology and the Subcommittee on Human Studies at the Massachusetts General Hospital. Subjects underwent GHRH and arginine stimulation testing in the morning after a 12-h fast.
Screening outpatient visit. After a 12-h overnight fast, subjects reported to the General Clinical Research Center for a screening visit, at which time eligibility was determined based on a fasting blood glucose level less than 126 mg/dl.
GH assessment
Subjects received standardized stimulation testing with GHRH-arginine [GHRH 1–29 (Geref, Serono, Inc., Norwell, MA) 1-μg/kg iv bolus along with simultaneous administration of arginine hydrochloride (0.5 g/kg) (maximum dose, 30 g) given iv over 30 min]. GH levels were collected at –15, 0, 15, 30, 45, 60, 90, and 120 min after GHRH administration.
Body composition analysis
Height, weight, and BMI were determined in the fasting state during the screen visit. Cross-sectional abdominal computed tomography (CT) scanning was performed to assess the distribution of sc and visceral abdominal fat. A lateral scout image was obtained to identify the level of the L4 pedicle, which served as a landmark for the single-slice image. Scan parameters for each image were standardized (144 cm table height, 80 kV, 70 mA, 2 sec, 1-cm slice thickness). Fat attenuation coefficients were at –50 Hansfield units as described by Borkan et al. Abdominal visceral (VAT) and sc fat areas (SAT) were then determined (6). Total fat and extremity fat were determined by dual-energy x-ray absorptiometry (DXA) (Hologic 4500, Hologic Inc., Waltham, MA) as previously described (7).
Biochemical and immunological function
Fasting glucose, insulin, IGF-I, IGF binding protein (IGFBP)-1, IGFBP-3, free fatty acids (FFAs), triglyceride, testosterone, TSH, CD4+ cell count, and viral load were determined after a 12-h fast before any stimulation testing. A 75-g oral glucose tolerance test was performed for glucose and insulin on a separate occasion after a 12-h fast.
Laboratory methods
GH was measured by two-site RIA with an intraassay coefficient of variation (CV) of 4.4% (Corning, Inc. Nichols Institute Diagnostics, San Juan Capistrano, CA). The interassay CV was 6.6%. The sensitivity of the assay was determined to be 0.05 ng/ml. IGF-I was measured by two-site RIA with an intraassay CV of 4.9% (Diagnostic Systems Laboratories, Inc., Webster, TX). The interassay CV was 5.1%. The sensitivity of the assay was determined to be 2.6 ng/ml. IGFBP-1 was measured by two-site RIA with sensitivity of 0.33 ng/ml and an intraassay CV of 4.2% (Diagnostic Systems Laboratories, Inc.). IGFBP-3 was measured by two-site RIA with sensitivity of 0.5 ng/ml and an intraassay CV of 2.9% (Diagnostic Systems Laboratories, Inc.) Serum TSH levels were run using a two-site RIA (DiaSorin, Inc., Stillwater, MN; intraassay CV average, 3.1–3.3%; sensitivity, 0.01 μIU/ml based on serial dilutions).
Nonesterified fatty acid concentrations were measured by an in vitro enzymatic colorimetric assay kit (Wako Chemicals USA, Inc., Richmond, VA). The intraassay CV for fatty acids ranged from 1.1–2.7%. The published normal range for fatty acids is 0.1–0.6 mmol/liter. Insulin concentrations were measured in serum by RIA (Diagnostic Products, Los Angeles). The intraassay and interassay CVs ranged from 4.7–7.7% and from 5.5–9.2%, respectively. Glucose and triglyceride concentrations were measured by standard techniques. Testosterone was measured by RIA (Diagnostic Products). The intraassay CV ranged from 5–11%, and the interassay CV ranged from 5.9–12%. The sensitivity for the assay was 4 ng/dl.
The CD4 count was determined by flow cytometry (Becton Dickinson Immunocytochemistry Systems, San Jose, CA), and the HIV viral load was determined by ultrasensitive assay (Amplicor HIV-1 Monitor Assay; Roche Molecular Systems, Indianapolis, IN), with limits of detection of 50–75,000 copies/ml.
Statistical analysis
Demographic, body composition, and biochemical indices were compared among the three groups by overall ANOVA for continuous variables and likelihood ratio for dichotomized variables. In stepwise regression modeling, peak GH response and GH area under the curve (AUC) were the dependent variables; and age, BMI, VAT, SAT, insulin, triglycerides, and FFA concentrations were tested for entry into the model as dependent variables at P = 0.10. Among the HIV-infected subjects, group (LIPO or NONLIPO) was also tested in stepwise regression modeling. Final P values and parameter estimates are shown for each model. All statistical analyses were made using SAS JMP Statistical Database Software (version 4; SAS Institute, Inc., Cary, NC). Statistical significance was defined as a two-tailed -value of P 0.05. Results are mean ± SEM unless otherwise indicated.
Results
Subject characteristics
Thirty-nine male HIV-infected LIPO patients and 17 NONLIPO patients were compared with sixteen healthy, HIV-negative male C subjects. The clinical characteristics of the study patients are shown in Table 1. The mean ages and BMI’s were similar among the three groups (Table 1). The LIPO subjects demonstrated higher WHR, visceral abdominal fat by CT, VAT:SAT ratio and trunk:lower extremity fat ratio (by DXA) than either the NONLIPO or C group. In contrast, lower extremity fat was reduced in the LIPO group (Table 1).
Triglyceride and insulin concentrations were significantly increased in the LIPO group compared with the NONLIPO and C groups (Table 1). Testosterone and TSH levels were not different among the groups and were normal in all subjects. FFA concentrations were higher in the LIPO group, but this difference did not reach statistical significance. IGF-I (P < 0.05, LIPO vs. NONLIPO) and IGFBP-3 were lowest in the LIPO group (P < 0.05, LIPO vs. NONLIPO and LIPO vs. C). CD4 count, viral load, and antiretroviral use were similar between the HIV groups (Table 1).
The rate of failure in response to the combined GHRH-arginine test was 18% in the LIPO group vs. 5.9% in the NONLIPO group and 0% in the C group, using a stringent cutoff of 3.3 ng/ml (P < 0.05 for LIPO vs. C). Using less stringent cutoffs, the failure rates in the LIPO group rose to 30.8% at 5.0 ng/ml and 38.5% at 7.5 ng/ml, with corresponding increases in the C group to 12.5% (P < 0.05 vs. NONLIPO at 5.0 ng/ml and P < 0.05 vs. NONLIPO and C at 7.5 ng/ml) (Fig. 1). At 9.0 ng/ml, 43.6% of the LIPO group vs. 17.7% and 25% of the NONLIPO and C groups, respectively, failed the stimulation testing (P = not significant).
Among the entire group of HIV-infected subjects, visceral fat area was the most significant predictor of peak GH response to GHRH and arginine (parameter estimate = –.09, P = 0.008), controlling for group (LIPO vs. NONLIPO), age, BMI, SAT, FFA concentration, fasting insulin, and triglycerides. In this model, fasting insulin was also a significant independent predictor of peak GH response (parameter estimate =–0.34, P = 0.03) (Table 2). Among the subjects in the LIPO group, visceral fat area (parameter estimate = –0.09, P = 0.008), basal FFA level (parameter estimate = –18.7, P = 0.04), and basal insulin level (parameter estimate = –0.33, P = 0.007) were significant inverse predictors of peak GH response to GHRH and arginine (Table 2). Among subjects in the C group, only sc abdominal fat predicted peak GH response to combined GHRH-arginine in regression modeling (parameter estimate = –0.24, P = 0.009) (Table 2). Generally, similar results were seen in modeling for GH AUC (Table 2).
Discussion
To our knowledge, this study is the first to evaluate GH rates of response to the GHRH-arginine test in HIV-infected patients. We used a standardized method of testing for GHD in HIV LIPO subjects in comparison with healthy male adult C subjects matched for age and BMI. Eighteen percent of LIPO patients failed the GHRH-arginine stimulation testing using a highly stringent cutoff, suggesting that a small, but significant, number of such subjects will fail a standardized GH stimulation test. Peak GH response is inversely related to visceral adiposity, such that lower GH peak responses are seen with increasing visceral fat accumulation.
The optimal testing paradigm and cutoff for inadequate GH response are controversial. Insulin testing to evoke hypoglycemia is traditionally regarded as the gold standard test to determine GHD but was not appropriate in our subjects because of the potential risk associated with this test. The insulin tolerance test (ITT) is useful in patients with hypothalamic and/or pituitary disease and evaluates the entire hypothalamic-somatotroph axis. However, the test is contraindicated in patients with hypoglycemia, seizures, and ischemic heart disease. It is unknown whether the GHRH-arginine is equivalent to the ITT in patients with HIV. Because the GHRH-arginine test has less risk and has been shown to be reliable in other populations to detect GHD (8), it was chosen for our study.
The GHRH-arginine test compares well with the insulin test in diagnosing GHD (8) and has been considered a reasonable alternative test. The GH response to GHRH-arginine is independent of age, and there is less inter- and intraindividual variability than with other stimulation tests. The combined GHRH-arginine test is considered by many to be the best diagnostic alternative to the ITT (9). The lowest limit of GH response to ITT in normal subjects has been reported as 5 ng/ml by some (but not other) authors (10, 11). The peak GH cutpoint of 9 ng/ml for the GHRH-arginine tests represents the first centile limit of the normal GH response in a large population of normal subjects (11). Furthermore, Aimaretti et al. (12) showed that 92.5% of hypopituitary adults had a GH peak below this limit.
However, because the optimal cutoff for inadequate GH response is unknown, we chose a highly stringent cutoff of 3.3 ng/ml, because this value has been previously shown to demonstrate 95% specificity, with respect to the ITT test, for diagnosing GHD (8) and to minimize the misclassification of subjects as GH deficient when this is not the case. A caveat in this regard is that the 3.3 ng/ml cutoff was previously determined in comparison with the ITT in patients with known pituitary disease, in contrast to the patients described in this study, with nutritional disturbances in GH response to GHRH-arginine. Nonetheless, there were significant differences between the LIPO and C groups using this cutoff, with 0% of the C subjects failing the test compared with 18% of the subjects in the LIPO group. With increasing cutoffs, as have been proposed by others in the literature (11, 12), e.g. to 9.0 ng/ml, rates of failure to GHRH-arginine increased as anticipated (up to 44%), but a relatively large percentage of C subjects also failed the test (25%). Significant differences were not seen between the groups at the 9.0 ng/ml cutoff. Further studies are necessary to determine the best threshold to define reduced GH response rates to the GHRH-arginine test in the HIV population; but our data, using a strict threshold of 3.3 ng/ml, demonstrate that approximately one in five such LIPO subjects will fail to stimulate adequately.
The mean serum IGF-I was significantly lower among subjects in the LIPO group. However, IGF-I was below the normal range in only 8% of the LIPO subjects compared with 0% the NONLIPO subjects or C subjects (P = not significant). IGF-I was not correlated with peak response to GHRH-arginine stimulation testing in any of the groups. IGF-I is a relatively poor test to diagnose GHD in adults and has been shown to be in the normal range, even among patients who fail ITT testing (13, 14, 15). In contrast, IGF-I might be more useful for the diagnosis of GHD in childhood-onset or young-adult-onset GHD patients (16). However, because different IGF-I assays may yield different results, and serum IGF-I concentrations may be decreased by a variety of causes, caution is generally used in applying low IGF-I diagnostic cutpoints to the diagnosis of adult GHD in clinical practice (17). Furthermore, in contrast to GH resistance in undernutrition, prior studies have shown that GH secretion and IGF-I can be dissociated in simple obesity and have suggested an increased sensitivity to GH in overnutrition and obesity (18). Relatively increased sensitivity to GH may also occur in HIV patients with visceral adiposity and normal weight, although this has not been formally investigated in the HIV population.
IGFBP-3 was also significantly lower, but in the normal range, in the HIV lipodystrophy group. IGFBP-3 is N-glycosylated and is the major IGF binding moiety in plasma, given that more than 75 percent of circulating IGF-I is bound to this protein. IGFBP-3 concentrations are increased by GH administration, and IGFBP-3 functions as the major GH-responsive carrier of IGF-I (19). Of the six IGFBPs, IGFBP-3 is the major serum carrier protein for IGF peptides and the most GH dependent (20). IGFBP-3 levels are less nutritionally dependent, and the normal range varies only modestly with age. In our study, IGFBP-3 levels were significantly lower in the LIPO group in comparison with the other C groups. In contrast to some reports among non-HIV-infected patients (21, 22), IGFBP-3 concentrations did not correlate with peak GH response to GHRH-arginine in the LIPO subjects. Although lower IGF-I and IGFBP-3 levels support the observation of reduced GH secretion in the LIPO group, neither test is specific enough to use for diagnostic purposes in this population.
GH secretion is suppressed in obesity, with 24-h GH levels in obese men reduced by 75% in comparison with age-matched normal-weight C subjects (5). In addition, decreased responsiveness to stimulation tests such as GHRH, arginine, and combined GHRH-arginine has been demonstrated in non-HIV-infected subjects with obesity (23). Veldhuis et al. (24) demonstrated that the degree of body fatness was associated inversely with indices of GH secretion. In contrast, in our study, visceral fat, not BMI, was the major determinant of GH responsiveness in LIPO subjects. Similar results were seen among the entire HIV group, controlling for group (e.g. LIPO or NONLIPO). In contrast, only sc abdominal fat predicted peak GH response to GHRH and arginine in the C group.
The rates of failure in response to GH stimulation testing in the LIPO group were not a simple function of obesity, because response rates differed in comparison to a C group of healthy subjects similar in BMI. Furthermore, the inadequate response was not due to HIV per se, because lower failure rates were seen in age- and BMI-matched NONLIPO subjects. Importantly, our results can only be generalized to the patient population we recruited, with excess visceral adiposity and simultaneous loss of sc and extremity fat. It is less likely that any differences in GH response rates would be seen among a population with more pure lipoatrophy, because total body fat and extremity fat did not correlate with peak GH response among patients in HIV patients.
What are the mechanisms of reduced GH in the LIPO population? In prior studies, we have shown a strong inverse association between overnight GH secretion rates and visceral adiposity in frequent sampling. We now extend this finding to show a strong inverse relationship between visceral adiposity and GH response to GHRH-arginine. In addition, elevated FFA and insulin levels were significant independent variables in the regression models for peak GH response to GHRH-arginine in LIPO subjects.
We have also shown decreased ghrelin as well as an inhibitory effect of increased FFA on GH response to GHRH alone (25). We now show that peak GH response to GHRH-arginine is also inversely associated with FFA concentration. FFA remained negatively associated with peak GH response to GHRH-arginine in stepwise regression analysis controlling for age, BMI, and visceral fat, suggesting a potential inhibitory role with respect to GH secretion in the LIPO population. In healthy, obese subjects, Cordido et al. (26) demonstrated improved GH secretion in response to acipimox and reduced FFA. We also recently showed improved GH response to GHRH alone using acipimox, suggesting that FFA may play an inhibitory role with respect to GH secretion in the HIV lipodystrophy population (25).
Are HIV patients who fail the GHRH-arginine test truly or functionally GH deficient? Subjects in this study did not have any risk factors for, or symptoms of, pituitary dysfunction, and testosterone levels were normal in all patients. Although we did not perform pituitary imaging, it is unlikely that pituitary disease per se would explain our findings of inadequate GH response rates in up to nearly 20% of LIPO patients. More likely, our subjects have a functional GHD related, in part, to excess visceral fat. Whether this represents true GHD is not clear, because recent studies suggest that GH levels can change significantly over time, with reduction in weight and changes in body composition in otherwise healthy subjects (27, 28). This study was limited to men, to control for the influence of gender on GH secretion. Further studies determining GH response in LIPO women are needed.
A number of prior studies have investigated GH in HIV patients, both with the wasting syndrome (29) and with LIPO subjects (30, 31). Most prior studies in LIPO patients have investigated relatively large, supraphysiological doses of GH. Reported side effects with supraphysiological GH dosing in the HIV population include impaired glucose tolerance and joint aches. Recent studies suggest that such effects may be minimized with lower dosing (32). In contrast, Johannsson et al. (33) reported on the use of low-dose GH in non-HIV-infected patients with abdominal obesity, suggesting that GH administration might improve insulin resistance and reduce visceral adiposity in this population. Similarly, physiological GH dosing may result in improved insulin resistance and abdominal fat but has not been investigated in the HIV population. Furthermore, prior studies have not targeted a specific subpopulation of HIV patients with abnormal GH responses to GH stimulation testing.
In conclusion, our study suggests that approximately 20% of HIV-infected patients with lipodystrophy and increased visceral adiposity will have inadequate GH stimulation to GHRH-arginine using a stringent cutoff of 3.3 ng/ml and may therefore be functionally GH deficient. Such patients may benefit from GH administration in terms of improved body fat and metabolic parameters, and further studies are necessary to test the efficacy of low-dose GH in this population. Use of a less stringent cutoff may increase the number of patients failing the GH stimulation test but decrease the specificity of the test. Further studies are needed to optimally identify HIV patients with decreased GH secretion and to assess whether such patients might benefit from physiologic GH replacement.
Acknowledgments
We acknowledge the nursing and bionutrition staffs of the General Clinical Research Center of the Massachusetts General Hospital and Massachusetts Institute of Technology for their dedicated patient care, and Dr. Anne Klibanski for helpful suggestions with the manuscript.
Footnotes
This work was supported in part by National Institutes of Health Grants R01 DK063639 and M01-RR01066.
First Published Online October 13, 2004
Abbreviations: AUC, Area under the curve; BMI, body mass index; C, control (group); CT, computed tomography; CV, coefficient of variation; DXA, dual-energy x-ray absorptiometry; FFA, free fatty acid; GHD, GH deficiency; IGFBP, IGF binding protein; ITT, insulin tolerance test; LIPO, HIV patients with lipodystrophy (i.e. with fat redistribution); NONLIPO, HIV patients without lipodystrophy; SAT, sc adipose tissue; VAT, visceral adipose tissue; WHR, waist to hip ratio.
Received July 15, 2004.
Accepted September 23, 2004.
References
Carr A, Samaras K, Chisholm DJ, Cooper DA 1998 Pathogenesis of HIV-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance. Lancet 351:1881–1883
Carr A, Samaras K, Burton S, Law M, Freund J, Chisholm DJ, Cooper DA 1998 A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 12:F51–F58
Hadigan C, Meigs JB, Corcoran C, Rietschel P, Piecuch S, Basgoz N, Davis B, Sax P, Stanley T, Wilson PW, D’Agostino RB, Grinspoon S 2001 Metabolic abnormalities and cardiovascular disease risk factors in adults with human immunodeficiency virus infection and lipodystrophy. Clin Infect Dis 32:130–139
Rietschel P, Hadigan C, Corcoran C, Stanley T, Neubauer G, Gertner J, Grinspoon S 2001 Assessment of growth hormone dynamics in human immunodeficiency virus-related lipodystrophy. J Clin Endocrinol Metab 86:504–510
Veldhuis JD, Iranmanesh A, Ho KK, Waters MJ, Johnson ML, Lizarralde G 1991 Dual defects in pulsatile growth hormone secretion and clearance subserve the hyposomatotropism of obesity in man. J Clin Endocrinol Metab 72:51–59
Borkan GA, Gerzof SG, Robbins AH, Hults DE, Silbert CK, Silbert JE 1982 Assessment of abdominal fat content by computed tomography. Am J Clin Nutr 36:172–177
Hadigan C, Miller K, Corcoran C, Anderson E, Basgoz N, Grinspoon S 1999 Fasting hyperinsulinemia and changes in regional body composition in human immunodeficiency virus-infected women. J Clin Endocrinol Metab 84:1932–1937
Biller BM, Samuels MH, Zagar A, Cook DM, Arafah BM, Bonert V, Stavrou S, Kleinberg DL, Chipman JJ, Hartman ML 2002 Sensitivity and specificity of six tests for the diagnosis of adult GH deficiency. J Clin Endocrinol Metab 87:2067–2079
Valetto MR, Bellone J, Baffoni C, Savio P, Aimaretti G, Gianotti L, Arvat E, Camanni F, Ghigo E 1996 Reproducibility of the growth hormone response to stimulation with growth hormone-releasing hormone plus arginine during lifespan. Eur J Endocrinol 135:568–572
de Boer H, Blok GJ, Van der Veen EA 1995 Clinical aspects of growth hormone deficiency in adults. Endocr Rev 16:63–86
Ghigo E, Aimaretti G, Gianotti L, Bellone J, Arvat E, Camanni F 1996 New approach to the diagnosis of growth hormone deficiency in adults. Eur J Endocrinol 134:352–356
Aimaretti G, Corneli G, Razzore P, Bellone S, Baffoni C, Arvat E, Camanni F, Ghigo E 1998 Comparison between insulin-induced hypoglycemia and growth hormone (GH)-releasing hormone + arginine as provocative tests for the diagnosis of GH deficiency in adults. J Clin Endocrinol Metab 83:1615–1618
Hilding A, Hall K, Wivall-Helleryd IL, Saaf M, Melin AL, Thoren M 1999 Serum levels of insulin-like growth factor I in 152 patients with growth hormone deficiency, aged 19–82 years, in relation to those in healthy subjects. J Clin Endocrinol Metab 84:2013–2019
Marzullo P, Di Somma C, Pratt KL, Khosravi J, Diamandis A, Lombardi G, Colao A, Rosenfeld RG 2001 Usefulness of different biochemical markers of the insulin-like growth factor (IGF) family in diagnosing growth hormone excess and deficiency in adults. J Clin Endocrinol Metab 86:3001–3008
Baum HB, Biller BM, Katznelson L, Oppenheim DS, Clemmons DR, Cannistraro KB, Schoenfeld DA, Best SA, Klibanski A 1996 Assessment of growth hormone (GH) secretion in men with adult-onset GH deficiency compared with that in normal men—a clinical research center study. J Clin Endocrinol Metab 81:84–92
GH Research Society 2000 Consensus quidelines for the diagnosis and treatment of growth hormone (GH) deficiency in childhood and adolescence: summary statement of the GH Research Society. J Clin Endocrinol Metab 85:3990–3993
Growth Hormone Research Society Workshop on Adult Growth Hormone Deficiency 1998 Consensus guidelines for the diagnosis, treatment of adults with growth hormone deficiency: summary statement of the Growth Hormone Research Society Workshop on Adult Growth Hormone Deficiency. J Clin Endocrinol Metab 83:379–381
Hochberg Z, Hertz P, Colin V, Ish-Shalom S, Yeshurun D, Youdim MB, Amit T 1992 The distal axis of growth hormone (GH) in nutritional disorders: GH-binding protein, insulin-like growth factor-I (IGF-I), and IGF-I receptors in obesity and anorexia nervosa. Metabolism 41:106–112
Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34
Baxter RC, Martin JL 1986 Radioimmunoassay of growth hormone-dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 78:1504–1512
Hasegawa Y, Hasegawa T, Aso T, Kotoh S, Tsuchiya Y, Nose O, Ohyama Y, Araki K, Tanaka T, Saisyo S 1992 Usefulness and limitation of measurement of insulin-like growth factor binding protein-3 (IGFBP-3) for diagnosis of growth hormone deficiency. Endocrinol Jpn 39:585–591
Blum WF, Ranke MB, Kietzmann K, Gauggel E, Zeisel HJ, Bierich JR 1990 A specific radioimmunoassay for the growth hormone (GH)-dependent somatomedin-binding protein: its use for diagnosis of GH deficiency. J Clin Endocrinol Metab 70:1292–1298
Cordido F, Dieguez C, Casanueva FF 1990 Effect of central cholinergic neurotransmission enhancement by pyridostigmine on the growth hormone secretion elicited by clonidine, arginine, or hypoglycemia in normal and obese subjects. J Clin Endocrinol Metab 70:1361–1370
Veldhuis JD, Liem AY, South S, Weltman A, Weltman J, Clemmons DA, Abbott R, Mulligan T, Johnson ML, Pincus S 1995 Differential impact of age, sex steroid hormones, and obesity on basal versus pulsatile growth hormone secretion in men as assessed in an ultrasensitive chemiluminescence assay. J Clin Endocrinol Metab 80:3209–3222
Koutkia P, Meininger G, Canavan B, Breu J, Grinspoon S 2004 Metabolic regulation of growth hormone by free fatty acids, somatostatin, and ghrelin in HIV-lipodystrophy. Am J Physiol Endocrinol Metab 286:E296–E303
Cordido F, Peino R, Penalva A, Alvarez CV, Casanueva FF, Dieguez C 1996 Impaired growth hormone secretion in obese subjects is partially reversed by acipimox-mediated plasma free fatty acid depression. J Clin Endocrinol Metab 81:914–918
Rasmussen MH, Hvidberg A, Juul A, Main KM, Gotfredsen A, Skakkebaek NE, Hilsted J, Skakkebae NE 1995 Massive weight loss restores 24-hour growth hormone release profiles and serum insulin-like growth factor-I levels in obese subjects. J Clin Endocrinol Metab 80:1407–1415
De Marinis L, Bianchi A, Mancini A, Gentilella R, Perrelli M, Giampietro A, Porcelli T, Tilaro L, Fusco A, Valle D, Tacchino RM 2004 Growth hormone secretion and leptin in morbid obesity before and after biliopancreatic diversion: relationships with insulin and body composition. J Clin Endocrinol Metab 89:174–180
Schambelan M, Mulligan K, Grunfeld C, Daar ES, LaMarca A, Kotler DP, Wang J, Bozzette SA, Breitmeyer JB 1996 Recombinant human growth hormone in patients with HIV-associated wasting. A randomized, placebo-controlled trial. Serostim Study Group. Ann Intern Med 125:873–882
Wanke C, Gerrior J, Kantaros J, Coakley E, Albrecht M 1999 Recombinant human growth hormone improves the fat redistribution syndrome (lipodystrophy) in patients with HIV. AIDS 13:2099–2103
Kotler DP, Muurahainen N, Grunfeld C, Wanke C, Thompson M, Saag M, Bock D, Simons G, Gertner JM, Serostim in Adipose Redistribution Syndrome Study Group 2004 Effects of growth hormone on abnormal visceral adipose tissue accumulation and dyslipidemia in HIV-infected patients. J Acquir Immune Defic Syndr 35:239–252
Lo JC, Mulligan K, Noor MA, Schwarz JM, Halvorsen RA, Grunfeld C, Schambelan M 2001 The effects of recombinant human growth hormone on body composition and glucose metabolism in HIV-infected patients with fat accumulation. J Clin Endocrinol Metab 86:3480–3487
Johannsson G, Marin P, Lonn L, Ottosson M, Stenlof K, Bjorntorp P, Sjostrom L, Bengtsson BA 1997 Growth hormone treatment of abdominally obese men reduces abdominal fat mass, improves glucose and lipoprotein metabolism, and reduces diastolic blood pressure. J Clin Endocrinol Metab 82:727–734(Polyxeni Koutkia, Bridget)
Prior studies suggest reduced overnight GH secretion in association with excess visceral adiposity among patients with HIV lipodystrophy (LIPO, i.e. with fat redistribution). We now investigate GH responses to standardized GHRH-arginine in LIPO patients (n = 39) in comparison with body mass index- and age-matched control groups [HIV patients without fat distribution (NONLIPO, n = 17)] and healthy subjects (C, n = 16). IGF-I [242 ± 17; 345 ± 38; 291 ± 27 ng/ml (P < 0.05 vs. NONLIPO)] was lowest in the LIPO group. Our data demonstrate failure rates of 18% for the LIPO group vs. 5.9% for the NONLIPO group and 0% for the C group, using a stringent criterion of 3.3 ng/ml for peak GH response to GHRH-arginine (P < 0.05 LIPO vs. C). Using less stringent cutoffs, the failure rate in the LIPO group rises to 38.5% at 7.5 ng/ml. Among the LIPO patients, the peak GH response to GHRH-arginine was significantly predicted by visceral adipose tissue (P = 0.008), free fatty acid (P = 0.04), and insulin level (P = 0.007) in regression modeling controlling for age, body mass index, sc fat area, and triglyceride level. These data demonstrate increased failure rates to standardized stimulation testing with GHRH-arginine in LIPO patients, in association with increased visceral adiposity. The effects of low-dose GH should be assessed in the large subset of LIPO patients with abnormal GH stimulation testing.
Introduction
HIV LIPODYSTROPHY IS characterized by changes in fat distribution and insulin resistance (1, 2, 3). Fat distribution changes are heterogeneous and can include reduced sc fat as well as increased visceral fat. We have previously shown decreased overnight GH secretion and pulse amplitude in patients with HIV lipodystrophy (LIPO, i.e. with fat redistribution) (4), but rates of response to standardized stimulation testing remain unknown.
The diagnosis of GH deficiency (GHD) in adults can be difficult because of the lack of a specific cutoff to establish the condition. Furthermore, GHD may be partial. Most often, the diagnosis of true GHD in the adult is made using the results from standardized stimulation testing in the context of a pituitary mass or prior insult, such as radiation therapy. However, increasingly, it is becoming recognized that nutritional status can affect GH secretion, and reduced GH secretion has been shown in obesity (5) and in relationship to excess visceral fat in HIV-infected patients (4). Whether such patients have true GHD or functional reduction in GH secretion and whether they might benefit from GH replacement remain controversial.
In this study, we investigated GH response rates to standardized GHRH-arginine stimulation testing among HIV patients with (LIPO) and without (NONLIPO)lipodystrophy, in comparison with C subjects of similar age and body mass index (BMI).
Subjects and Methods
Clinical research protocol
Inclusion and exclusion criteria. Seventy-two male subjects [HIV-infected (n = 56) and healthy C subjects (n = 16) similar in age and BMI] were enrolled in the study between October 2001 and December 2003. HIV-infected subjects were characterized at study entry as LIPO (n = 39) and NONLIPO (n = 17), based on evidence of fat redistribution as previously described. All HIV-infected subjects were required to be on a stable antiretroviral regimen for at least 6 wk before entrance into the study. Subjects were categorized as LIPO based on a waist to hip ratio (WHR) of at least 0.90 and evidence of fat redistribution, including increased fat under the chin, at the back of the neck, or in the abdominal, chest, or breast areas or decreased fat in the arms, legs, or face. A score for fat change (between 0 and 2) was graded for each site, and subjects with an increased WHR and lipodystrophy score of at least 2.0 were characterized as LIPO. HIV-infected subjects with a lipodystrophy score of less than 2.0 (without significant fat redistribution) were characterized as NONLIPO patients. Similar criteria were used in prior studies from our group to identify LIPO patients (4). Subjects with diabetes mellitus, BMI less than 20 kg/m2, hemoglobin less than 9 g/dl, and use of GH, GHRH, oral, or parenteral glucocorticoids, megesterol acetate, or antidiabetic agents within the 3 months before study initiation were excluded. Subjects with symptoms of pituitary disease, known history of pituitary disease, or history of radiation were excluded.
Written informed consent was obtained from each subject before testing, in accordance with the Committee on the Use of Humans as Experimental Subjects of the Massachusetts Institute of Technology and the Subcommittee on Human Studies at the Massachusetts General Hospital. Subjects underwent GHRH and arginine stimulation testing in the morning after a 12-h fast.
Screening outpatient visit. After a 12-h overnight fast, subjects reported to the General Clinical Research Center for a screening visit, at which time eligibility was determined based on a fasting blood glucose level less than 126 mg/dl.
GH assessment
Subjects received standardized stimulation testing with GHRH-arginine [GHRH 1–29 (Geref, Serono, Inc., Norwell, MA) 1-μg/kg iv bolus along with simultaneous administration of arginine hydrochloride (0.5 g/kg) (maximum dose, 30 g) given iv over 30 min]. GH levels were collected at –15, 0, 15, 30, 45, 60, 90, and 120 min after GHRH administration.
Body composition analysis
Height, weight, and BMI were determined in the fasting state during the screen visit. Cross-sectional abdominal computed tomography (CT) scanning was performed to assess the distribution of sc and visceral abdominal fat. A lateral scout image was obtained to identify the level of the L4 pedicle, which served as a landmark for the single-slice image. Scan parameters for each image were standardized (144 cm table height, 80 kV, 70 mA, 2 sec, 1-cm slice thickness). Fat attenuation coefficients were at –50 Hansfield units as described by Borkan et al. Abdominal visceral (VAT) and sc fat areas (SAT) were then determined (6). Total fat and extremity fat were determined by dual-energy x-ray absorptiometry (DXA) (Hologic 4500, Hologic Inc., Waltham, MA) as previously described (7).
Biochemical and immunological function
Fasting glucose, insulin, IGF-I, IGF binding protein (IGFBP)-1, IGFBP-3, free fatty acids (FFAs), triglyceride, testosterone, TSH, CD4+ cell count, and viral load were determined after a 12-h fast before any stimulation testing. A 75-g oral glucose tolerance test was performed for glucose and insulin on a separate occasion after a 12-h fast.
Laboratory methods
GH was measured by two-site RIA with an intraassay coefficient of variation (CV) of 4.4% (Corning, Inc. Nichols Institute Diagnostics, San Juan Capistrano, CA). The interassay CV was 6.6%. The sensitivity of the assay was determined to be 0.05 ng/ml. IGF-I was measured by two-site RIA with an intraassay CV of 4.9% (Diagnostic Systems Laboratories, Inc., Webster, TX). The interassay CV was 5.1%. The sensitivity of the assay was determined to be 2.6 ng/ml. IGFBP-1 was measured by two-site RIA with sensitivity of 0.33 ng/ml and an intraassay CV of 4.2% (Diagnostic Systems Laboratories, Inc.). IGFBP-3 was measured by two-site RIA with sensitivity of 0.5 ng/ml and an intraassay CV of 2.9% (Diagnostic Systems Laboratories, Inc.) Serum TSH levels were run using a two-site RIA (DiaSorin, Inc., Stillwater, MN; intraassay CV average, 3.1–3.3%; sensitivity, 0.01 μIU/ml based on serial dilutions).
Nonesterified fatty acid concentrations were measured by an in vitro enzymatic colorimetric assay kit (Wako Chemicals USA, Inc., Richmond, VA). The intraassay CV for fatty acids ranged from 1.1–2.7%. The published normal range for fatty acids is 0.1–0.6 mmol/liter. Insulin concentrations were measured in serum by RIA (Diagnostic Products, Los Angeles). The intraassay and interassay CVs ranged from 4.7–7.7% and from 5.5–9.2%, respectively. Glucose and triglyceride concentrations were measured by standard techniques. Testosterone was measured by RIA (Diagnostic Products). The intraassay CV ranged from 5–11%, and the interassay CV ranged from 5.9–12%. The sensitivity for the assay was 4 ng/dl.
The CD4 count was determined by flow cytometry (Becton Dickinson Immunocytochemistry Systems, San Jose, CA), and the HIV viral load was determined by ultrasensitive assay (Amplicor HIV-1 Monitor Assay; Roche Molecular Systems, Indianapolis, IN), with limits of detection of 50–75,000 copies/ml.
Statistical analysis
Demographic, body composition, and biochemical indices were compared among the three groups by overall ANOVA for continuous variables and likelihood ratio for dichotomized variables. In stepwise regression modeling, peak GH response and GH area under the curve (AUC) were the dependent variables; and age, BMI, VAT, SAT, insulin, triglycerides, and FFA concentrations were tested for entry into the model as dependent variables at P = 0.10. Among the HIV-infected subjects, group (LIPO or NONLIPO) was also tested in stepwise regression modeling. Final P values and parameter estimates are shown for each model. All statistical analyses were made using SAS JMP Statistical Database Software (version 4; SAS Institute, Inc., Cary, NC). Statistical significance was defined as a two-tailed -value of P 0.05. Results are mean ± SEM unless otherwise indicated.
Results
Subject characteristics
Thirty-nine male HIV-infected LIPO patients and 17 NONLIPO patients were compared with sixteen healthy, HIV-negative male C subjects. The clinical characteristics of the study patients are shown in Table 1. The mean ages and BMI’s were similar among the three groups (Table 1). The LIPO subjects demonstrated higher WHR, visceral abdominal fat by CT, VAT:SAT ratio and trunk:lower extremity fat ratio (by DXA) than either the NONLIPO or C group. In contrast, lower extremity fat was reduced in the LIPO group (Table 1).
Triglyceride and insulin concentrations were significantly increased in the LIPO group compared with the NONLIPO and C groups (Table 1). Testosterone and TSH levels were not different among the groups and were normal in all subjects. FFA concentrations were higher in the LIPO group, but this difference did not reach statistical significance. IGF-I (P < 0.05, LIPO vs. NONLIPO) and IGFBP-3 were lowest in the LIPO group (P < 0.05, LIPO vs. NONLIPO and LIPO vs. C). CD4 count, viral load, and antiretroviral use were similar between the HIV groups (Table 1).
The rate of failure in response to the combined GHRH-arginine test was 18% in the LIPO group vs. 5.9% in the NONLIPO group and 0% in the C group, using a stringent cutoff of 3.3 ng/ml (P < 0.05 for LIPO vs. C). Using less stringent cutoffs, the failure rates in the LIPO group rose to 30.8% at 5.0 ng/ml and 38.5% at 7.5 ng/ml, with corresponding increases in the C group to 12.5% (P < 0.05 vs. NONLIPO at 5.0 ng/ml and P < 0.05 vs. NONLIPO and C at 7.5 ng/ml) (Fig. 1). At 9.0 ng/ml, 43.6% of the LIPO group vs. 17.7% and 25% of the NONLIPO and C groups, respectively, failed the stimulation testing (P = not significant).
Among the entire group of HIV-infected subjects, visceral fat area was the most significant predictor of peak GH response to GHRH and arginine (parameter estimate = –.09, P = 0.008), controlling for group (LIPO vs. NONLIPO), age, BMI, SAT, FFA concentration, fasting insulin, and triglycerides. In this model, fasting insulin was also a significant independent predictor of peak GH response (parameter estimate =–0.34, P = 0.03) (Table 2). Among the subjects in the LIPO group, visceral fat area (parameter estimate = –0.09, P = 0.008), basal FFA level (parameter estimate = –18.7, P = 0.04), and basal insulin level (parameter estimate = –0.33, P = 0.007) were significant inverse predictors of peak GH response to GHRH and arginine (Table 2). Among subjects in the C group, only sc abdominal fat predicted peak GH response to combined GHRH-arginine in regression modeling (parameter estimate = –0.24, P = 0.009) (Table 2). Generally, similar results were seen in modeling for GH AUC (Table 2).
Discussion
To our knowledge, this study is the first to evaluate GH rates of response to the GHRH-arginine test in HIV-infected patients. We used a standardized method of testing for GHD in HIV LIPO subjects in comparison with healthy male adult C subjects matched for age and BMI. Eighteen percent of LIPO patients failed the GHRH-arginine stimulation testing using a highly stringent cutoff, suggesting that a small, but significant, number of such subjects will fail a standardized GH stimulation test. Peak GH response is inversely related to visceral adiposity, such that lower GH peak responses are seen with increasing visceral fat accumulation.
The optimal testing paradigm and cutoff for inadequate GH response are controversial. Insulin testing to evoke hypoglycemia is traditionally regarded as the gold standard test to determine GHD but was not appropriate in our subjects because of the potential risk associated with this test. The insulin tolerance test (ITT) is useful in patients with hypothalamic and/or pituitary disease and evaluates the entire hypothalamic-somatotroph axis. However, the test is contraindicated in patients with hypoglycemia, seizures, and ischemic heart disease. It is unknown whether the GHRH-arginine is equivalent to the ITT in patients with HIV. Because the GHRH-arginine test has less risk and has been shown to be reliable in other populations to detect GHD (8), it was chosen for our study.
The GHRH-arginine test compares well with the insulin test in diagnosing GHD (8) and has been considered a reasonable alternative test. The GH response to GHRH-arginine is independent of age, and there is less inter- and intraindividual variability than with other stimulation tests. The combined GHRH-arginine test is considered by many to be the best diagnostic alternative to the ITT (9). The lowest limit of GH response to ITT in normal subjects has been reported as 5 ng/ml by some (but not other) authors (10, 11). The peak GH cutpoint of 9 ng/ml for the GHRH-arginine tests represents the first centile limit of the normal GH response in a large population of normal subjects (11). Furthermore, Aimaretti et al. (12) showed that 92.5% of hypopituitary adults had a GH peak below this limit.
However, because the optimal cutoff for inadequate GH response is unknown, we chose a highly stringent cutoff of 3.3 ng/ml, because this value has been previously shown to demonstrate 95% specificity, with respect to the ITT test, for diagnosing GHD (8) and to minimize the misclassification of subjects as GH deficient when this is not the case. A caveat in this regard is that the 3.3 ng/ml cutoff was previously determined in comparison with the ITT in patients with known pituitary disease, in contrast to the patients described in this study, with nutritional disturbances in GH response to GHRH-arginine. Nonetheless, there were significant differences between the LIPO and C groups using this cutoff, with 0% of the C subjects failing the test compared with 18% of the subjects in the LIPO group. With increasing cutoffs, as have been proposed by others in the literature (11, 12), e.g. to 9.0 ng/ml, rates of failure to GHRH-arginine increased as anticipated (up to 44%), but a relatively large percentage of C subjects also failed the test (25%). Significant differences were not seen between the groups at the 9.0 ng/ml cutoff. Further studies are necessary to determine the best threshold to define reduced GH response rates to the GHRH-arginine test in the HIV population; but our data, using a strict threshold of 3.3 ng/ml, demonstrate that approximately one in five such LIPO subjects will fail to stimulate adequately.
The mean serum IGF-I was significantly lower among subjects in the LIPO group. However, IGF-I was below the normal range in only 8% of the LIPO subjects compared with 0% the NONLIPO subjects or C subjects (P = not significant). IGF-I was not correlated with peak response to GHRH-arginine stimulation testing in any of the groups. IGF-I is a relatively poor test to diagnose GHD in adults and has been shown to be in the normal range, even among patients who fail ITT testing (13, 14, 15). In contrast, IGF-I might be more useful for the diagnosis of GHD in childhood-onset or young-adult-onset GHD patients (16). However, because different IGF-I assays may yield different results, and serum IGF-I concentrations may be decreased by a variety of causes, caution is generally used in applying low IGF-I diagnostic cutpoints to the diagnosis of adult GHD in clinical practice (17). Furthermore, in contrast to GH resistance in undernutrition, prior studies have shown that GH secretion and IGF-I can be dissociated in simple obesity and have suggested an increased sensitivity to GH in overnutrition and obesity (18). Relatively increased sensitivity to GH may also occur in HIV patients with visceral adiposity and normal weight, although this has not been formally investigated in the HIV population.
IGFBP-3 was also significantly lower, but in the normal range, in the HIV lipodystrophy group. IGFBP-3 is N-glycosylated and is the major IGF binding moiety in plasma, given that more than 75 percent of circulating IGF-I is bound to this protein. IGFBP-3 concentrations are increased by GH administration, and IGFBP-3 functions as the major GH-responsive carrier of IGF-I (19). Of the six IGFBPs, IGFBP-3 is the major serum carrier protein for IGF peptides and the most GH dependent (20). IGFBP-3 levels are less nutritionally dependent, and the normal range varies only modestly with age. In our study, IGFBP-3 levels were significantly lower in the LIPO group in comparison with the other C groups. In contrast to some reports among non-HIV-infected patients (21, 22), IGFBP-3 concentrations did not correlate with peak GH response to GHRH-arginine in the LIPO subjects. Although lower IGF-I and IGFBP-3 levels support the observation of reduced GH secretion in the LIPO group, neither test is specific enough to use for diagnostic purposes in this population.
GH secretion is suppressed in obesity, with 24-h GH levels in obese men reduced by 75% in comparison with age-matched normal-weight C subjects (5). In addition, decreased responsiveness to stimulation tests such as GHRH, arginine, and combined GHRH-arginine has been demonstrated in non-HIV-infected subjects with obesity (23). Veldhuis et al. (24) demonstrated that the degree of body fatness was associated inversely with indices of GH secretion. In contrast, in our study, visceral fat, not BMI, was the major determinant of GH responsiveness in LIPO subjects. Similar results were seen among the entire HIV group, controlling for group (e.g. LIPO or NONLIPO). In contrast, only sc abdominal fat predicted peak GH response to GHRH and arginine in the C group.
The rates of failure in response to GH stimulation testing in the LIPO group were not a simple function of obesity, because response rates differed in comparison to a C group of healthy subjects similar in BMI. Furthermore, the inadequate response was not due to HIV per se, because lower failure rates were seen in age- and BMI-matched NONLIPO subjects. Importantly, our results can only be generalized to the patient population we recruited, with excess visceral adiposity and simultaneous loss of sc and extremity fat. It is less likely that any differences in GH response rates would be seen among a population with more pure lipoatrophy, because total body fat and extremity fat did not correlate with peak GH response among patients in HIV patients.
What are the mechanisms of reduced GH in the LIPO population? In prior studies, we have shown a strong inverse association between overnight GH secretion rates and visceral adiposity in frequent sampling. We now extend this finding to show a strong inverse relationship between visceral adiposity and GH response to GHRH-arginine. In addition, elevated FFA and insulin levels were significant independent variables in the regression models for peak GH response to GHRH-arginine in LIPO subjects.
We have also shown decreased ghrelin as well as an inhibitory effect of increased FFA on GH response to GHRH alone (25). We now show that peak GH response to GHRH-arginine is also inversely associated with FFA concentration. FFA remained negatively associated with peak GH response to GHRH-arginine in stepwise regression analysis controlling for age, BMI, and visceral fat, suggesting a potential inhibitory role with respect to GH secretion in the LIPO population. In healthy, obese subjects, Cordido et al. (26) demonstrated improved GH secretion in response to acipimox and reduced FFA. We also recently showed improved GH response to GHRH alone using acipimox, suggesting that FFA may play an inhibitory role with respect to GH secretion in the HIV lipodystrophy population (25).
Are HIV patients who fail the GHRH-arginine test truly or functionally GH deficient? Subjects in this study did not have any risk factors for, or symptoms of, pituitary dysfunction, and testosterone levels were normal in all patients. Although we did not perform pituitary imaging, it is unlikely that pituitary disease per se would explain our findings of inadequate GH response rates in up to nearly 20% of LIPO patients. More likely, our subjects have a functional GHD related, in part, to excess visceral fat. Whether this represents true GHD is not clear, because recent studies suggest that GH levels can change significantly over time, with reduction in weight and changes in body composition in otherwise healthy subjects (27, 28). This study was limited to men, to control for the influence of gender on GH secretion. Further studies determining GH response in LIPO women are needed.
A number of prior studies have investigated GH in HIV patients, both with the wasting syndrome (29) and with LIPO subjects (30, 31). Most prior studies in LIPO patients have investigated relatively large, supraphysiological doses of GH. Reported side effects with supraphysiological GH dosing in the HIV population include impaired glucose tolerance and joint aches. Recent studies suggest that such effects may be minimized with lower dosing (32). In contrast, Johannsson et al. (33) reported on the use of low-dose GH in non-HIV-infected patients with abdominal obesity, suggesting that GH administration might improve insulin resistance and reduce visceral adiposity in this population. Similarly, physiological GH dosing may result in improved insulin resistance and abdominal fat but has not been investigated in the HIV population. Furthermore, prior studies have not targeted a specific subpopulation of HIV patients with abnormal GH responses to GH stimulation testing.
In conclusion, our study suggests that approximately 20% of HIV-infected patients with lipodystrophy and increased visceral adiposity will have inadequate GH stimulation to GHRH-arginine using a stringent cutoff of 3.3 ng/ml and may therefore be functionally GH deficient. Such patients may benefit from GH administration in terms of improved body fat and metabolic parameters, and further studies are necessary to test the efficacy of low-dose GH in this population. Use of a less stringent cutoff may increase the number of patients failing the GH stimulation test but decrease the specificity of the test. Further studies are needed to optimally identify HIV patients with decreased GH secretion and to assess whether such patients might benefit from physiologic GH replacement.
Acknowledgments
We acknowledge the nursing and bionutrition staffs of the General Clinical Research Center of the Massachusetts General Hospital and Massachusetts Institute of Technology for their dedicated patient care, and Dr. Anne Klibanski for helpful suggestions with the manuscript.
Footnotes
This work was supported in part by National Institutes of Health Grants R01 DK063639 and M01-RR01066.
First Published Online October 13, 2004
Abbreviations: AUC, Area under the curve; BMI, body mass index; C, control (group); CT, computed tomography; CV, coefficient of variation; DXA, dual-energy x-ray absorptiometry; FFA, free fatty acid; GHD, GH deficiency; IGFBP, IGF binding protein; ITT, insulin tolerance test; LIPO, HIV patients with lipodystrophy (i.e. with fat redistribution); NONLIPO, HIV patients without lipodystrophy; SAT, sc adipose tissue; VAT, visceral adipose tissue; WHR, waist to hip ratio.
Received July 15, 2004.
Accepted September 23, 2004.
References
Carr A, Samaras K, Chisholm DJ, Cooper DA 1998 Pathogenesis of HIV-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance. Lancet 351:1881–1883
Carr A, Samaras K, Burton S, Law M, Freund J, Chisholm DJ, Cooper DA 1998 A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors. AIDS 12:F51–F58
Hadigan C, Meigs JB, Corcoran C, Rietschel P, Piecuch S, Basgoz N, Davis B, Sax P, Stanley T, Wilson PW, D’Agostino RB, Grinspoon S 2001 Metabolic abnormalities and cardiovascular disease risk factors in adults with human immunodeficiency virus infection and lipodystrophy. Clin Infect Dis 32:130–139
Rietschel P, Hadigan C, Corcoran C, Stanley T, Neubauer G, Gertner J, Grinspoon S 2001 Assessment of growth hormone dynamics in human immunodeficiency virus-related lipodystrophy. J Clin Endocrinol Metab 86:504–510
Veldhuis JD, Iranmanesh A, Ho KK, Waters MJ, Johnson ML, Lizarralde G 1991 Dual defects in pulsatile growth hormone secretion and clearance subserve the hyposomatotropism of obesity in man. J Clin Endocrinol Metab 72:51–59
Borkan GA, Gerzof SG, Robbins AH, Hults DE, Silbert CK, Silbert JE 1982 Assessment of abdominal fat content by computed tomography. Am J Clin Nutr 36:172–177
Hadigan C, Miller K, Corcoran C, Anderson E, Basgoz N, Grinspoon S 1999 Fasting hyperinsulinemia and changes in regional body composition in human immunodeficiency virus-infected women. J Clin Endocrinol Metab 84:1932–1937
Biller BM, Samuels MH, Zagar A, Cook DM, Arafah BM, Bonert V, Stavrou S, Kleinberg DL, Chipman JJ, Hartman ML 2002 Sensitivity and specificity of six tests for the diagnosis of adult GH deficiency. J Clin Endocrinol Metab 87:2067–2079
Valetto MR, Bellone J, Baffoni C, Savio P, Aimaretti G, Gianotti L, Arvat E, Camanni F, Ghigo E 1996 Reproducibility of the growth hormone response to stimulation with growth hormone-releasing hormone plus arginine during lifespan. Eur J Endocrinol 135:568–572
de Boer H, Blok GJ, Van der Veen EA 1995 Clinical aspects of growth hormone deficiency in adults. Endocr Rev 16:63–86
Ghigo E, Aimaretti G, Gianotti L, Bellone J, Arvat E, Camanni F 1996 New approach to the diagnosis of growth hormone deficiency in adults. Eur J Endocrinol 134:352–356
Aimaretti G, Corneli G, Razzore P, Bellone S, Baffoni C, Arvat E, Camanni F, Ghigo E 1998 Comparison between insulin-induced hypoglycemia and growth hormone (GH)-releasing hormone + arginine as provocative tests for the diagnosis of GH deficiency in adults. J Clin Endocrinol Metab 83:1615–1618
Hilding A, Hall K, Wivall-Helleryd IL, Saaf M, Melin AL, Thoren M 1999 Serum levels of insulin-like growth factor I in 152 patients with growth hormone deficiency, aged 19–82 years, in relation to those in healthy subjects. J Clin Endocrinol Metab 84:2013–2019
Marzullo P, Di Somma C, Pratt KL, Khosravi J, Diamandis A, Lombardi G, Colao A, Rosenfeld RG 2001 Usefulness of different biochemical markers of the insulin-like growth factor (IGF) family in diagnosing growth hormone excess and deficiency in adults. J Clin Endocrinol Metab 86:3001–3008
Baum HB, Biller BM, Katznelson L, Oppenheim DS, Clemmons DR, Cannistraro KB, Schoenfeld DA, Best SA, Klibanski A 1996 Assessment of growth hormone (GH) secretion in men with adult-onset GH deficiency compared with that in normal men—a clinical research center study. J Clin Endocrinol Metab 81:84–92
GH Research Society 2000 Consensus quidelines for the diagnosis and treatment of growth hormone (GH) deficiency in childhood and adolescence: summary statement of the GH Research Society. J Clin Endocrinol Metab 85:3990–3993
Growth Hormone Research Society Workshop on Adult Growth Hormone Deficiency 1998 Consensus guidelines for the diagnosis, treatment of adults with growth hormone deficiency: summary statement of the Growth Hormone Research Society Workshop on Adult Growth Hormone Deficiency. J Clin Endocrinol Metab 83:379–381
Hochberg Z, Hertz P, Colin V, Ish-Shalom S, Yeshurun D, Youdim MB, Amit T 1992 The distal axis of growth hormone (GH) in nutritional disorders: GH-binding protein, insulin-like growth factor-I (IGF-I), and IGF-I receptors in obesity and anorexia nervosa. Metabolism 41:106–112
Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34
Baxter RC, Martin JL 1986 Radioimmunoassay of growth hormone-dependent insulin-like growth factor binding protein in human plasma. J Clin Invest 78:1504–1512
Hasegawa Y, Hasegawa T, Aso T, Kotoh S, Tsuchiya Y, Nose O, Ohyama Y, Araki K, Tanaka T, Saisyo S 1992 Usefulness and limitation of measurement of insulin-like growth factor binding protein-3 (IGFBP-3) for diagnosis of growth hormone deficiency. Endocrinol Jpn 39:585–591
Blum WF, Ranke MB, Kietzmann K, Gauggel E, Zeisel HJ, Bierich JR 1990 A specific radioimmunoassay for the growth hormone (GH)-dependent somatomedin-binding protein: its use for diagnosis of GH deficiency. J Clin Endocrinol Metab 70:1292–1298
Cordido F, Dieguez C, Casanueva FF 1990 Effect of central cholinergic neurotransmission enhancement by pyridostigmine on the growth hormone secretion elicited by clonidine, arginine, or hypoglycemia in normal and obese subjects. J Clin Endocrinol Metab 70:1361–1370
Veldhuis JD, Liem AY, South S, Weltman A, Weltman J, Clemmons DA, Abbott R, Mulligan T, Johnson ML, Pincus S 1995 Differential impact of age, sex steroid hormones, and obesity on basal versus pulsatile growth hormone secretion in men as assessed in an ultrasensitive chemiluminescence assay. J Clin Endocrinol Metab 80:3209–3222
Koutkia P, Meininger G, Canavan B, Breu J, Grinspoon S 2004 Metabolic regulation of growth hormone by free fatty acids, somatostatin, and ghrelin in HIV-lipodystrophy. Am J Physiol Endocrinol Metab 286:E296–E303
Cordido F, Peino R, Penalva A, Alvarez CV, Casanueva FF, Dieguez C 1996 Impaired growth hormone secretion in obese subjects is partially reversed by acipimox-mediated plasma free fatty acid depression. J Clin Endocrinol Metab 81:914–918
Rasmussen MH, Hvidberg A, Juul A, Main KM, Gotfredsen A, Skakkebaek NE, Hilsted J, Skakkebae NE 1995 Massive weight loss restores 24-hour growth hormone release profiles and serum insulin-like growth factor-I levels in obese subjects. J Clin Endocrinol Metab 80:1407–1415
De Marinis L, Bianchi A, Mancini A, Gentilella R, Perrelli M, Giampietro A, Porcelli T, Tilaro L, Fusco A, Valle D, Tacchino RM 2004 Growth hormone secretion and leptin in morbid obesity before and after biliopancreatic diversion: relationships with insulin and body composition. J Clin Endocrinol Metab 89:174–180
Schambelan M, Mulligan K, Grunfeld C, Daar ES, LaMarca A, Kotler DP, Wang J, Bozzette SA, Breitmeyer JB 1996 Recombinant human growth hormone in patients with HIV-associated wasting. A randomized, placebo-controlled trial. Serostim Study Group. Ann Intern Med 125:873–882
Wanke C, Gerrior J, Kantaros J, Coakley E, Albrecht M 1999 Recombinant human growth hormone improves the fat redistribution syndrome (lipodystrophy) in patients with HIV. AIDS 13:2099–2103
Kotler DP, Muurahainen N, Grunfeld C, Wanke C, Thompson M, Saag M, Bock D, Simons G, Gertner JM, Serostim in Adipose Redistribution Syndrome Study Group 2004 Effects of growth hormone on abnormal visceral adipose tissue accumulation and dyslipidemia in HIV-infected patients. J Acquir Immune Defic Syndr 35:239–252
Lo JC, Mulligan K, Noor MA, Schwarz JM, Halvorsen RA, Grunfeld C, Schambelan M 2001 The effects of recombinant human growth hormone on body composition and glucose metabolism in HIV-infected patients with fat accumulation. J Clin Endocrinol Metab 86:3480–3487
Johannsson G, Marin P, Lonn L, Ottosson M, Stenlof K, Bjorntorp P, Sjostrom L, Bengtsson BA 1997 Growth hormone treatment of abdominally obese men reduces abdominal fat mass, improves glucose and lipoprotein metabolism, and reduces diastolic blood pressure. J Clin Endocrinol Metab 82:727–734(Polyxeni Koutkia, Bridget)