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Departments of Endocrinology and Cardiology (J.L.M.O., M.H.A.-O., A.D., R.M.C.P., C.R.P.O., C.T.F., J.A.B.-F., F.D.A.-A., C.M.-S., A.C.N.-J., E.O.A., F.T.O. V.C.C., R.X.), Federal University of Sergipe, Aracaju, SE Brazil 49060-100; and The Sidney Kimmel Comprehensive Cancer Center (A.B., G.P.) and the Division of Endocrinology (R.S.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
Address all correspondence and requests for reprints to: Roberto Salvatori, M.D., Division of Endocrinology, The Johns Hopkins University School of Medicine, 1830 East Monument Street, Suite 333, Baltimore, Maryland 21287. E-mail: salvator{at}jhmi.edu.
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Abstract |
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Methods: We have studied the effects of 6-month GH replacement therapy in 20 adult members of a large Brazilian kindred with lifelong severe and isolated GHD due to a homozygous mutation in GHRH receptor gene (46 ± 14.5 yr; 122 ± 7.7 cm; 36.7 ± 5.4 kg; 10 men). Subjects were studied at baseline, after 6-month bimonthly depot GH injections (Nutropin Depot; Genentech, Inc., South San Francisco, CA) [post GH (pGH)], and after 6- and 12-month washout.
Results: Despite modest trough serum IGF-I increase, GH replacement therapy caused a decrease in skinfolds and in waist-hip ratio, with a rebound increase at 12 months. Total and low-density lipoprotein cholesterol were reduced pGH and returned to baseline at 6 months. High-density lipoprotein cholesterol increased pGH, but at 12 months was lower than baseline. A progressive increase in left ventricular mass index, posterior wall, and septum thickness occurred from pGH to 12 months, and of carotid intima-media thickness, from 6 to 12 months. Individuals were 6, 16, and 52 times more likely to have an atherosclerotic carotid plaque at pGH, 6 and 12 months, respectively, when compared with baseline.
Conclusion: In patients with lifetime isolated GHD, 6-month treatment with GH has reversible beneficial effects on body composition and metabolic profile, but it causes a progressive increase in intima-media thickness and in the number of atherosclerotic carotid plaques.
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Introduction |
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We have identified a large extended pedigree with approximately 100 affected individuals, with familial autosomal recessive IGHD, residing in Itabaianinha County, in the Northeastern Brazilian state of Sergipe. They all carry a homozygous null mutation in the splice donor site of intron 1 (IVS1 + 1G
A) of the GHRH receptor gene (8). This is the largest cohort of patients with IGHD described to date. GH-naive adults and children from this kindred have very low serum GH and IGF-I, reduced fat free mass, increase in percentage of fat mass, increased waist-hip (W/H) ratio, total and LDL-C, CRP levels, and systolic blood pressure (SBP), but no insulin resistance and no evidence of premature atherosclerosis (9, 10, 11, 12). We have shown that in children, GH replacement therapy (GHRT) exerts beneficial effects in lipids and body composition (13).
In this work we assessed the effects of 6-month GHRT with a depot preparation in 20 GH-naive adults on body composition, metabolic and inflammatory profile. Moreover, we investigated the effect of GHRT on cardiac morphology and function, and on carotid artery walls. We demonstrate that, despite beneficial effects on traditional risk factors, GHRT caused thickening of the carotid walls and increase in the number of atherosclerotic plaques.
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Subjects and Methods |
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A total of 20 adult GHD subjects (10 men and 10 women; age, 46 ± 14.5 yr; height, 122.1 ± 7.7 cm; weight, 36.7 ± 5.4 kg) were recruited by advertising in the local health clinic and by word of mouth. None of the subjects had any evidence, based on medical history, of cardiovascular or other systemic disease. Eight of the GHD patients (36%) were receiving captopril therapy for hypertension and had been previously instructed to follow a reduced salt diet by their treating physicians. Subjects were studied at baseline, after 6-month bimonthly depot GH, Nutropin Depot [post GH (pGH)], and after 6- and 12-month washout. The initial and final doses were 0.33 and 0.38 mg/kg in women and 0.25 and 0.35 mg/kg in men. Dosing was based on previous work showing an increase in both GH and IGF-I lasting 14–17 d in adults after a single 0.25 or 0.5 mg/kg dose (14). A similar starting dose (0.3 mg/kg) was used in the only clinical trial that has shown efficacy of this depot GH preparation in adults (15). We had initially planned to titrate the dose according to serum IGF-I level and patients’ symptomatology. However, due to the limited availability of the drug (whose manufacturing was discontinued immediately before the start of this study), after the fourth dose, we administered one vial (13.5 mg) per subject. None of the female subjects was taking oral or transdermal estrogen therapy, known to cause resistance to GH (16). Injections were given sc at the subject’s residence by the same operator. Both the University of Sergipe and The Johns Hopkins University institutional review boards approved these studies, and all subjects gave written informed consent.
Study protocol
Anthropometric measurements. The subjects’ height and body weight were measured with a portable stadiometer and portable scale; body mass index (BMI) was calculated using the formula: weight (kilograms)/height (meters)2. Triceps, biceps, subscapular, and suprailiac skin folds were measured (in millimeters) with the subjects standing erect using the Lange Skin fold Caliper (Cambridge, MD) on the right side of the body. Hip and waist circumferences were measured (in centimeters), and W/H ratio was calculated.
Laboratory assessment. Total cholesterol, triglycerides, and glucose were measured by the enzymatic Trinder colorimetric test. The high-density lipoprotein cholesterol (HDL-C) was separated using the phosphotungstic acid/magnesium chloride method, and the LDL-C concentration was calculated indirectly (Friedewald formula). Glycosylated hemoglobin (HbA1c) was measured by HPLC by the Bio-Rad variant II turbo (Bio-Rad Laboratories, Hercules, CA). Insulin was measured by a solid-phase, two-site chemiluminescent immunometric assay, with the sensitivity of 2 µIU/ml (Diagnostic Product Corp., Los Angeles, CA), and the intraassay and interassay variabilities were 4.26 and 5.43%, respectively; IGF-I was measured by a immunoradiometric assay, with double extraction and an assay sensitivity of 0.8 ng/ml (5600; Diagnostic Systems Laboratories, Inc., Webster, TX). The intraassay and interassay variabilities were 2.25 and 2.6%, respectively. SD values for serum IGF-I were calculated by subtracting the mean of IGF-I level of the age from the individual value and dividing this value by the SD of the respective mean age given by the manufacturer.
Insulin resistance was estimated using the homeostasis model assessment of insulin resistance with the formula: fasting serum insulin (µU/ml) x fasting plasma glucose (mmol/liter)/22.5 (17). Ultrasensitive CRP (CRPus) and lipoprotein(a) [Lp(a)] were measured by nephelometry (Dade Behring Marburg GmbH Marburg, Germany, and Image Immunochemistry System, Beckman Coulter, Inc., Brea, CA, respectively). CRPus and Lp(a) sensitivities were 0.175 mg/liter and 2 mg/dl, respectively. The intraassay and the interassay variabilities were 3.5 and 3.4, and 5.0 and 6.5%, respectively.
CRPus and IGF-I were measured in serum after storage at –20 C. Lp(a) was measured in serum after storage at 4 C. Samples were assayed together after the end of all the collections, except for insulin, and lipid profiles and serum glucose levels were measured on the day of blood collection in the clinical Laboratory of the Hospital of the Federal University of Sergipe.
Assessment of carotid intima-media thickness (IMT). Longitudinal ultrasonographic scans of the carotid arteries were all performed by the same trained observer (R.X.). An ATL Ultramark (Advanced Technology Laboratories, Signal Hill, CA) equipped with a 10.0 MHz linear-array transducer was used. Subjects were examined in the supine position, and the neck was in slight hyperextension. The arterial wall was observed in longitudinal sections. With this technique, the layers of the arterial wall are represented as parallel echogenic lines separated by a hypoechoic space (double line). The distance between the beginning of the first echogenic line and the end of the second echogenic line was considered to be the combined thickness of the intima and media layers. These measurements were done in both carotids, 1.0-cm below the bifurcation. Two approaches were used (anterolateral and posterolateral), and the images were focused on the posterior wall of the vessel. The width was calculated for each subject as the mean of three measurements for each segment. In addition, both the right and left common, internal, and external carotid arteries were examined in multiple projections to identify the presence of atherosclerotic plaques. An artery was classified as having a plaque if there was a localized thickening more than or equal to 1.3 mm that did not uniformly involve the whole carotid with or without flow disturbance (18).
Resting echocardiography. Echocardiographic studies were performed with a commercial machine (HP-Sonos 5500; Hewlett-Packard Co., Palo Alto, CA) according to standard procedures. M-mode echocardiography of the left ventricle was performed according to the American Society of Echocardiography recommendations. Only frames with optimal visualization of interfaces and simultaneously visible septum, left ventricular (LV) internal diameters, and posterior wall were used for calculations. LV mass was calculated according to Devereux et al. (19), and normalized according to body surface area and height. Relative wall thickness was calculated as 2 x posterior wall thickness/LV internal radius.
Exercise echocardiography. The preexercise standing SBP and diastolic blood pressure (DBP) and heart rate (HR) were measured just before the exercise test. Patients underwent symptom-limited treadmill exercise testing according to the standard Bruce protocol. Two-dimensional echocardiographic images were obtained from the parasternal and apical windows before and immediately after exercise. Both digitized and digital video display DVD images were used for interpretation of the studies. Regional wall motion was assessed semi quantitatively by the same experienced echocardiographer (J.L.M.O.). Wall motion at rest and with exercise was scored on a five-point scale, with a score of: 1, indicating normal; 2, mild hypokinesis (decrease of movement and systolic thickening); 3, severe hypokinesis; 4, akinesis (absence of movement and systolic thinning); and 5, dyskinesis (paradoxical outward movement and possible systolic thinning) according to a 16-segment model. Wall motion score index (WMSI) was determined at rest and peak exercise as sum of the segmental scores divided by the number of visualized segments (20). The development of new or worsening wall motion was considered indicative of myocardial ischemia. A wall motion abnormality present at rest and unchanged with exercise was classified as "fixed." Exercise echocardiography results were defined as abnormal if there was ischemia or fixed wall motion abnormalities. The exercise electrocardiogram was considered positive for ischemia if there was horizontal or down-sloping ST-segment depression of more than 1 mm at 80 msec after the J point, nondiagnostic if the baseline ST-segment was abnormal, and negative for ischemia in the absence of these criteria.
Statistical analysis. The changes in anthropometric measurements, laboratory assessments, carotid IMT, and resting and exercise echocardiography measurements were explored using box plots at each measurement. HbA1c and insulin were not measured in 12-month washout. Some of the variables were skewed, so appropriate transformations (i.e. log, square root) were used to achieve normality. The changes from baseline (pGH vs. baseline, 6 months vs. baseline, and 12 months vs. baseline) were estimated for each measurement outcome using linear regression models with generalized estimating equations to account for the correlation between measures within individuals. The presence of plaques at each time point was modeled similarly using logistic regression. A signed rank test was used to compare changes in IGF-I from baseline to 6 months. Analyses were completed with software SPSS/PC 11.5 (SPSS, Inc., Chicago, IL) and freeware R (http://www.r-project.org). Probability values less than or equal to 0.05 were considered statistically significant.
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Results |
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Trough serum IGF-I in the 20 individuals increased significantly from basal to pGH and returned to basal values at 6 and 12 months (Table 1
). The increase of IGF-I at 6 months in comparison to basal was significant in both sexes (females: median difference = 35 ng/ml, P = 0.006; and males: median difference = 14 ng/ml, P = 0.036), indicating no GH resistance in our female group. GHRT caused a decrease in all the skin folds and in W/H, with a rebound increase after 12 months of washout. GHRT caused a reversible increase in DBP, whereas a trend toward increase in SBP was observed pGH and 6 months, which became significant at 12 months (Table 2). Weight and BMI did not change after GHRT. Total cholesterol and LDL-C were reduced pGH and returned to baseline at 6-month washout. HDL-C increased pGH, but at 12 months was lower than baseline. There was a nonsignificant trend toward decrease in CRPus (P = 0.06) and increase in Lp(a) (P = 0.06) at pGH. Serum triglycerides and fasting plasma glucose did not change (Table 1).
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A progressive increase in carotid IMT occurred from pGH to 6–12 months (Fig. 1). The number of individuals with carotid plaques was: one at baseline; five after GH [odds ratio = 6.3 (0.99, 40.4); P = 0.05 vs. baseline]; nine at 6 months [odds ratio = 16.3 (2.2, 124.0); P = 0.006 vs. baseline]; and 14 at 12 months [odds ratio = 51.9 (6.2, 437.0); P = 0.0002 vs. baseline].
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A progressive increase in LV mass index (LVMI), posterior wall thickness, and septum thickness occurred from baseline to 12 months (Table 2
and Fig. 1). Ejection fraction and fractional shortening (expression of systolic function) did not change with GH replacement. Among the parameters of diastolic function [E/A (tissue Doppler-derived diastolic velocities) and E'/A' wave ratio], only E/A changed transiently at the 6-month point.
Exercise echocardiography
During the Bruce protocol, no symptoms or electrocardiogram changes suggestive of myocardial ischemia were observed. The preexercise standing SBP and DBP and HR were similar all the time. The magnitude of the SBP, DBP, and HR responses to treadmill exercise did not differ.
Exercise echocardiogram was normal in all subjects. Rest and/or exercise WMSIs did not show any abnormality (Table 2).
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Discussion |
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In a previous study, we have shown that the IGHD individuals from the Itabaianinha kindred, who have never received GHRT, have a higher percentage of fat mass, central obesity, increase of total cholesterol and LDL serum levels, and elevated systolic arterial pressure, but no evidence of premature atherosclerosis (10, 11). This population offers an ideal model to study the effects of GHRT. In consideration of logistical factors (these subjects live in a rural area, and most of them do not have access to refrigeration), we used a form of depot GH that would allow optimal compliance. Due to lack of telephones and transportation means, we could only collect their blood every 2 wk, immediately before the depot GH injection. Therefore, we cannot determine that the peak serum IGF-I reached normal levels in the immediate days after the injections. However, previous pharmacokinetic studies have shown that a similar dose causes an increase in serum IGF-I that lasts approximately 2 wk after doses that are comparable to the one we used (14). Indeed, a long-term (32 wk) study showed normalization of serum IGF-I in GHD adults using a starting dose (in males > 35) that was lower than the one we have used (0.3 mg/kg) (15). However, in that study most patients had pretherapy serum IGF-I levels that were less markedly reduced than our subjects. Therefore, less GH may have been needed to normalize serum IGF-I. Regrettably, the limited amount of GH depot did not allow us titration of the drug according to the trough serum IGF-I. However, despite the modest increases in trough serum IGF-I, in agreement with the literature generated in adult onset GHD, GHRT caused an improvement in the metabolic cardiovascular risk profile followed by a return to baseline after the interruption of therapy. We found only a nonsignificant trend toward decrease in CRP and increase in Lp(a). This may be due to the submaximal GH dosage we have used.
Despite the apparently favorable effects on cardiovascular risk profile (cholesterol and CRP levels), the mean carotid IMT and the number of individuals with carotid atherosclerotic plaques increased significantly. This increase persisted and progressed into the 12-month washout. This result paralleled a trend toward progressive increase in SBP, which became statistically significant at 12 months. This result is surprising because it contradicts all the previously published literature generated in adults with acquired GHD (29, 30, 31). We can only speculate on the reasons for this contradictory finding. There are many differences between our IGHD individuals and the adult-onset GHD patients studied by the others investigators. Our subjects have lower IGF-I levels than observed in acquired hypopituitarism. Moreover, they have never been exposed to GHRT and, thus, differ from the acquired GHD patients who have normal GH levels during a long period of their lives. Atherosclerosis is a complex and multifactorial disease commonly associated with traditional cardiovascular risk factors. Recent epidemiological studies demonstrate that low serum IGF-I levels are associated with coronary atherosclerosis and acute myocardial infarction (32). Hypopituitary patients have endothelial dysfunction that is reversed by GHRT (33). These observations suggest the hypothesis that the low serum IGF-I levels contribute to increase in cardiovascular disease. On the other hand, a recent Japanese study has shown that higher serum IGF-I is positively correlated with IMT (34). Interestingly, experimental studies in animals suggest an association between IGF-I deficiency and increased longevity (35). The final effect of GH and IGF-I in the vascular wall is likely to result from multiple atherosclerotic and anti-atherosclerotic functions of these two hormones. It was suggested then that the IGF-I could have a double influence on the atherosclerotic physiopathology, promoting atherogenesis, by stimulating vascular smooth muscle proliferation and migration but also inducing vasodilatation by stimulating nitric oxide production (36). It is possible that different degrees of deficiency of IGF-I result in different final effects in the vascular wall, with a very severe decrease in IGF-I protecting against atherosclerosis, whereas a milder decrease being detrimental. It is possible that a submaximal GH treatment may have transformed our subjects in a GHD model similar to the adult GHD. We also found that the increase in IMT and in the number of plaques continued through the 12th month after the discontinuation of the drug, long after IGF-I serum levels had returned to baseline values. This is difficult to explain because we expected that the progression of atherosclerosis would slow down after GHRT was stopped. However, it has to be noted that HDL levels increased after 6-month treatment, but a rebound decrease was observed after 12-month washout, possibly contributing, at least in part, to the persistent pro-atherosclerotic effects of 6-month GHRT. Finally, it is well known that the risk of carotid atherosclerosis correlates with SBP rather than DBP (37). We have observed a progressive increase in SBP that continued during the washout. Although the mechanism of such increase is unknown, it is likely that this has contributed to the worsening of the carotid status.
GH and IGF-I are also well known to affect directly myocardial contractility, by increasing intracellular calcium content and enhancing calcium sensitivity of myofilaments in the cardiomyocytes (38). IGF-I also has a central role in determining LV mass. In a cohort of obese hypertensive patients, it was shown that besides blood pressure, IGF-I and insulin were independent determinants of LV mass (39). In addition, in some, but not all, studies GHRT has increased cardiac mass (40). In GHD adults GHRT induced a 26% increase in the LVMI and a 12% increase in the LV ejection fraction (41). Conversely, in a 10-yr follow-up study, no difference in cardiac size over baseline measurements was observed in adults who had received short- or long-term GH treatment (42). In agreement with most studies, we have observed an increase in all the parameters related to LV mass after GH treatment without return to baseline at 12 months. The E/A wave ratio (expression of diastolic function) changed transiently at 6 months, remaining in the range of normality. The persistency of the effects on cardiac morphology is unlikely to be an effect of the persistent presence of circulating GH because kinetic studies of this depot preparation show that GH is completely cleared within 1 month (43). Indeed, a persistent effect (lasting at least 3 months after discontinuation of GH therapy) on LV mass has been previously reported in subjects with adult onset GHD treated with GH for 1 yr (44). Contrarily to our subjects, the patients reported in that study had normal pre-GH LV mass. Because we do not have follow-up beyond 12 months, we cannot comment on whether such changes are permanent or if the heart morphology would eventually return to baseline.
Our study has the important limitation of a lack of an untreated control group. Therefore, we cannot exclude with certainty that the changes observed after GH treatment are not just a reflection of aging. However, the rapid increase in the number of atherosclerotic plaques and the expected effects on cardiac morphology make this possibility overall unlikely. Indeed, in a high-risk population with type 2 diabetes patients of age comparable to our subjects, a 2-yr period did not cause a detectable change in IMT, even if they did not receive hypocholesterolemic agents (45).
In conclusion, in adult individuals with lifetime congenital and severe IGHD, a 6-month treatment with depot GH, despite only a modest increase in serum through IGF-I levels, induced changes in fat distribution, lipid profile, and in cardiac morphology. The changes in fat distribution and lipid profile were reversible, but the ones in cardiac morphology lasted for at least 1 yr after treatment completion. In addition, GHRT caused an increase in SBP and the development of carotid atherosclerosis in a large percentage of the study subjects. Our data suggest a word of caution on the universally assumed positive effect of GHRT on cardiovascular status.
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Acknowledgments |
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Footnotes |
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Clinical trial.gov identifier NCT00149708.
Disclosure Statement: J.L.M.O., M.H.A.-O., A.D., R.M.C.P., C.R.P.O., C.T.F., J.A.B.-F., F.D.A.-A., C.M.-S., A.C.N.-J., E.O.A., F.T.O. V.C.C., R.X., A.B., and G.P. have nothing to declare. R.S. receives grant support from the Genentech Center for Clinical Research (August 1, 2006 - July 31, 2008).
First Published Online October 2, 2007
Abbreviations: BMI, Body mass index; CRP, C-reactive protein; CRPus, ultrasensitive CRP; DBP, diastolic blood pressure; E/A, tissue Doppler-derived diastolic velocities; GHD, GH deficiency; GHRT, GH replacement therapy; HbA1c, glycosylated hemoglobin; HDL, high-density lipoprotein; HDL-C, HDL cholesterol; HR, heart rate; IGHD, isolated GHD; IMT, intima-media thickness; LDL, low-density lipoprotein; LDL-C, LDL cholesterol; Lp(a), lipoprotein(a); LV, left ventricular; LVMI, LV mass index; pGH, post GH; SBP, systolic blood pressure; W/H, waist-hip; WMSI, wall motion score index.
Received July 23, 2007.
Accepted September 25, 2007.
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