Oktavianus 200 *Department of Biochemistry, Faculty of Medicine, Trisakti University **Department of Biology, Faculty of Medicine, Trisakti University ***Department of Histology, Faculty of Medicine, Trisakti University Correspondence dr. Suweino, Sp.Bkm Department of Biochemistry, Faculty of Medicine, Trisakti University Jl. Kyai Tapa No. 260 Grogol Jakarta 11440 Phone: +6221-5672731 ext. 2204 Email: suweino@trisakti.ac.id Univ Med 2012;31:200-207 ABSTRACT UNIVERSA MEDICINA September-December, 2012September-December, 2012September-December, 2012September-December, 2012September-December, 2012 Vol.31 - No.3 Vol.31 - No.3 Vol.31 - No.3 Vol.31 - No.3 Vol.31 - No.3 BACKGROUND Total testosterone (TT) levels in males decrease with age. There has been a vigorous debate on the extent to which low testosterone causally contributes to diabetes and its complications. The aim of the present study was to determine the relationship between sex hormones and blood glucose levels in adult males. METHODS A cross-sectional study involving 259 males aged 41 - 70 years was conducted at Cilandak Subdistrict, South Jakarta. Sex hormone binding globulin (SHBG) and testosterone levels were measured by means of electro–chemiluminescent immunoassay (ECLIA), while blood glucose levels were measured enzymatically using a spectrophotometer. Free testosterone index (FTI) and body massa index (BMI) were calculated. Inter-variable relationships were tested by Pearson correlation analysis, followed by multiple linear regression analysis to determine the most influential factor on fasting blood glucose levels. RESULTS BMI was positively correlated with fasting blood glucose, but the correlation was statistically not significant (r=0.105; p=0.106). In contrast, total testosterone (TT) (r=-0.258; p=0.000) and SHBG (r=-0.193; p=0.02) had a significant negative correlation with fasting blood glucose level. Multiple linear regression showed that TT was the most influential factor on fasting blood glucose level (â=-0.044; p=0.008). CONCLUSIONS Low total testosterone level may increase fasting blood glucose level in adult males. SHBG levels did not predict fasting blood glucose levels.Assessment of testosterone in middle-aged men may allow early intervention for diabetes mellitus. Keywords: Total testosterone, sex hormone binding globulin, blood glucose, male Low testosterone level increases fasting blood glucose level in adult males Suweino*, ML Edy Parwanto**, and David Tjahjadi*** 201 Univ Med Vol. 31 No.3 Kadar testosteron yang rendah meningkatkan kadar gula darah puasa pada laki-laki dewasa LATAR BELAKANG Pada laki-laki, seiring dengan meningkatnya usia terjadi penurunan kadar kadar testosteron total (TT). Masih banyak perbedaan pendapat mengenai kontribusi kausal dari kadar testosteron yang rendah terhadap diabetes dan komplikasinya. Penelitian ini bertujuan untuk menentukan adanya hubungan antara hormon seks dan kadar glukosa darah puasa pada pria dewasa. METODE Studi potong lintang dengan mengikut sertakan 259 pria yang berumur 41 – 70 tahun dilakukan di Kecamatan Cilandak, Jakarta Selatan. Pengukuran kadar sex hormone binding globulin (SHBG) dan testosteron dilakukan dengan teknik electro–chemiluminescent immunoassay (ECLIA), dan glukosa diukur secara enzimatis menggunakan spektrofotometer. Dilakukan perhitungan indeks testosteron bebas (ITB) dan indeks massa tubuh (IMT). Hubungan antar variabel diuji menggunakan analisis korelasi Pearson, yang dilanjutkan dengan analisis regresi ganda linear untuk menetukan faktor yang paling berperan terhadap kadar glukosa darah puasa. HASIL IMT berkorelasi positif dengan kadar gula darah puasa, tetapi secara statistik tidak bermakna (r=0.105; p=0.106). Sebaliknya TT (r=-0.258; p=0.000) dan SHBG (r=-0.193; p=0.02) berkorelasi negatif secara bermakna dengan kadar gula darah puasa. Analisis regresi ganda linear menunjukkan kadar TT yang paling berperan secara bermakna terhadap kadar gula darah puasa (â=-0.044; p=0.008). KESIMPULAN Kadar testosteron total yang rendah dapat meningkatkan kadar gula darah puasa pada pria dewasa. SHBG tidak dapat memprediksi kadar gula darah puasa. Pengukuran testosteron pada pria setengah baya memungkinkan intervensi dini terhadap diabetes melitus. Kata kunci: Testosteron total, sex hormone binding globulin, glukosa darah, pria ABSTRAK INTRODUCTION In males, the age-related reduction in total testosterone and adrenal androgens such as dehydroepiandrosterone sulfate (DHEAS) has been documented and linked to changes in physiological functions, including abdominal obesity, insulin resistence, lung function, and cardiovascular disease.(1-3) Testosterone levels in males are low before puberty and increase with puberty, reaching a peak in the age group of 40 – 49 years and decreasing gradually with advancing age.(4) Testosterone is synthesized by Leydig cells in the testis and is the main sex hormone in males. Part of the testosterone in the testes is used for spermatogenesis and the rest is secreted into the circulation. The total amount of testosterone in the circulation is called total testosterone. In general, total testosterone (TT) in the circulation is divided into 4 main parts, viz. testosterons bound to sex hormone binding globulin (SHBG) (44%), t e s t o s t e r o n e b o u n d t o a l b u m i n ( 5 0 % ) , testosterone bound to cortisol binding globulin (4%), and free or unbound testosterone (2%). One third of US men older than 65 years have type 2 diabetes, and a similar percentage have low or subnormal testosterone levels, compared 202 with reference ranges based on healthy young men.(5) Dhindsa et al.(6) reported that 43% of males with type 2 diabetes had low total testosterone levels, with a decrease in 57% of the free testosterone. On the other hand, among patients with type 1 diabetes, 1.7% had decreased TT and 20% had low free testosterone levels. A high prevalence of hypogonadism was reportedly found in patients with type 2 diabetes. The variable prevalences of hypogonadism found in several studies was presumably caused by differences in diagnostic criteria and the instruments used to determine testosterone levels.(7) One study reported a high prevalence of hypogonadism in type 2 diabetes, on the basis of free testosterone, and showed that one-third of males with type 2 diabetes (aged between 31 – 75 years) had a low free testosteron level.(7) Supplementation of testosterone have been found to improve body composition and insulin sensitivity.(8) In hypogonadal male diabetics older than 30 years, testosterone improved insulin resistance, glycemic control, and visceral obesity. O n t h e o t h e r h a n d , a b r u p t c e s s a t i o n o f testosterone supplementation in men with idiopathic hypogonadotropic hypogonadism caused worsening of insulin sensitivity, without detectable changes in body fat content (9) Another study reported that lower testosterone levels may promote insulin resistance through impairment of mitochondrial function.(10) Because is is still uncertain whether insulin resistance and diabetes may be causing male hypogonadism or vice versa, the mechanisms regulating any preventive effect of conserved testosterone levels need to be investigated further. It has been observed that low testosterone is inversely associated to both diabetes and the metabolic syndrome, and may be a predictor of these disorders. These associations are of moderate strength, corresponding to a decrease of 2–3 nmol/L in total testosterone.(11) Total testosterone has a stronger association with diabetes than does free testosterone. Since changes in total testosterone (but not free testosterone) are proportional to those of SHBG, it may be suggested that SHBG also plays a causal role in the above associations. However, the possibility of a reverse causation or a common third factor cannot be excluded, because of the strong inverse association of SHBG with insulin resistance and visceral adiposity. Further study is required to SHBG levels add additional information to conventional risk factors for diabetes and related complications and whether S H B G m e d i a t e s r i s k v i a a n d r o g e n i c o r testosterone-independent mechanisms. In the meta-analytical study conducted by Ding et al.(12) on 20 cross sectional studies, involving 850 diabetic men and 2000 nondiabetic controls, it was found that in each study, total testosterone levels were lower in diabetic men compared with nondiabetic controls. However, the meta-analysis did not address the association of free testosterone levels with future diabetes, this has been reported by six prospective studies.(13) Based on the variable results of the abovementioned studies, it was felt necessary to conduct a study aiming to determine any relationships between sex hormone levels and other determinant factors, such as BMI and SHBG, and blood glucose levels in adult males. METHODS Study design This study used a cross-sectional design and was conducted between February 2011 and October 2011 at Cilandak Subdistrict, South Jakarta. Study subjects The study subjects were healthy males who were at least 40 years old and were residents of Cilandak Subdistrict, South Jakarta. Inclusion criteria were a good health status as evidenced by physical examination by the investigators, and normal serum albumin level (3.5 - 5.0 g/dL). Exclusion criteria were: i) liver cirrhosis, hypogonadal or hypergonadal status; ii) consumption of drugs related to liver functions, Suweino, Parwanto, Tjahjadi Testosteron increases blood glucose 203 Univ Med Vol. 31 No.3 e.g. phenytoin; iii) consumption of drugs affecting reproductive organs, e.g. androgens, growth hormones, high-dose glucocorticoids; iv) c o n s u m p t i o n o f d r u g s a ff e c t i n g s t e r o i d production and metabolism, e.g. diazoxide; and v) consumption of drugs affecting insulin production, e.g. epinephrine, cortisol, progestins, sulfonylureas. The subjects gave written informed consent before participating in the study. Subjects were selected by cluster random s a m p l i n g o f t h e k e l u r a h a n ( v i l l a g e s ) i n Cilandak Subdistrict, South Jakarta. In each selected RW (hamlet), subject selection was by simple random sampling. Measurement of body mass index (BMI) Height and weight were measured by means of weighing scales with a built-in device for measuring height. BMI was computed as the ratio of weight to the square of height (kg/ m2). The anthropometry was performed in standing subjects wearing light clothes and no shoes. Laboratory measurements Venous blood samples were collected in the morning from the subjects after a 10-hour fast. Fasting blood samples were drawn from the cubital vein in the supine position. Serum aliquots were prepared for immediate analysis and for storage at - 80°C for further analysis. B l o o d g l u c o s e l e v e l s w e r e m e a s u r e d enzymatically using a spectrophotometer and were expressed in mg/dL. Total testosterone levels were determined by electrochemiluminescence (ECLIA) immunoassay (Elecsys 2010; Roche Diagnostics, Indianapolis, IN, USA), and levels of SHBG were measured by chemiluminescent immunometric assay (Immulite, DPC, Los Angeles, CA, USA). Free testosterone index (FTI) values were calculated using the formula TT/SHBG x 100 and were expressed in percentages, in accordance with Vermeulen’s method. (14) FTI was used to determine the testosterone status, to indicate binding abnormalities between SHBG and testosterone. Determination of SHBG and total testosterone levels was performed at the Prodia Laboratories, Jakarta. Testosterone levels were expressed in nmol/L. Ethical clearance The study protocol was approved by the Research Ethical Committee, Faculty of Medicine, Trisakti University, Jakarta. Data analysis Descriptive statistics, means and SD for continuous variables were used to describe the study population. The correlations between age, BMI, serum hormone levels and blood glucose levels were tested by Pearson’s test. Multivariate regression linear analysis was conducted in order to identify the most influential variable on fasting blood glucose levels. All p-values were two- tailed, and p< 0.05 was considered statistically significant. All analyses were performed with SPSS 17.0. RESULTS A total of 237 males participated in this study, with mean age of 53.19 ± 8.39 years and mean BMI of 24.35 ± 3.74 kg/m2. Mean fasting blood glucose level was 106.59 ± 33.78 mg/dL, TT 529.76 ± 190.75 nmol/dL, and SHBG 40.59 ± 17.58 nmol/L (Table 1). Characteristics All subjects (n = 428) Age (years) 53.19 ± 8.39 BMI (kg/m2) 24.35 ± 3.74 Glucose (mg/dL) 106.59 ± 33.78 TT (nmol/dL) 529.76 ± 190.75 SHBG (nmol/L) 40.59 ± 17.58 FTI (%) 48.17 ± 14.15 Table 1. Demographic and clinical characteristics of the study subjects (n=237) Data are expressed as mean ± SD; BMI : body mass index; TT: total testosterone; SHBG: sex hormone binding globulin; FTI: free testosterone index 204 Ta b l e 2 a b o v e s h o w s t h a t B M I w a s positively correlated with fasting blood glucose, b u t s t a t i s t i c a l l y t h e c o r r e l a t i o n w a s n o t significant (r=0.105; p=0.106). In contrast, TT (r=-0.258) and SHBG (r=-0.193; p=0.002) had a significant negative correlation with fasting blood glucose. It may be concluded that high TT and SHBG levels will result in decreased fasting blood glucose levels, while low TT and SHBG levels will result in increased fasting blood glucose levels. Although BMI did not show a significant relationship with fasting blood glucose level, theoretically BMI affects fasting blood glucose level, which was the reason for its inclusion in the multiple linear regreassion model. Multiple linear regression analysis showed that TT was the most infuential factor on fasting blood glucose, in comparison with BMI and SHBG levels (â=-0.044; p=0.008). This means that low TT levels result in high fasting blood glucose levels (Table 3). Table 2 . Correlation between several covariates and fasting blood glucose level Covariate Fasting blood glucose p Age BMI TT SHBG FTI 0.077 0.105 -0.258 -0.193 -0.068 0.224 0.106 0.000* 0.002* 0.283 BMI: body mass index; TT: total testosterone; SHBG: sex hormone binding globulin; FTI: free testosterone index Table 3. Multiple linear regression analysis of several covariates and fasting blood sugar level BMI: body mass index; TT: total testosterone; SHBG: sex hormone binding globulin DISCUSSION Our study results showed reduced TT levels in the study subjects, accompanied by increased fasting blood glucose levels. This is consistent with the results of other studies reporting that decreases in the levels of androgenic hormones, i.c. testosterone, is one of the causal factors for increased blood glucose levels in the subjects,(15) with zinc deficiency and adipokines (such as leptin and tumor necrosis f a c t o r a l p h a ) b e i n g t w o o f t h e p o s s i b l e causes.(16) Increased insulin levels decrease SHBG levels leading to a decrease in TT. The correlation between low testosterone levels and diabetes may also be the result of decreased functioning of Leydig cells. Reductions in TT and SHBG levels are closely associated with male abdominal obesity.(17,18) Although its mechanism is uncertain, abdominal osetity in males may be caused by increased lipid uptake and increased accumulation of visceral fat. O t h e r s t u d i e s f o u n d t h a t t e s t o s t e r o n e replacement therapy significantly decreases insulin resistence and improves blood glucose levels. In addition, it has been documented that men with impaired glucose tolerance (IGT) have low TT levels and that TT levels are inversely associated to fasting blood glucose.(19) TT levels in male diabetics were lower than those in normal males, possibly because free testosterone (FT) levels in the male subjects with diabetes were also lower than in normal males. Furthermore, FT has autoregulatory functions for a feedback mechanism to both the hypothalamus and the pituitary gland. Suweino, Parwanto, Tjahjadi Testosteron increases blood glucose 205 Univ Med Vol. 31 No.3 Our findings that SHBG levels were inversely related to fasting blood glucose are consistent with the results of the studies of Ding et al.(20) as well as Lakshman et al.(21) Similar results were obtained in male diabetics, showing a negative correlation of SHBG with TT.(22,23) The current evidence about the impact of SHBG on the prediction of later diabetes suggests that previous data on the impact of TT on later diabetes might have been confounded by SHBG. Therefore, we repeated the analyses intending to explore the independent contribution from total testosterone and SHBG when included in the same multivariate models. TT but not SHBG was still independently predictive of subsequent diabetes. Our study showed that BMI was not correlated with blood glucose level. Kaplan et al.(23) clearly demonstrated that obesity and fasting hyperglycemia are the key components of the metabolic syndrome that exacerbates the age-related decrease in TT levels. It is well recognized that obesity and type 2 DM are both conditions associated with low TT levels. (5,25) Decreased testosterone levels are known to be associated with increased glucose concentration, and our study confirmed this association. The clinical implication of our findings is that administration of testosterone may be beneficial to males. Testosterone replacement therapy may have beneficial effects on insulin sensitivity in older diabetic men with late hypogonadism. Since testosterone replacement therapy results in reduced fat mass and consequently decreased circulating free faty acids, the end result may be improved insulin sensitivity.(16) However, routine administrtaion of testosterone therapy to diabetic men with low-normal testosterone levels should await the outcomes of well-conducted clinical trials. Males with subnormal testosterone levels should implement lifestyle measures such as weight reduction and exercise, which may raise testosterone levels and provide multiple health benefits. Some limitations should be recognized in the current study. First, FT were calculated from TT and SHBG, using the Vermeulen’s equation. Second, we used a single blood sample to determine TT and SHBG levels. Increasing the number of samples would have yielded greater precision in measurement. Daily testosterone levels may fluctuate greatly, and therefore repeated tests are indicated for a diagnosis of hypogonadism. Our use of a single blood sample did not allow us to account for the intra- i n d i v i d u a l d a i l y v a r i a t i o n i n o u r s t u d y. Observational studies of cross-sectional design as used in our study cannot explain the temporal nature of the association between testosterone and blood glucose levels. CONCLUSIONS To t a l t e s t o s t e r o n e l e v e l i s t h e m o s t influential factor on fasting blood glucose level. A low total testosterone level may increase fasting blood glucose level in adult males. SHBG levels did not predict fasting blood glucose levels. However, a follow-up study is needed to confirm these associations in men. Assessment of testosterone in middle-aged men may allow early intervention for diabetes mellitus. ACKNOWLEDGEMENTS We would like to express our gratutude to the Dean and Vice-Deans of the Faculty of Medicine, Trisakti University for providing the funding of this study. 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