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Effect of testosterone replacement therapy on insulin sensitivity and body composition in congenital hypogonadism: A prospective longitudinal follow-up study K CO Reddy, SB YadavDepartment of Endocrinology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/jpgm.JPGM_887_20
Keywords: β-cell function, insulin resistance, lean body mass, low testosterone, metabolic syndrome, total body fat
Numerous studies have described hypogonadism in patients with metabolic syndrome (MS).[1–5] However, there are few studies on MS in young adults with congenital hypogonadism, and its improvement on testosterone treatment.[6–9] An increased prevalence of MS and truncal obesity has been reported in subjects with Klinefelter syndrome and idiopathic gonadotropin deficiency.[7] Sönmez et al. reported increased prevalence of MS in congenital hypogonadotropic hypogonadism, though they observed an increased waist circumference and BMI after testosterone replacement.[6] Naharci et al. reported increased insulin resistance at baseline in idiopathic hypogonadotropic hypogonadism and improvement of insulin sensitivity and body fat mass after replacement.[8] Previous studies on aging men with hypogonadism predicted the development of MS and diabetes.[10–13] However, very few Indian studies have been conducted in patients with congenital hypogonadism to study MS prevalence and testosterone replacement effects.[5],[14],[15] A Study by Tripathy et al. had studied 10 patients of idiopathic hypogonadotropic hypogonadism with injection testosterone enanthate for 12 weeks and found no significant improvement in insulin sensitivity.[15] Other studies by Singh et al. and Gopal et al. had seen effect of testosterone replacement therapy in diabetic patient with hypogonadism.[5],[14] Male hypogonadism typically was treated with testosterone replacement to restore testosterone levels to normal. Various factors influence the choice of regimen, including patient preference, expense, and convenience. In India, commonly preferred regimen was Injection testosterone undecanoate with a dose of 1,000 mg administered as a deep IM injection, with a second dose 6 weeks after the first and subsequent doses every 12 weeks.[16] Indian subjects have a higher prevalence of MS and its components at an early age.[17] Often, diagnosis of congenital hypogonadism is delayed, may resulting with more severe metabolic abnormalities prior to diagnosis. The reversibility of metabolic abnormalities with current therapy with testosterone are not well established in our populations. The primary objective of this study was to determine prevalence of metabolic syndrome and abnormality in whole-body compositions in young adult males with congenital hypogonadism and compared these with age and BMI matched healthy controls and in subset of patients of hypogonadism we prospectively studied the effects of injectable testosterone undecanoate in physiological doses on parameters of metabolic syndrome and whole-body composition.
Patients and controls It was a single arm prospective longitudinal intervention study in patient with congenital hypogonadism. Thirty three out of 48 male patients who met the study inclusion and exclusion criteria were enrolled from the endocrinology clinic of Sanjay Gandhi Post Graduate Institute of Medical Science, Lucknow over a period of one and half years [Figure 1]. We included all patients who had clinical features of hypogonadism (failure to undergo spontaneous puberty before 18 years of age) and a fasting morning total serum total testosterone <8.6 nmol/l (measured twice). Subjects with hypergonadotropic hypogonadism diagnosed with elevated gonadotropins [follicular stimulating hormone (FSH), Luteinising hormone (LH)] and hypo gonadotropic hypogonadism by low or inappropriately normal gonadotropins (normal FSH 1.5–12.4 IU/L: LH 1.7–8.6 IU/L) with low testosterone. Patients with chronic illness or on glucocorticoid therapy were excluded from the study. Patients with hypogonadotropic hypogonadism associated with other pituitary hormone deficiencies (cortisol and thyroid axis abnormality) were excluded from the study.
Thirty-three healthy eugonadal men with history of spontaneous puberty, matched for age, and body mass index (BMI) of patients, were recruited as controls. Control subjects with chronic illness or steroid use were excluded from study. Controls were enrolled from community. Their age was matched within 5 years and BMI within 2 kg/m2. The sample size was determined by assuming the proportion of the discordant (patients who reported change in status between pre-and post-observations) was 34% (worst to good) and 1% (good to worst), respectively. At minimum two-sided 95% confidence interval and 80% power of the study, an estimated sample size of the paired observations was 23. The sample size was estimated using software power analysis and sample size version-16 (PASS-16, NCSS, LLC. Kaysville, Utah, USA). The Institutional Ethics Committee of Sanjay Gandhi Post Graduate Institute of Medical Science had reviewed and approved the study. Consent has been obtained from each patient and control after full explanation of the purpose and nature of all procedures used. Study protocol The patients underwent a thorough clinical examination. Height, weight, arm span, waist and hip circumference, upper and lower segment, testicular volume, and blood pressure were measured at the onset of study. Height was measured using a Harpendon stadiometer (Holtain Ltd., Cambridge MD, UK), while weight was recorded using electronic weighing machine (Omron HN 289, OMRON Healthcare India PVT Ltd. Gurgaon, Haryana, sensitivity of scales was up to 100 g). The circumference of the waist (WC) was determined at the midpoint between the lowest point of the costal margin and the highest point of the iliac crest with the subject standing at the end of the normal expiration. The hip circumference was measured at the level of the greater trochanters, with the subject wearing minimal clothing. BMI (in kg/m2) was calculated with formula [BMI = weight (kg)/height (m2)]. MS was defined according to the Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) i.e., NCEP-ATP ΙΙΙ criteria.[18] Patients with at least three of the five criteria [elevated waist circumference (>90 cm), elevated triglycerides (≥150 mg/dl), reduced high density lipoprotein (HDL) cholesterol (<40 mg/dl), elevated blood pressure (≥130/85 mm Hg) and elevated fasting plasma glucose (≥100 mg/dl)] were diagnosed as MS. Blood pressure was measured using the typical electronic BP instrument (Omron HEM 7120, OMRON Healthcare India PVT Ltd. Gurgaon, Haryana) in the right arm in the sitting position after at least 5 min. of rest. It was repeated after 5 min. and the second reading was used for analysis. Hypertension was diagnosed according to JNC 7 criteria.[19] After an overnight fast of 8 h, serum sample was taken for fasting plasma glucose, lipid profile, insulin, proinsulin, C-peptide, high sensitive C-reactive protein (hs-CRP), total testosterone, LH and FSH. An oral glucose tolerance test with 75-gm anhydrous glucose was then performed and samples were collected in fluoride tubes after 2 h for plasma glucose. The diagnosis of diabetes or impaired glucose tolerance was made according to American Diabetes Association guidelines.[20] Prospective study Twenty-one patients with hypogonadism, who had previously never been treated with testosterone, were recruited for this single arm prospective longitudinal intervention study. Patients were given injection 1,000 mg testosterone undecanoate, long-acting injectable (Cernos, Sun Pharmaceuticals Industries Ltd, Mumbai, India) at baseline, repeated after 6 weeks, and subsequently every 12 weeks. All patients were provided oral calcium carbonate (1,000 mg/day) and vitamin D3 (cholecalciferol 60,000 units/month). Patients were seen in follow-up every 3 months to check for side effects and compliance with medicines. Anthropometric measurement, biochemistry (fasting plasma glucose, lipid profile, insulin, proinsulin, C-peptide, high sensitive C-reactive protein), total testosterone, LH and FSH, and whole-body composition were assessed at the start and after 9 months of treatment. All investigations, treatment with testosterone, follow-ups were as per standard of care and data was collected in proforma.[16] Analytical methods Fasting plasma glucose, lipid profile was analyzed by an autoanalyzer (RX Imola; Randox Laboratories, United Kingdom). Plasma insulin, C-peptide, testosterone, LH, FSH and hs-CRP were analyzed by electro-chemiluminescence immunoassay (COBAS 410 Analyzer, Roche Diagnostics, Mannheim, Germany). The assays had a functional sensitivity as follows: insulin 0.2 µu/ml, testosterone 0.416 nmol/l, hs-CRP 0.3 mg/l. Serum proinsulin was analysed ELISA (DRG International, city, USA). The assay had a functional sensitivity was 0.5 pmol/l. Insulin resistance (IR) was calculated by the following homeostasis model assessment of insulin resistance (HOMA-IR) formula: HOMA-IR = FPG (mg/dL) × immunoreactive insulin (IRI) (µu/ml)/405.[21] Homeostatic model for assessment of insulin sensitivity (HOMA%S) and Homeostatic model assessment for beta cell function (HOMA%β, which were used to measure insulin sensitivity and β- cell function) were calculated using the HOMA2 Calculator v 2.2.3 (http://www.dtu.ox.ac.uk/homacalculator/).[22] Whole-body composition Whole-body composition was measured by Hologic dual-energy X-ray absorptiometry (DXA) scanner (model QDR 4500A; software version 12.4: Hologic, Waltham, MA). The coefficient of variation for various parameters in whole body composition were 1.39% for total body fat, 2.42% for truncal fat, 0.83% for lean body mass, 1.4% for fat mass index (Single subject and single technician and 5 times repeated). Statistical analysis The data were tested for normality using the Shapiro–Wilk test. Continuous variables such as age, weight, BMI, waist and hip circumference, systolic, lipid profiles and body composition were presented as mean with standard deviation. Since data of serum proinsulin, HOMA%S, hs-CRP were not normally distributed, therefore the median and interquartile ranges (IQR) were reported. The prevalence of MS was determined by the number of subjects with MS, divided by the total number of subjects in the case or control group. Proportions like MS and hypertension were compared by using the Chi-square tests. Independent t-test was used to compare parametric data of two classes. Continuous variables like BMI, WC, lipid profile, C-peptide, beta cell function, hs-CRP, and parameters of body compositions were further tested using Pearson correlation test to establish association with HOMA-IR at baseline. The effect of testosterone replacement after 9 months was compared using paired sample t-test for variables like C-peptide, insulin, HOMA-IR, HOMA% β, testosterone, and whole-body composition while Wilcoxon signed-rank tests used for variables like proinsulin, HOMA%S, hs-CRP. Per protocol analysis done in intervention arm of study. The difference was considered significant at P value <0.05. International Business Machines Statistical Package for Social Science (IBM SPSS) Statistics (IBM SPSS version 24, Armonk, NY, USA) was used for data analysis.
Baseline metabolic and whole-body compositions variables We studied 33 [17 idiopathic hypo gonadotropic and 16 hyper gonadotropic (8 patients 47XXY, 3 patients 46 XY, 5 patients karyotype not available)] male patients with congenital hypogonadism and age, BMI matched healthy controls. The mean age of patients with hypogonadism [26.6 ± 5.1 years (range 20–39 years) vs. 27.6 ± 2.4 years (range 23–36 years)] and BMI (22.9 ± 4.5 vs. 24.3 ± 2.9, P = 0.15) were comparable with controls. Characteristics of patients with hypogonadism and healthy controls described in [Table 1].
There was no difference in prevalence of metabolic syndrome in hypogonadism patients and control subjects 27.3% [95% CI (14.8–44.7) vs. 9.1% [95% CI (3.0–24.7)]. The mean fasting blood sugar was higher in the patients compared to control subjects (87 ± 19 mg/dl vs. 77 ± 16 mg/dl, P < 0.05). The prevalence of diabetes (3%) and IFG (9%) in hypogonadism patients was comparable to the control population. Hypertension prevalence was higher in hypogonadism relative to controls (33.3% vs. 3.0%; P < 0.01). There was no difference in the hypogonadism patients and control population in waist circumference, serum triglycerides, serum high density lipoprotein, and truncal fat and total body fat. Patients had lower lean body mass than control population (45,909 ± 7,970 gm/cm2 vs. 50,713 ± 7,458 gm/cm2; P < 0.05) [Table 1]. Pearson Correlation was done with HOMA-IR at baseline in patient with hypogonadism and found significantly correlated with BMI (r = 0.53, P < 0.01), waist circumference (r = 0.50, P < 0.01), C-peptide (r = 1.0, P < 0.001 ), β-cell function (r = 0.54, P < 0.01), lean body mass (r = 0.41, P < 0.05), truncal fat (r = 0.51, P < 0.01), total body fat (r = 0.54, P < 0.01) [Table 2].
Effect of testosterone replacement The mean testosterone level was higher than the level of testosterone before replacement (23.2 ± 13.2 nmol/l vs. 2.9 ± 2.8 nmol/l, P < 0.001; normal range 8.6–29 nmol/l). There was significant decrease in waist circumference (88.6 ± 13.1 cm vs. 83.9 ± 12.9 cm, P < 0.01) and hip circumference (89.6 ± 9.1 cm vs. 87.1 ± 8.9 cm, P < 0.01) after testosterone replacement. The prevalence of metabolic syndrome, IFG, and diabetes were not changed. There was no change in lipid parameters. [Table 3].
Serum C-peptide (2.2 ± 0.79 nmol/l vs. 0.68 ± 0.23 nmol/l, P < 0.001) and serum proinsulin levels (1.43 pmol/l vs. 0.5 pmol/l, P < 0.001) decreased after testosterone replacement. HOMA-IR (4.7 ± 1.7 vs. 0.5 ± 0.2, P < 0.001) was decreased significantly and HOMA% β (330 ± 104 vs. 67 ± 20, P < 0.001) and HOMA%S [21 (12–65) vs. 206 (125–714), P < 0.001)] were improved significantly following testosterone replacement. Total body fat percent (29.6 ± 7.0 vs. 27.6 ± 5.7, P < 0.05) truncal fat percent (25.9 ± 7.3 vs. 24.0 ± 6.3, P < 0.05) and fat mass index (7.4 ± 2.3 vs. 6.7 ± 2.4, P < 0.001) decreased significantly after testosterone replacement. In addition, there was a significant increase in lean body mass (46,906 ± 8,876 gm vs. 50,083 ± 7,590 gm, P < 0.001). There was no change in hs-CRP levels [Table 3]. During follow of 9 months all patient experienced mild pain at injection site which subsided over 24–36 h, and no significant increased gynecomastia observed. Hematocrit increased significantly (40.3 ± 3.5% vs. 43.8 ± 3.6%, P < 0.01) but none had increased >48%. Two patient noticed worsening of obstructive sleep apnoea and one patient had mild pulmonary oil microembolization which resolved within 10 minutes of injection.
MS consists of pathological disorders including insulin resistance, arterial hypertension, obesity and dyslipidaemia; related abnormalities include inflammation and endothelial dysfunction.[23],[24] There were few studies showing the effect of testosterone replacement therapy various component of MS and body composition in congenital hypogonadism men.[8],[9] In this study, we reported significant improvement in parameters related to body composition after testosterone replacement in congenital hypogonadism. In addition, patients improved insulin sensitivity, beta-cell function, and insulin resistance. Almost 27% patients fulfilled the NCEP ATP III criteria for the metabolic syndrome, whereas only 10% of the control subjects did, even though difference was not statistically significant. This was different from previous studies which have reported high prevalence of metabolic syndrome in hypogonadism patients compared to control population.[7],[8],[25] This may be because of the high prevalence of MS in Indian population.[17],[26] In congenital hypogonadism patient's total body fat, truncal fat, and fat index mass was similar but lean body mass was less as compare to control subject. This could be probably explained because of 25% high body fat in young healthy Indian (20–39 years) as compared to white populations.[27] While the prevalence of MS (NCEP ATP III) has not decreased; though testosterone replacement therapy decreased its component like waist circumference, and hypertension. There was a moderate change in the prevalence of MS after testosterone replacement therapy in the current study; this may be because of small number of patients, normal glycemia and normal lipid profile in most of patients at recruitment, and short duration of treatment. Similar results were reported by Bojesen et al. in his study of 35 Klinefelter's syndrome patients who received low dose of testosterone replacement.[9] We documented at baseline HOMA-IR was significantly correlated with BMI, WC, C-peptide. Further, testosterone replacement led to an improvement in biomarkers of MS like insulin sensitivity, as manifested by decreased HOMA-IR, improved HOMA%β and lower serum C-peptide and proinsulin levels. Naharci et al. studied 24 patients with idiopathic hypogonadotropic hypogonadism and reported improvement in insulin sensitivity improves and body fat mass decreases after testosterone replacement therapy.[8] Similarly a study by Kapoor et al. also reported decreased in HOMA-IR, visceral adiposity as assessed by waist circumference in 24 patients over 3 months of primary and secondary hypogonadism.[28] Aversa et al. in their study with 52 elderly hypogonadism found significant improvement in HOMA-index, waist circumference over 12 months.[29] Similar findings including weight and BMI were also reported from observation registry by Saad et al.[4] Study by Singh et al. from north India, in small subset of hypogonadism who were received oral testosterone for 3 months showed improvement of weight, waist circumference, and insulin sensitivity.[5] In contrast Bojesen et al. reported no improvement in waist circumference and insulin sensitivity in Klinefelter syndrome patients following testosterone replacement therapy; this may be because of inadequate testosterone replacement.[9] Insulin resistance has assumed rising significance as a risk factor for cardiovascular disease and most studies has shown improvement of HOMA-IR after testosterone replacement. The effect of testosterone on HOMA-IR and insulin sensitivity was mediated through changes in body fat and our study also showed a reduction in waist circumference, and fat mass.[30] These data highlight the value of systematic metabolic assessment including biomarkers and care in men with hypogonadism. Current study observed no difference in weight and BMI following testosterone replacement despite significant decrease in total body fat, truncal fat, and fat mas index this may be because of increase in lean body mass. We also noticed baseline HOMA-IR was significantly correlated with lean mass and body fat. Sheffield-Moore et al. in their randomized study done in 24 elderly men with low testosterone showed total lean body mass was increased and percent fat was reduced after 5 months of testosterone replacement. They also noticed no significant changes in weight and BMI.[31] The effects of testosterone therapy on increased lean body mass and reduced fat mass were consistently reported in most studies, irrespective of testosterone formulations used or duration of testosterone treatment.[29],[32],[33] Even if obtained in uncontrolled studies, these results indicate that testosterone can be a physiological modulator of body composition. This was explained by the animal research conducted by Singh et al. on-androgen pathways for altering body composition. These investigators stated that androgens induced the differentiation of mesenchymal pluripotent cells via the myogenic pathway and inhibited the progression of the adipogenic pathway. This mechanism was accepted as an explanation for improvements in muscle and fat mass observed during testosterone supplementation.[34] Nonetheless, testosterone can influence the body's composition, which is correlated with insulin sensitivity. It is also possible that testosterone can have an indirect impact on insulin sensitivity through its effects on body composition.[35] There was a trend toward decrease in total cholesterol and LDL cholesterol, though there was no difference in serum triglycerides and HDL cholesterol. Contrary to this, a study by Haider et al. in their research work of beneficial effects of 2 years of administration of parenteral testosterone undecanoate showed significant improvement of various parameters of lipid profile.[36] In long term registry of 255 hypogonadism patients by Traish et al. also reported improvement in total cholesterol, LDL cholesterol, triglycerides, and increased HDL cholesterol.[37] But in another case-control study by Shigehara et al. demonstrated no change in total cholesterol and HDL cholesterol with testosterone therapy over 1 year among 33 Japanese hypogonadal men.[38] In our study duration of treatment was 9 months and probably too short to demonstrate the beneficial effects on lipid profiles and a larger and longer study is needed to prove this effect conclusively. There was no improvement in high sensitive C-reactive protein which have been posited as a predictor for the development of CVD after testosterone replacement in current study. A study by Kapoor et al. in 20 patients with type 2 diabetes and hypogonadism treated with injection testosterone for 3 months. They found that there was no significant effect of testosterone treatment on CRP levels.[39] Another study by Singh et al. found that 20 weeks of testosterone replacement given to healthy men resulted in no change in hs-CRP.[40] Both these studies, similar to the present study, were short-duration studies over 3–6 months and the long-term effect of testosterone treatment on CRP has not been investigated. In this brief period of 36 weeks of testosterone administration, there were no adverse events with regard to significant increase in the haematocrit or prostate pathology. Our study had strengths and limitations. The study was prospective nonrandomized intervention study done in congenital hypogonadism patients. This was the first study in India to do whole-body composition in hypogonadism patients. Limitation of the study was small sample size, and lack of subject with hypogonadism as controls. This was because, in intervention study we cannot denied standard care to patients, so it was ethically inappropriate to use subject with hypogonadism without testosterone as control. Other limitations were, less duration of therapy with testosterone replacement, and HOMA-IR and insulin sensitivity indices were not calculated for healthy controls.
We describe number of components of the MS such as high fasting blood sugar, hypertension, and low lean body mass were significantly present in congenital hypogonadism patients as compared to healthy controls. In prospective study, 36 weeks of testosterone replacement significantly decreased waist circumference and truncal fat causing improved insulin sensitivity as evidenced by decreased serum C-peptide and serum proinsulin. Despite decreased total body fat and truncal fat, there was no reduction weight and BMI, this may be due to increased lean body mass. Levels of glucose, lipids and inflammatory markers have not changed significantly. However, longer-term clinical endpoint studies are required to assess if these changes lead to a reduced risk of developing cardiovascular disease. Acknowledgements We gratefully acknowledge senior technical officers Mr. Rajesh Srivastava and Mr. Manoj Dubey for their help in doing whole body DXA. Financial support and sponsorship The financial support provided by Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, (PGI/DIR/RC/213/2015) as an intra-mural grant to Dr SB Yadav. Conflicts of interest There are no conflicts of interest.
[Figure 1]
[Table 1], [Table 2], [Table 3]
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