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Testosterone deficiency impairs glucose oxidation through defective insulin and its receptor gene expression in target tissues of adult male rats
Life Sciences 81 (2007) 534 – 542 www.elsevier.com/locate/lifescie
Testosterone deficiency impairs glucose oxidation through defective insulin and its receptor gene expression in target tissues of adult male rats Thirupathi Muthusamy a , Sivakumar Dhevika a , Palaniappan Murugesan b , Karundevi Balasubramanian a,⁎ a
b
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600 113, Tamil Nadu, India Department of Obstetrics and Gynaecology, University of Michigan Medical School, Ann Arbor, Michigan-48109, USA Received 23 January 2007; accepted 15 June 2007
Abstract Testosterone and insulin interact in their actions on target tissues. Most of the studies that address this issue have focused on the physiological concentration of testosterone, which maintains normal insulin sensitivity but has deleterious effects on the same when the concentration of testosterone is out of this range. However, molecular basis of the action of testosterone in the early step of insulin action is not known. The present study has been designed to assess the impact of testosterone on insulin receptor gene expression and glucose oxidation in target tissues of adult male rat. Adult male albino rats were orchidectomized and supplemented with testosterone (100 μg/100 g b. wt., twice daily) for 15 days from the 11th day of post orchidectomy. On the day after the last treatment, animals were euthanized and blood was collected for the assay of plasma glucose, serum testosterone and insulin. Skeletal muscles, such as gracilis and quadriceps, liver and adipose tissue were dissected out and used for the assay of various parameters such as insulin receptor concentration, insulin receptor mRNA level and glucose oxidation. Testosterone deprivation due to orchidectomy decreased serum insulin concentration. In addition to this, insulin receptor number and its mRNA level and glucose oxidation in target tissues were significantly decreased (p b 0.05) when compared to control. However, testosterone replacement in orchidectomized rats restored all these parameters to control level. It is concluded from this study that testosterone deficiency-induced defective glucose oxidation in skeletal muscles, liver and adipose tissue is mediated through impaired expression of insulin and its receptor gene. © 2007 Elsevier Inc. All rights reserved. Keywords: Adipose tissue; Glucose oxidation; Insulin receptor; Liver; Orchidectomy; Skeletal muscles; Testosterone
Introduction Low levels of testosterone predict the development of type-2 diabetes in men (Haffner et al., 1996; Tibblin et al., 1996; Stellato et al., 2000; Oh et al., 2002; Laaksonen et al., 2004; Svartberg et al., 2004) and, in addition, aging is accompanied by insulin resistance and decline in the levels of testosterone (Morley et al., 1997; Harman et al., 2001; Feldman et al., 2002). The pathogenesis of type-2 diabetes involves impairment of insulin secretion in pancreatic β-cell and insulin resistance in ⁎ Corresponding author. Professor, Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600113, India. Tel.: +91 44 24540784; fax: +91 44 24540709. E-mail address: [email protected] (K. Balasubramanian). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.06.009
the skeletal muscles, liver, and adipose tissue (Ashcroft and Rorsman, 2004; Panunti et al., 2004). In men, low level of plasma testosterone is associated with obesity, upper body fat distribution, increased levels of glucose and insulin (Haffner et al., 1994), and strongly associated with insulin resistance and metabolic syndrome (Kapoor et al., 2005). Holmang and Bjorntorp (1992) have shown that insulin resistance is associated with the castration of male rats and the condition is reversed with subsequent testosterone replacement. Therefore, there appears to be a link between testosterone deficiency and diabetes (Barrett-Connor, 1992; Andersson et al., 1994). Physiological level of testosterone maintains normal insulin sensitivity, whereas both excess and deficiency of testosterone promote insulin resistance (Holmang and Bjorntorp, 1992). However, the molecular mechanisms involved in such actions of testosterone on target tissues have not yet been identified.
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The effects of insulin are mediated by efficient signal transduction process, which is initiated by its binding to the extracellular domain of the insulin receptor (Taha and Klip, 1999). The binding of insulin to the α-subunits induces a transmembrane conformational change that activates the βsubunit tyrosine kinase domain. Subsequently, the β-subunits undergo a series of autophosphorylation reactions at specific tyrosine residues (Franco, 1992). The insulin receptor phosphorylates many proximal intracellular target molecules that serve as docking sites for effector proteins (White, 1998). Tyrosine-phosphorylated insulin receptor substrate provides SH2-domain binding sites for the regulatory subunit of phosphotidylinositol-3-kinase (PI3-kinase) (Shepherd et al., 1998). Two classes of serine/threonine kinases are known to act downstream of PI3-kinase, namely, PKB and a typical PKC whereby their activation is brought about by phosphoinositidedependent protein kinase (PDK-1), which leads to glucose uptake by the cell through activation and translocation of GLUT4 from cytosol to plasma membrane (Ishiki et al., 2005). In this event, the initial interaction between insulin and its receptor on target cell surface is followed by a series of surface and intracellular steps that participate in the control of insulin action such as glucose clearance from circulation. Abnormalities in any of these steps could result in defective modulation of receptor number on the cell surface and thus inappropriate cell sensitivity to the hormone (Carpentier, 1994). Skeletal muscles, liver and adipose tissue are insulin responsive target organs, which have specific receptors for testosterone (Eagon et al., 2001; Carson et al., 2002). There is a wealth of clinical and experimental data, demonstrating that the testosterone and insulin interact in their actions on target tissues (Livingstone and Collison, 2002). However, specific effects of testosterone on insulin receptor gene expression and glucose oxidation in target tissues of insulin are unknown. On the basis of the available information, it is hypothesized that testosterone may be an important regulator for insulin receptor expression and glucose oxidation in target tissues such as skeletal muscles, liver and adipose tissue. This study is designed to test the above hypothesis using adult male rats.
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Animals Animals were maintained as per the National Guidelines and Protocols, approved by the Institutional Animal Ethical Committee (IAEC No. 03/016/05). Healthy adult male albino rats of Wistar strain (Rattus norvegicus) weighing 180–200 g (90 days old) were used in the present study. Animals were housed in clean polypropylene cages and maintained in an airconditioned animal house with constant 12 h light and 12 h dark schedule. They were fed with standard rat pellet diet (Lipton India Ltd., Mumbai, India) and clean drinking water was made available ad libitum. Experimental design
Materials and methods
Rats were divided into three groups. Each group consists of six animals. Group-I: Intact adult; Group-II: Orchidectomized (ORD); and Group-III: Orchidectomized and treated with testosterone (ORD + T). Orchidectomy was performed under ether anesthesia. Group-III rats were treated with testosterone (dissolved in propylene glycol) 10 days after orchidectomy at a dose of 100 μg /100 g body weight (∼ 200 μl per rat), subcutaneously, twice daily (at 8.00 a.m. and 6 p.m.) for 15 days. The dose was selected based on the previous report (Holmang and Bjorntorp, 1992). At the end of treatment, animals were killed by decapitation, blood was collected, sera separated and stored at − 80 °C until use. Skeletal muscles, such as quadriceps and gracilis, liver and subcutaneous adipose tissue were dissected out and used for the measurements of glucose oxidation and insulin receptor. Quadriceps and Gracilis muscles are anaerobic and glycolytic type. These muscles have high glucose utilization capacity (Holloszy and Coyle, 1984; Thayer et al., 1993). Therefore, quadriceps and gracilis muscles were considered for the present study. Subcutaneous white adipose tissue is abundant in the abdomen and excessive central adiposity (especially intraabdominal) has been linked to the metabolic syndrome, which includes insulin resistance, dyslypidemia and increased risk of cardiovascular diseases (Collins et al., 2005). Therefore, in the present study subcutaneous adipose tissue was considered.
Chemicals
Collection of blood samples
All chemicals and reagents used in the present study were of molecular biology and analytical grade, and they were purchased from Sigma Chemical Company (St. Louis, MO, USA) and Amersham Biosciences Ltd. (UK). Glucose estimation kit was supplied by CPC diagnostics (Spain). 14C-glucose and [125 I] were purchased from Board of Radiation and Isotope Technology (BRIT, Mumbai, India). Radioimmunoassay kits for the assay of insulin and testosterone were obtained from Diasorin (Italy). Total RNA isolation reagent (TRIR), one-step reverse transcriptase-polymerase chain reaction (RT-PCR) kit and primers were purchased from ABgene (UK), Qiagen (Germany) and Integrated DNA technologies, Inc. (Coralville, IA), respectively.
One day prior to the last injection, blood samples were collected after overnight fasting in microfuge tubes containing EDTA by puncturing the orbital sinus with the help of heparinized microhematocrit capillary tubes (Riley, 1960). Plasma glucose Plasma was separated from blood by centrifugation for 10 min at 800 ×g at 4 °C within 30 min to prevent autoglycolysis by leukocytes. Plasma glucose was estimated by glucose oxidase–peroxidase method (CPC diagnostics, Spain). The coefficient of variations was 1.8%. Results are expressed as mg/dl.
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reactivity of the testosterone antiserum to other steroids such as 5α-dihydrotestosterone and androstenedione is 6.9% and 1.1%, respectively. Intra-assay coefficients of variation (CV) was b 8% and inter-assay CV was b 7.6%. Results are expressed as ng/ml. Serum insulin was assayed using 125I-labeled RIA kit obtained from Diasorin (Italy). The maximum binding was 47% and the sensitivity of the assay was 4 μU/ml. The percentage cross reactivity of insulin antibody to c-peptide was b0.01%. Intra-assay coefficient of variation (CV) was b10.6% and inter-assay CV was b 10.8%. Results are expressed as μU/ml. 14
C-Glucose oxidation 14
Fig. 1. Serum insulin (a), testosterone (b) and plasma glucose (c) levels in control, orchidectomized (ORD) and testosterone-replaced orchidectomized (ORD + T) rats. Serum insulin and testosterone were assayed using solid-phase RIA kit. Plasma glucose was estimated by glucose oxidase–peroxidase method. Each value represents mean ± SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD.
C-Glucose oxidation was estimated as per the standard method (Johnson and Turner, 1971; Kraft and Johnson, 1972). Briefly, 10 mg tissue was weighed and placed in a 2 ml ampoule containing 170 μl DMEM (Dulbecco's modified Eagle's medium) (pH 7.4), 10 IU penicillin in 10 μl DMEM and 0.5 μCi 14Cglucose. Then, the ampoules were aerated with a gas mixture (5% CO2, 95% air) for 30 s and tightly covered with rubber cork containing CO2 trap. A piece of filter paper was inserted into the rubber cork and 0.1 ml of diethanolamine buffer (pH 9.5) was applied to the filter paper before closing the ampoule. This closed system with CO2 trap was placed in an incubator at 37 °C. CO2 trap was replaced every 2 h. After removing the second trap, 0.01 ml of 1 N H2SO4 was added to halt further metabolism and release of any residual CO2 from the sample. The system was again closed for 1 h before the third and final trap is removed. All the CO2 traps were placed in the scintillation vials containing 10 ml of scintillation fluid and counted in a Beta counter. Results are expressed as CPM of 14CO2 released/mg tissue.
Radioimmunoassay Insulin receptor assay Serum testosterone was assayed using solid-phase RIA kit obtained from Diasorin (Italy). The maximum binding was 47% and sensitivity of the assay was 0.05 ng/ml. The percentage cross
Insulin receptors were quantified as per the method published by Torlinska et al. (2000). Briefly, 100 mg of each
Fig. 2. Effects of orchidectomy (ORD) and testosterone replacement (ORD + T) on insulin binding in skeletal muscles [gracilis (a), quadriceps (b)], liver (c) and adipose tissue (d) of adult male rats. Insulin receptor assay was performed using 100 μg membrane from each tissue. Samples were incubated at 4 °C for 16 h with increasing concentration of 125I-labeled porcine insulin. Nonspecific binding was determined in the presence of excess unlabeled insulin. Bound and free fractions of insulin were separated by centrifugation and then the radioactivity of the pellets was determined using a gamma counter. Total number of receptors as a binding capacity was determined by the Scatchard analysis. Each value represents mean ± SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD.
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Fig. 3. Effects of orchidectomy (ORD) and testosterone replacement (ORD + T) on insulin receptor (IR) mRNA expression in skeletal muscles [gracilis and quadriceps (a)], liver (b) and adipose tissue (c) of adult male rats. RT-PCR was performed on 2 μg of total RNA from the samples. A 224 bp fragment corresponding to insulin receptor was amplified and separated on a 2% agarose gel. Gels were densitometrically scanned and cDNAs were normalized to that of RPS16, which was coamplified along with the cDNA of interest. Data are expressed as mean + SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD. L1-100 bp Ladder. Gracilis : L2-Control; L3-ORD; L4-ORD + T. Quadriceps : L5-Control ; L6-ORD; L7-ORD + T. Liver : L1-100 bp Ladder ; L2-Control; L3ORD; L4-ORD + T. Adipose tissue: L1-100 bp Ladder ; L2-Control; L3-ORD; L4-ORD + T.
tissue (skeletal muscles, liver and adipose tissue) was homogenized in 2.5-fold 0.001 M NaHCO3 and centrifuged at 600 ×g for 30 min. The supernatant was centrifuged for 30 min at 20,000 ×g. The membrane was washed twice with 0.001 M NaHCO3. The final pellet was resuspended in 0.04 M Tris–HCl buffer (pH 7.4) containing 0.1% BSA. All procedures mentioned above were carried out at 4 °C. Protein concentration was estimated (Lowry et al., 1951) using BSA as a standard.
Membrane preparation (100 μg protein) was incubated at 4 °C for 16 h with increasing concentration of 125 I-labeled porcine insulin in a final volume of 0.5 ml of 0.04 M Tris buffer (pH 7.4) containing 0.1% BSA. Nonspecific binding was determined in the presence of excess (1000-fold) unlabeled insulin. Bound and free fractions of insulin were separated by centrifugation at 20,000 ×g for 10 min and then the radioactivity of the pellets was determined using a gamma counter. Total number of
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receptors as a binding capacity was determined by the Scatchard analysis (Scatchard, 1949). The receptor concentration is expressed as fmol/mg protein. Isolation of total RNA Total RNA was isolated from control and experimental samples using TRIR kit (total RNA isolation reagent) from ABgene, UK. Briefly, 100 mg fresh tissue was homogenized with 1 ml TRIR and the homogenates were transferred immediately to a microfuge tube and kept at 4 °C for 5 min to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 ml of chloroform was added, vortexed vigorously for 15 s and placed on ice at 4 °C for 10 min. The homogenates were centrifuged at 12,000 ×g for 15 min at 4 °C. The aqueous phase was carefully transferred to a fresh microfuge tube and an equal volume of isopropanol was added and stored for 10 min at 4 °C. The samples were centrifuged at 12,000 ×g for 10 min. The supernatant was removed and RNA pellet was washed twice with 75% ethanol by vortexing and subsequent centrifugation for 5 min at 7500 ×g (4 °C). RNA pellets were mixed with 50 μl of autoclaved Milli-Q water. The concentration and purity of RNA were determined spectrophotometrically at A 260/280 nm. The purity of RNA obtained was N 1.75. The yield of RNA is expressed in microgram (μg).
primer is 5′-GCC ATC CCG AAA GCG AAG ATC-3′ and the antisense primer is 5′-TCT GGG GAG TCC TGA TTG CAT-3′. The predicted size of the amplified fragment by RT-PCR is 224 bp. One 20-mer oligonucleotide primer and one 19-mer oligonucleotide primer for RPS-16 (Pubmed Nucleotide Accession No. XM341815) were selected as described previously (Shan et al., 1995). The sense primer is 5′-AAG TCT TCG GAC GCA AGA AA-3′ and the antisense is 5′-TTG CCC AGA AGC AGA ACA G-3′. The predicted size of the amplified fragment by RT-PCR is 148 bp. For the first strand cDNA synthesis, 2 μg of RNA template was added with mastermix containing 10 μl of RT-PCR buffer, 2.0 μl of dNTP mix, 2.0 μl of RT-PCR enzyme, appropriate volume of 0.6 μM primers and made up to 50 μl with RNasefree water. RT-PCR was performed using the thermal cycler (Eppendorf) programmed as RT reaction at 50 °C for 30 min, initial PCR activation at 95 °C for 15 min, denaturation for 1.5 min at 94 °C, annealing for 1.5 min at 58 °C, extension for 3.0 min at 72 °C. Thirty-five cycles were performed and final extension at 72 °C for 10 min. Finally, the reaction mixture containing PCR products were separated by 2% agarose gel electrophoresis along with 100 bp marker DNA. Gels were densitometrically (Bio-Rad) scanned and the cDNAs were normalized to that of the house keeping gene or internal control (RPS-16), which was co-amplified along with the cDNA of interest.
Reverse transcriptase-polymerase chain reaction Statistical Analysis Total RNA was used for the synthesis of cDNA. The Qiagen one-step RT-PCR kit was used for the generation of cDNA. The following specific oligonucleotide primers were used for the generation of cDNAs. Two 21-mer oligonucleotide primers for insulin receptor (Pubmed Nucleotide Accession No. M29014) were selected as described previously (Gonzalez et al., 2002). The sense
Data are expressed as mean ± SEM. The data were subjected to statistical analysis using one-way analysis of variance (ANOVA) and Duncan's multiple range test to assess the significance of individual variations between the control and treatment groups using a SPSS® statistical software package. In
Fig. 4. 14C-glucose oxidation in skeletal muscles [gracilis (a), quadriceps (b)], liver (c) and adipose tissue (d) of control, orchidectomized (ORD) and testosterone-replaced orchidectomized (ORD+ T) rats. 14C-glucose oxidation was performed using 10 mg of each fresh tissue by incubating with DMEM containing 0.5 μCi of 14C-glucose at 37 °C. CO2 released was captured with the help of Whatman filter paper containing diethanolamine buffer and placed in scintillation fluid. Radioactivity of the CO2 trap was determined using a Beta counter. Each value represents mean ± SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD.
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Duncan's test, the significance was considered at the level of p b 0.05. Results Testosterone deficiency due to orchidectomy significantly (p b 0.05) reduced the serum insulin and testosterone levels (Fig. 1a and b). These hormonal changes resulted in a significant increase (p b 0.05) in plasma glucose level (Fig. 1c). However, administration of testosterone restored these changes to normal level in orchidectomized rat. Orchidectomy significantly (p b 0.05) diminished the insulin receptor number in gracilis (Fig. 2a) and quadriceps muscles (Fig. 2b), liver (Fig. 2c) and adipose tissue (Fig. 2d). However, insulin receptors were recovered due to testosterone replacement. Similar to that of insulin receptor, a significant decrease (p b 0.05) in insulin receptor mRNA level was also recorded in skeletal muscles (gracilis and quadriceps) (Fig. 3a), liver (Fig. 3b) and adipose tissue (Fig. 3c) as a result of orchidectomy. Despite the fact that the administration of testosterone significantly (p b 0.05) increased the insulin receptor mRNA level to that of control in skeletal muscles and liver, adipose tissue insulin receptor mRNA level was not brought back to normal level. Skeletal muscles showed a maximum ability to oxidize glucose when compared to liver and adipose tissue (Fig. 4). Orchidectomy caused a remarkable decrease (p b 0.05) in glucose oxidation in gracilis (Fig. 4a) and quadriceps muscles (Fig. 4b), liver (Fig. 4c) and adipose tissue (Fig. 4d). Nevertheless, administration of testosterone restored the rate of glucose oxidation in skeletal muscles, liver and adipose tissue to that of control. Discussion Hormones are the major regulators of metabolic activities in skeletal muscles, liver and adipose tissue. Testosterone, an anabolic steroid, plays an important role in this direction (Lemoine et al., 2002; Livingstone and Collison, 2002; Boyanov et al., 2003; Tsai et al., 2004; Mayes and Watson, 2004; Inaba et al., 2005; Lee et al., 2005). Therefore, metabolic activities of tissues are likely to change with experimental or pathological manipulation of hormones (Mainwaring and Mangan, 1973). Among the hormones, insulin is the major regulator of glucose homeostasis. Studies have shown that testosterone and insulin interact in their actions at the level of target organs (Rajkhowa et al., 1994; Legro et al., 1999; Livingstone and Collison, 2002; Sheffield-Moore and Urban, 2004). Conversely, the exact molecular mechanism underlying the specific influence of testosterone on insulin receptor expression is not known. Serum insulin level following testosterone replacement was significantly increased when compared to castrated rats, suggesting that normal circulating level of testosterone is essential to maintain optimum insulin concentration in serum. In accordance with this, Morimoto et al. (2001) have shown that testosterone modify serum insulin
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levels through the direct effect on pancreatic islet function by favoring insulin gene expression and release. Castration-induced reduction in insulin binding and restoration of the same in testosterone-replaced rats imply the importance of physiological level of testosterone in maintaining insulin receptors in target tissues. In support of this, Sesti et al. (1992) have shown that androgens increase insulin binding and insulin responsiveness in HEp-2 larynx carcinoma cells. It appears that the testosterone deficiency-induced deterioration of glucose homeostasis is mediated through deficiency in circulating levels of insulin, its receptors and their sensitivity in target organs. To identify the molecular mechanism responsible for insulin receptor deficiency, mRNA expression of insulin receptor was quantified. Changes in insulin receptor in castrated and testosterone-replaced rats were found to be associated with corresponding changes in insulin receptor mRNA expression. However, adipose tissue insulin receptor mRNA level was not brought back to normal level. In this regard, Cohen (1999) and Moretti et al. (2005) have reported that testosterone deficiency or hypogonadism is associated with a doubling of fat mass. With the increasing fatty-tissue accumulation, there is an increase of aromatase activity that is associated with a greater conversion of testosterone to estradiol. In the present study, bioavailability of testosterone in adipose tissue of testosterone-replaced castrated rats may be insufficient to evoke any change in insulin receptor mRNA levels. These findings tempt to propose the involvement of testosterone in the insulin receptor gene expression. Effect of testosterone on gene expression is known to be mediated through the activation of its receptor (Cato and Peterziel, 1998). Insulin target tissues have receptor for androgen (Lemoine et al., 2002; Mayes and Watson, 2004). The fact that testosterone replacement was able to increase insulin receptor number and its mRNA level suggests that testosterone may have a direct effect on insulin receptor gene transcription as well as translation of mRNA. Although the presence of androgen response element has not been identified in the promoter region, it is possible that androgen receptor may interact with basal transcription machinery (McEwan and Gustafsson, 1997) or other coactivators that bind to the androgen receptor (Yeh and Chang, 1996; Aarnisalo et al., 1998) could be involved in the mechanism by which testosterone increases insulin receptor gene expression. In this regard, studies on insulin receptor gene promoter region would be of great interest. Insulin receptor promoter contains DNA sequences responsive to glucose and insulin. This was demonstrated in the primary culture of rat hepatocytes transfected with plasmids containing the DNA-regulatory sequences −618 to −593 of the rat insulin receptor promoter fused to the luciferase gene. When three copies of the nucleotides −618 to −593 of the insulin receptor promoter were transfected, the reporter activity was significantly increased in the presence of glucose and more increased in the presence of glucose and insulin, indicating the regulatory role of these two factors (Fukuda et al., 2001). Insulin receptor synthesis can also contribute to the cell surface receptor population (Virkamaki et al., 1999). In the present study, castrated rats showed a significant
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decrease in serum insulin level, insulin receptor and its mRNA levels in target tissues. It is therefore suggested that the castrationinduced deficiency in insulin may be one of the reasons for impaired insulin receptor gene expression. Orchidectomy-induced impairment in glucose oxidation may be the result of low level of serum insulin as well as its defective action. Skeletal muscles, liver and adipose tissue are the major site of insulin-mediated glucose metabolism (Bajaj and Defronzo, 2003; Defronzo and Mandarino, 2003; Kahn and Saltiel, 2005). Studies suggest that normal serum insulin is essential to maintain glucose homeostasis by enhancing glycolysis and glycogen synthesis in skeletal muscles (Richter et al., 1984; Mandarino et al., 1987) with concomitant decrease in glycogenolysis in liver and skeletal muscles (Shimazu, 1987). Fukuda et al. (2001) reported that insulin regulates its own receptor gene expression. Insulin receptor is essential for insulin action such as glucose metabolism. In the present study, serum insulin and its receptor expression in skeletal muscles, liver and adipose tissue were decreased due to testosterone deficiency. Therefore, orchidectomy-induced impairment in glucose oxidation in skeletal muscles, liver and adipose tissue appears to be the effects of subnormal serum insulin and its receptors. Recently, Chen et al. (2006) have shown that testosterone has bidirectional effects on the cellular expression of insulin receptor substrate-1 (IRS-1) and GLUT4, which increase with low dose and decrease with higher dose in cultured adipocytes and skeletal muscle cells. These findings implicate the role of testosterone deficiency to bring about changes recorded in this study. Testosterone-deficient animals showed a significant elevation in plasma glucose concentration, which appears to be the result of testosterone deficiency-mediated impairment of glucose oxidation. In this regard, Holmang and Bjorntorp (1992) have reported that castration was followed by marked insulin resistance in the clamp experiments. This was also paralleled by a diminished insulin stimulation of glucose incorporation into glycogen down to about 50% of control values. Substitution of castrated rats with testosterone (which is equivalent to the dose employed in the present study) completely abolished these perturbations of insulin sensitivity. Plasma glucose in testosterone-replaced animals was similar to that of normal rats reinforcing the importance of this steroid in glucose homeostasis. In this respect, it is worth to mention the report of Thomas et al. (2003), which showed a significant inverse correlation between testosterone and fasting blood glucose. These observations attest the involvement of testosterone in the regulation of glucose homeostasis, which may explain the testosterone deficiency-induced impairment of glucose oxidation in target tissues. In addition to testosterone, corticosterone and estradiol were found to play an important role in glucose homeostasis. Though the circulating levels of estradiol and corticosterone were not measured in the present study, the available reports indicate an increase in corticosterone (Kitay, 1963; Seale et al., 2004) and a decrease in estradiol (Banu et al., 2002) following gonadectomy in male rats. Excess glucocorticoids have also been shown to decrease the number of insulin receptors and affinity in rat liver and adipocytes (Kahn et al., 1978; Buren et al., 2002). Likewise,
low levels of estradiol were shown to be associated with insulin resistance in men (Dunajska et al., 2004). In view of these findings, it is proposed that castration-induced changes in corticosterone and estradiol could have contributed for the changes recorded in the present study. It is concluded from the present study that testosterone deficiency-induced defective glucose oxidation in skeletal muscles, liver and adipose tissue is mediated through impaired serum insulin level and its receptor gene expression. Acknowledgement Financial assistance from the Indian Council of Medical Research (ICMR), New Delhi in the form of Senior Research Fellowship (SRF) to T. Muthusamy (Award No. 3/1/2/15/05RHN dated 9-8-2005), DST-FIST, UGC-SAP-DRS-II and UGC-ASIST programmes are gratefully acknowledged. References Aarnisalo, P., Palvimo, J.J., Janne, O.A., 1998. CREB-binding protein in androgen receptor-mediated signaling. Proceedings of the National Academy of Sciences of the United States of America 95 (5), 2122–2127. Andersson, B., Marin, P., Lissner, L., Vermeulen, A., Bjorntorp, P., 1994. Testosterone concentrations in women and men with NIDDM. Diabetes Care 17 (5), 405–411. Ashcroft, F.M., Rorsman, P., 2004. Molecular defects in insulin secretion in type-2 diabetes. Reviews in Endocrine and Metabolic Disorders 5 (2), 135–142. Bajaj, M., Defronzo, R.A., 2003. Metabolic and molecular basis of insulin resistance. Journal of Nuclear Cardiology 10 (3), 311–323. Banu, S.K., Govindarajulu, P., Aruldhas, M.M., 2002. Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo. Steroids 67 (13–14), 1007–1014. Barrett-Connor, E., 1992. Lower endogenous androgen levels and dyslipidemia in men with non insulin-dependent diabetes mellitus. Annals of Internal Medicine 117 (10), 807–811. Boyanov, M.A., Boneva, Z., Christov, V.G., 2003. Testosterone supplementation in men with type-2 diabetes, visceral obesity and partial androgen deficiency. Aging Male 6 (1), 1–7. Buren, J., Liu, H.X., Jensen, J., Eriksson, J.W., 2002. Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes. European Journal of Endocrinology 146 (3), 419–429. Carpentier, J.L., 1994. Insulin receptor internalization: molecular mechanisms and physiopathological implications. Diabetologia 37 (Suppl 2), S117–S124. Carson, J.A., Lee, W.J., Mc Clung, J., Hand, G.A., 2002. Steroid receptor concentration in aged rat hindlimb muscle: effect of anabolic steroid administration. Journal of Applied Physiology 93 (1), 242–250. Cato, A.C.B., Peterziel, H., 1998. The androgen receptor as mediator of gene expression and signal transduction pathways. Trends in Endocrinology and Metabolism 9 (4), 150–154. Chen, X., Li, X., Huang, H.Y., Li, X., Lin, J.F., 2006. Effects of testosterone on insulin receptor substrate-1 and glucose transporter 4 expression in cells sensitive to insulin. Zhonghua Yi Xue Za Zhi 86 (21), 1474–1477. Cohen, P.G., 1999. The hypogonadal-obesity cycle: role of aromatase in modulating the testosterone-estradiol shunt-a major factor in the genesis of morbid obesity. Medical Hypotheses 52 (1), 49–51. Collins, S., Ahima, R.S., Kahn, B.B., 2005. Biology of adipose tissue. In: Kahn, C.R., Weir, G.C., King, G.L., Jacobson, A.M., Moses, A.C., Smith, R.J. (Eds.), Joslin's diabetes Mellitus. A Wolters Klunwer Company, Indian edition by BI publication, India, pp. 207–226. Defronzo, R.A., Mandarino, L.J., 2003. Insulin action. In: Henry, H.L., Norman, A.W. (Eds.), Encyclopedia of hormones. Elseivier Inc, California, pp. 333–347.
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Testosterone deficiency impairs glucose oxidation through defective insulin and its receptor gene expression in target tissues of adult male rats Thirupathi Muthusamy a , Sivakumar Dhevika a , Palaniappan Murugesan b , Karundevi Balasubramanian a,⁎ a
b
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600 113, Tamil Nadu, India Department of Obstetrics and Gynaecology, University of Michigan Medical School, Ann Arbor, Michigan-48109, USA Received 23 January 2007; accepted 15 June 2007
Abstract Testosterone and insulin interact in their actions on target tissues. Most of the studies that address this issue have focused on the physiological concentration of testosterone, which maintains normal insulin sensitivity but has deleterious effects on the same when the concentration of testosterone is out of this range. However, molecular basis of the action of testosterone in the early step of insulin action is not known. The present study has been designed to assess the impact of testosterone on insulin receptor gene expression and glucose oxidation in target tissues of adult male rat. Adult male albino rats were orchidectomized and supplemented with testosterone (100 μg/100 g b. wt., twice daily) for 15 days from the 11th day of post orchidectomy. On the day after the last treatment, animals were euthanized and blood was collected for the assay of plasma glucose, serum testosterone and insulin. Skeletal muscles, such as gracilis and quadriceps, liver and adipose tissue were dissected out and used for the assay of various parameters such as insulin receptor concentration, insulin receptor mRNA level and glucose oxidation. Testosterone deprivation due to orchidectomy decreased serum insulin concentration. In addition to this, insulin receptor number and its mRNA level and glucose oxidation in target tissues were significantly decreased (p b 0.05) when compared to control. However, testosterone replacement in orchidectomized rats restored all these parameters to control level. It is concluded from this study that testosterone deficiency-induced defective glucose oxidation in skeletal muscles, liver and adipose tissue is mediated through impaired expression of insulin and its receptor gene. © 2007 Elsevier Inc. All rights reserved. Keywords: Adipose tissue; Glucose oxidation; Insulin receptor; Liver; Orchidectomy; Skeletal muscles; Testosterone
Introduction Low levels of testosterone predict the development of type-2 diabetes in men (Haffner et al., 1996; Tibblin et al., 1996; Stellato et al., 2000; Oh et al., 2002; Laaksonen et al., 2004; Svartberg et al., 2004) and, in addition, aging is accompanied by insulin resistance and decline in the levels of testosterone (Morley et al., 1997; Harman et al., 2001; Feldman et al., 2002). The pathogenesis of type-2 diabetes involves impairment of insulin secretion in pancreatic β-cell and insulin resistance in ⁎ Corresponding author. Professor, Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai-600113, India. Tel.: +91 44 24540784; fax: +91 44 24540709. E-mail address: [email protected] (K. Balasubramanian). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.06.009
the skeletal muscles, liver, and adipose tissue (Ashcroft and Rorsman, 2004; Panunti et al., 2004). In men, low level of plasma testosterone is associated with obesity, upper body fat distribution, increased levels of glucose and insulin (Haffner et al., 1994), and strongly associated with insulin resistance and metabolic syndrome (Kapoor et al., 2005). Holmang and Bjorntorp (1992) have shown that insulin resistance is associated with the castration of male rats and the condition is reversed with subsequent testosterone replacement. Therefore, there appears to be a link between testosterone deficiency and diabetes (Barrett-Connor, 1992; Andersson et al., 1994). Physiological level of testosterone maintains normal insulin sensitivity, whereas both excess and deficiency of testosterone promote insulin resistance (Holmang and Bjorntorp, 1992). However, the molecular mechanisms involved in such actions of testosterone on target tissues have not yet been identified.
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The effects of insulin are mediated by efficient signal transduction process, which is initiated by its binding to the extracellular domain of the insulin receptor (Taha and Klip, 1999). The binding of insulin to the α-subunits induces a transmembrane conformational change that activates the βsubunit tyrosine kinase domain. Subsequently, the β-subunits undergo a series of autophosphorylation reactions at specific tyrosine residues (Franco, 1992). The insulin receptor phosphorylates many proximal intracellular target molecules that serve as docking sites for effector proteins (White, 1998). Tyrosine-phosphorylated insulin receptor substrate provides SH2-domain binding sites for the regulatory subunit of phosphotidylinositol-3-kinase (PI3-kinase) (Shepherd et al., 1998). Two classes of serine/threonine kinases are known to act downstream of PI3-kinase, namely, PKB and a typical PKC whereby their activation is brought about by phosphoinositidedependent protein kinase (PDK-1), which leads to glucose uptake by the cell through activation and translocation of GLUT4 from cytosol to plasma membrane (Ishiki et al., 2005). In this event, the initial interaction between insulin and its receptor on target cell surface is followed by a series of surface and intracellular steps that participate in the control of insulin action such as glucose clearance from circulation. Abnormalities in any of these steps could result in defective modulation of receptor number on the cell surface and thus inappropriate cell sensitivity to the hormone (Carpentier, 1994). Skeletal muscles, liver and adipose tissue are insulin responsive target organs, which have specific receptors for testosterone (Eagon et al., 2001; Carson et al., 2002). There is a wealth of clinical and experimental data, demonstrating that the testosterone and insulin interact in their actions on target tissues (Livingstone and Collison, 2002). However, specific effects of testosterone on insulin receptor gene expression and glucose oxidation in target tissues of insulin are unknown. On the basis of the available information, it is hypothesized that testosterone may be an important regulator for insulin receptor expression and glucose oxidation in target tissues such as skeletal muscles, liver and adipose tissue. This study is designed to test the above hypothesis using adult male rats.
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Animals Animals were maintained as per the National Guidelines and Protocols, approved by the Institutional Animal Ethical Committee (IAEC No. 03/016/05). Healthy adult male albino rats of Wistar strain (Rattus norvegicus) weighing 180–200 g (90 days old) were used in the present study. Animals were housed in clean polypropylene cages and maintained in an airconditioned animal house with constant 12 h light and 12 h dark schedule. They were fed with standard rat pellet diet (Lipton India Ltd., Mumbai, India) and clean drinking water was made available ad libitum. Experimental design
Materials and methods
Rats were divided into three groups. Each group consists of six animals. Group-I: Intact adult; Group-II: Orchidectomized (ORD); and Group-III: Orchidectomized and treated with testosterone (ORD + T). Orchidectomy was performed under ether anesthesia. Group-III rats were treated with testosterone (dissolved in propylene glycol) 10 days after orchidectomy at a dose of 100 μg /100 g body weight (∼ 200 μl per rat), subcutaneously, twice daily (at 8.00 a.m. and 6 p.m.) for 15 days. The dose was selected based on the previous report (Holmang and Bjorntorp, 1992). At the end of treatment, animals were killed by decapitation, blood was collected, sera separated and stored at − 80 °C until use. Skeletal muscles, such as quadriceps and gracilis, liver and subcutaneous adipose tissue were dissected out and used for the measurements of glucose oxidation and insulin receptor. Quadriceps and Gracilis muscles are anaerobic and glycolytic type. These muscles have high glucose utilization capacity (Holloszy and Coyle, 1984; Thayer et al., 1993). Therefore, quadriceps and gracilis muscles were considered for the present study. Subcutaneous white adipose tissue is abundant in the abdomen and excessive central adiposity (especially intraabdominal) has been linked to the metabolic syndrome, which includes insulin resistance, dyslypidemia and increased risk of cardiovascular diseases (Collins et al., 2005). Therefore, in the present study subcutaneous adipose tissue was considered.
Chemicals
Collection of blood samples
All chemicals and reagents used in the present study were of molecular biology and analytical grade, and they were purchased from Sigma Chemical Company (St. Louis, MO, USA) and Amersham Biosciences Ltd. (UK). Glucose estimation kit was supplied by CPC diagnostics (Spain). 14C-glucose and [125 I] were purchased from Board of Radiation and Isotope Technology (BRIT, Mumbai, India). Radioimmunoassay kits for the assay of insulin and testosterone were obtained from Diasorin (Italy). Total RNA isolation reagent (TRIR), one-step reverse transcriptase-polymerase chain reaction (RT-PCR) kit and primers were purchased from ABgene (UK), Qiagen (Germany) and Integrated DNA technologies, Inc. (Coralville, IA), respectively.
One day prior to the last injection, blood samples were collected after overnight fasting in microfuge tubes containing EDTA by puncturing the orbital sinus with the help of heparinized microhematocrit capillary tubes (Riley, 1960). Plasma glucose Plasma was separated from blood by centrifugation for 10 min at 800 ×g at 4 °C within 30 min to prevent autoglycolysis by leukocytes. Plasma glucose was estimated by glucose oxidase–peroxidase method (CPC diagnostics, Spain). The coefficient of variations was 1.8%. Results are expressed as mg/dl.
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reactivity of the testosterone antiserum to other steroids such as 5α-dihydrotestosterone and androstenedione is 6.9% and 1.1%, respectively. Intra-assay coefficients of variation (CV) was b 8% and inter-assay CV was b 7.6%. Results are expressed as ng/ml. Serum insulin was assayed using 125I-labeled RIA kit obtained from Diasorin (Italy). The maximum binding was 47% and the sensitivity of the assay was 4 μU/ml. The percentage cross reactivity of insulin antibody to c-peptide was b0.01%. Intra-assay coefficient of variation (CV) was b10.6% and inter-assay CV was b 10.8%. Results are expressed as μU/ml. 14
C-Glucose oxidation 14
Fig. 1. Serum insulin (a), testosterone (b) and plasma glucose (c) levels in control, orchidectomized (ORD) and testosterone-replaced orchidectomized (ORD + T) rats. Serum insulin and testosterone were assayed using solid-phase RIA kit. Plasma glucose was estimated by glucose oxidase–peroxidase method. Each value represents mean ± SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD.
C-Glucose oxidation was estimated as per the standard method (Johnson and Turner, 1971; Kraft and Johnson, 1972). Briefly, 10 mg tissue was weighed and placed in a 2 ml ampoule containing 170 μl DMEM (Dulbecco's modified Eagle's medium) (pH 7.4), 10 IU penicillin in 10 μl DMEM and 0.5 μCi 14Cglucose. Then, the ampoules were aerated with a gas mixture (5% CO2, 95% air) for 30 s and tightly covered with rubber cork containing CO2 trap. A piece of filter paper was inserted into the rubber cork and 0.1 ml of diethanolamine buffer (pH 9.5) was applied to the filter paper before closing the ampoule. This closed system with CO2 trap was placed in an incubator at 37 °C. CO2 trap was replaced every 2 h. After removing the second trap, 0.01 ml of 1 N H2SO4 was added to halt further metabolism and release of any residual CO2 from the sample. The system was again closed for 1 h before the third and final trap is removed. All the CO2 traps were placed in the scintillation vials containing 10 ml of scintillation fluid and counted in a Beta counter. Results are expressed as CPM of 14CO2 released/mg tissue.
Radioimmunoassay Insulin receptor assay Serum testosterone was assayed using solid-phase RIA kit obtained from Diasorin (Italy). The maximum binding was 47% and sensitivity of the assay was 0.05 ng/ml. The percentage cross
Insulin receptors were quantified as per the method published by Torlinska et al. (2000). Briefly, 100 mg of each
Fig. 2. Effects of orchidectomy (ORD) and testosterone replacement (ORD + T) on insulin binding in skeletal muscles [gracilis (a), quadriceps (b)], liver (c) and adipose tissue (d) of adult male rats. Insulin receptor assay was performed using 100 μg membrane from each tissue. Samples were incubated at 4 °C for 16 h with increasing concentration of 125I-labeled porcine insulin. Nonspecific binding was determined in the presence of excess unlabeled insulin. Bound and free fractions of insulin were separated by centrifugation and then the radioactivity of the pellets was determined using a gamma counter. Total number of receptors as a binding capacity was determined by the Scatchard analysis. Each value represents mean ± SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD.
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Fig. 3. Effects of orchidectomy (ORD) and testosterone replacement (ORD + T) on insulin receptor (IR) mRNA expression in skeletal muscles [gracilis and quadriceps (a)], liver (b) and adipose tissue (c) of adult male rats. RT-PCR was performed on 2 μg of total RNA from the samples. A 224 bp fragment corresponding to insulin receptor was amplified and separated on a 2% agarose gel. Gels were densitometrically scanned and cDNAs were normalized to that of RPS16, which was coamplified along with the cDNA of interest. Data are expressed as mean + SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD. L1-100 bp Ladder. Gracilis : L2-Control; L3-ORD; L4-ORD + T. Quadriceps : L5-Control ; L6-ORD; L7-ORD + T. Liver : L1-100 bp Ladder ; L2-Control; L3ORD; L4-ORD + T. Adipose tissue: L1-100 bp Ladder ; L2-Control; L3-ORD; L4-ORD + T.
tissue (skeletal muscles, liver and adipose tissue) was homogenized in 2.5-fold 0.001 M NaHCO3 and centrifuged at 600 ×g for 30 min. The supernatant was centrifuged for 30 min at 20,000 ×g. The membrane was washed twice with 0.001 M NaHCO3. The final pellet was resuspended in 0.04 M Tris–HCl buffer (pH 7.4) containing 0.1% BSA. All procedures mentioned above were carried out at 4 °C. Protein concentration was estimated (Lowry et al., 1951) using BSA as a standard.
Membrane preparation (100 μg protein) was incubated at 4 °C for 16 h with increasing concentration of 125 I-labeled porcine insulin in a final volume of 0.5 ml of 0.04 M Tris buffer (pH 7.4) containing 0.1% BSA. Nonspecific binding was determined in the presence of excess (1000-fold) unlabeled insulin. Bound and free fractions of insulin were separated by centrifugation at 20,000 ×g for 10 min and then the radioactivity of the pellets was determined using a gamma counter. Total number of
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receptors as a binding capacity was determined by the Scatchard analysis (Scatchard, 1949). The receptor concentration is expressed as fmol/mg protein. Isolation of total RNA Total RNA was isolated from control and experimental samples using TRIR kit (total RNA isolation reagent) from ABgene, UK. Briefly, 100 mg fresh tissue was homogenized with 1 ml TRIR and the homogenates were transferred immediately to a microfuge tube and kept at 4 °C for 5 min to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 ml of chloroform was added, vortexed vigorously for 15 s and placed on ice at 4 °C for 10 min. The homogenates were centrifuged at 12,000 ×g for 15 min at 4 °C. The aqueous phase was carefully transferred to a fresh microfuge tube and an equal volume of isopropanol was added and stored for 10 min at 4 °C. The samples were centrifuged at 12,000 ×g for 10 min. The supernatant was removed and RNA pellet was washed twice with 75% ethanol by vortexing and subsequent centrifugation for 5 min at 7500 ×g (4 °C). RNA pellets were mixed with 50 μl of autoclaved Milli-Q water. The concentration and purity of RNA were determined spectrophotometrically at A 260/280 nm. The purity of RNA obtained was N 1.75. The yield of RNA is expressed in microgram (μg).
primer is 5′-GCC ATC CCG AAA GCG AAG ATC-3′ and the antisense primer is 5′-TCT GGG GAG TCC TGA TTG CAT-3′. The predicted size of the amplified fragment by RT-PCR is 224 bp. One 20-mer oligonucleotide primer and one 19-mer oligonucleotide primer for RPS-16 (Pubmed Nucleotide Accession No. XM341815) were selected as described previously (Shan et al., 1995). The sense primer is 5′-AAG TCT TCG GAC GCA AGA AA-3′ and the antisense is 5′-TTG CCC AGA AGC AGA ACA G-3′. The predicted size of the amplified fragment by RT-PCR is 148 bp. For the first strand cDNA synthesis, 2 μg of RNA template was added with mastermix containing 10 μl of RT-PCR buffer, 2.0 μl of dNTP mix, 2.0 μl of RT-PCR enzyme, appropriate volume of 0.6 μM primers and made up to 50 μl with RNasefree water. RT-PCR was performed using the thermal cycler (Eppendorf) programmed as RT reaction at 50 °C for 30 min, initial PCR activation at 95 °C for 15 min, denaturation for 1.5 min at 94 °C, annealing for 1.5 min at 58 °C, extension for 3.0 min at 72 °C. Thirty-five cycles were performed and final extension at 72 °C for 10 min. Finally, the reaction mixture containing PCR products were separated by 2% agarose gel electrophoresis along with 100 bp marker DNA. Gels were densitometrically (Bio-Rad) scanned and the cDNAs were normalized to that of the house keeping gene or internal control (RPS-16), which was co-amplified along with the cDNA of interest.
Reverse transcriptase-polymerase chain reaction Statistical Analysis Total RNA was used for the synthesis of cDNA. The Qiagen one-step RT-PCR kit was used for the generation of cDNA. The following specific oligonucleotide primers were used for the generation of cDNAs. Two 21-mer oligonucleotide primers for insulin receptor (Pubmed Nucleotide Accession No. M29014) were selected as described previously (Gonzalez et al., 2002). The sense
Data are expressed as mean ± SEM. The data were subjected to statistical analysis using one-way analysis of variance (ANOVA) and Duncan's multiple range test to assess the significance of individual variations between the control and treatment groups using a SPSS® statistical software package. In
Fig. 4. 14C-glucose oxidation in skeletal muscles [gracilis (a), quadriceps (b)], liver (c) and adipose tissue (d) of control, orchidectomized (ORD) and testosterone-replaced orchidectomized (ORD+ T) rats. 14C-glucose oxidation was performed using 10 mg of each fresh tissue by incubating with DMEM containing 0.5 μCi of 14C-glucose at 37 °C. CO2 released was captured with the help of Whatman filter paper containing diethanolamine buffer and placed in scintillation fluid. Radioactivity of the CO2 trap was determined using a Beta counter. Each value represents mean ± SEM of 6 animals. Significance at p b 0.05. a — compared with control; b — compared with ORD.
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Duncan's test, the significance was considered at the level of p b 0.05. Results Testosterone deficiency due to orchidectomy significantly (p b 0.05) reduced the serum insulin and testosterone levels (Fig. 1a and b). These hormonal changes resulted in a significant increase (p b 0.05) in plasma glucose level (Fig. 1c). However, administration of testosterone restored these changes to normal level in orchidectomized rat. Orchidectomy significantly (p b 0.05) diminished the insulin receptor number in gracilis (Fig. 2a) and quadriceps muscles (Fig. 2b), liver (Fig. 2c) and adipose tissue (Fig. 2d). However, insulin receptors were recovered due to testosterone replacement. Similar to that of insulin receptor, a significant decrease (p b 0.05) in insulin receptor mRNA level was also recorded in skeletal muscles (gracilis and quadriceps) (Fig. 3a), liver (Fig. 3b) and adipose tissue (Fig. 3c) as a result of orchidectomy. Despite the fact that the administration of testosterone significantly (p b 0.05) increased the insulin receptor mRNA level to that of control in skeletal muscles and liver, adipose tissue insulin receptor mRNA level was not brought back to normal level. Skeletal muscles showed a maximum ability to oxidize glucose when compared to liver and adipose tissue (Fig. 4). Orchidectomy caused a remarkable decrease (p b 0.05) in glucose oxidation in gracilis (Fig. 4a) and quadriceps muscles (Fig. 4b), liver (Fig. 4c) and adipose tissue (Fig. 4d). Nevertheless, administration of testosterone restored the rate of glucose oxidation in skeletal muscles, liver and adipose tissue to that of control. Discussion Hormones are the major regulators of metabolic activities in skeletal muscles, liver and adipose tissue. Testosterone, an anabolic steroid, plays an important role in this direction (Lemoine et al., 2002; Livingstone and Collison, 2002; Boyanov et al., 2003; Tsai et al., 2004; Mayes and Watson, 2004; Inaba et al., 2005; Lee et al., 2005). Therefore, metabolic activities of tissues are likely to change with experimental or pathological manipulation of hormones (Mainwaring and Mangan, 1973). Among the hormones, insulin is the major regulator of glucose homeostasis. Studies have shown that testosterone and insulin interact in their actions at the level of target organs (Rajkhowa et al., 1994; Legro et al., 1999; Livingstone and Collison, 2002; Sheffield-Moore and Urban, 2004). Conversely, the exact molecular mechanism underlying the specific influence of testosterone on insulin receptor expression is not known. Serum insulin level following testosterone replacement was significantly increased when compared to castrated rats, suggesting that normal circulating level of testosterone is essential to maintain optimum insulin concentration in serum. In accordance with this, Morimoto et al. (2001) have shown that testosterone modify serum insulin
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levels through the direct effect on pancreatic islet function by favoring insulin gene expression and release. Castration-induced reduction in insulin binding and restoration of the same in testosterone-replaced rats imply the importance of physiological level of testosterone in maintaining insulin receptors in target tissues. In support of this, Sesti et al. (1992) have shown that androgens increase insulin binding and insulin responsiveness in HEp-2 larynx carcinoma cells. It appears that the testosterone deficiency-induced deterioration of glucose homeostasis is mediated through deficiency in circulating levels of insulin, its receptors and their sensitivity in target organs. To identify the molecular mechanism responsible for insulin receptor deficiency, mRNA expression of insulin receptor was quantified. Changes in insulin receptor in castrated and testosterone-replaced rats were found to be associated with corresponding changes in insulin receptor mRNA expression. However, adipose tissue insulin receptor mRNA level was not brought back to normal level. In this regard, Cohen (1999) and Moretti et al. (2005) have reported that testosterone deficiency or hypogonadism is associated with a doubling of fat mass. With the increasing fatty-tissue accumulation, there is an increase of aromatase activity that is associated with a greater conversion of testosterone to estradiol. In the present study, bioavailability of testosterone in adipose tissue of testosterone-replaced castrated rats may be insufficient to evoke any change in insulin receptor mRNA levels. These findings tempt to propose the involvement of testosterone in the insulin receptor gene expression. Effect of testosterone on gene expression is known to be mediated through the activation of its receptor (Cato and Peterziel, 1998). Insulin target tissues have receptor for androgen (Lemoine et al., 2002; Mayes and Watson, 2004). The fact that testosterone replacement was able to increase insulin receptor number and its mRNA level suggests that testosterone may have a direct effect on insulin receptor gene transcription as well as translation of mRNA. Although the presence of androgen response element has not been identified in the promoter region, it is possible that androgen receptor may interact with basal transcription machinery (McEwan and Gustafsson, 1997) or other coactivators that bind to the androgen receptor (Yeh and Chang, 1996; Aarnisalo et al., 1998) could be involved in the mechanism by which testosterone increases insulin receptor gene expression. In this regard, studies on insulin receptor gene promoter region would be of great interest. Insulin receptor promoter contains DNA sequences responsive to glucose and insulin. This was demonstrated in the primary culture of rat hepatocytes transfected with plasmids containing the DNA-regulatory sequences −618 to −593 of the rat insulin receptor promoter fused to the luciferase gene. When three copies of the nucleotides −618 to −593 of the insulin receptor promoter were transfected, the reporter activity was significantly increased in the presence of glucose and more increased in the presence of glucose and insulin, indicating the regulatory role of these two factors (Fukuda et al., 2001). Insulin receptor synthesis can also contribute to the cell surface receptor population (Virkamaki et al., 1999). In the present study, castrated rats showed a significant
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decrease in serum insulin level, insulin receptor and its mRNA levels in target tissues. It is therefore suggested that the castrationinduced deficiency in insulin may be one of the reasons for impaired insulin receptor gene expression. Orchidectomy-induced impairment in glucose oxidation may be the result of low level of serum insulin as well as its defective action. Skeletal muscles, liver and adipose tissue are the major site of insulin-mediated glucose metabolism (Bajaj and Defronzo, 2003; Defronzo and Mandarino, 2003; Kahn and Saltiel, 2005). Studies suggest that normal serum insulin is essential to maintain glucose homeostasis by enhancing glycolysis and glycogen synthesis in skeletal muscles (Richter et al., 1984; Mandarino et al., 1987) with concomitant decrease in glycogenolysis in liver and skeletal muscles (Shimazu, 1987). Fukuda et al. (2001) reported that insulin regulates its own receptor gene expression. Insulin receptor is essential for insulin action such as glucose metabolism. In the present study, serum insulin and its receptor expression in skeletal muscles, liver and adipose tissue were decreased due to testosterone deficiency. Therefore, orchidectomy-induced impairment in glucose oxidation in skeletal muscles, liver and adipose tissue appears to be the effects of subnormal serum insulin and its receptors. Recently, Chen et al. (2006) have shown that testosterone has bidirectional effects on the cellular expression of insulin receptor substrate-1 (IRS-1) and GLUT4, which increase with low dose and decrease with higher dose in cultured adipocytes and skeletal muscle cells. These findings implicate the role of testosterone deficiency to bring about changes recorded in this study. Testosterone-deficient animals showed a significant elevation in plasma glucose concentration, which appears to be the result of testosterone deficiency-mediated impairment of glucose oxidation. In this regard, Holmang and Bjorntorp (1992) have reported that castration was followed by marked insulin resistance in the clamp experiments. This was also paralleled by a diminished insulin stimulation of glucose incorporation into glycogen down to about 50% of control values. Substitution of castrated rats with testosterone (which is equivalent to the dose employed in the present study) completely abolished these perturbations of insulin sensitivity. Plasma glucose in testosterone-replaced animals was similar to that of normal rats reinforcing the importance of this steroid in glucose homeostasis. In this respect, it is worth to mention the report of Thomas et al. (2003), which showed a significant inverse correlation between testosterone and fasting blood glucose. These observations attest the involvement of testosterone in the regulation of glucose homeostasis, which may explain the testosterone deficiency-induced impairment of glucose oxidation in target tissues. In addition to testosterone, corticosterone and estradiol were found to play an important role in glucose homeostasis. Though the circulating levels of estradiol and corticosterone were not measured in the present study, the available reports indicate an increase in corticosterone (Kitay, 1963; Seale et al., 2004) and a decrease in estradiol (Banu et al., 2002) following gonadectomy in male rats. Excess glucocorticoids have also been shown to decrease the number of insulin receptors and affinity in rat liver and adipocytes (Kahn et al., 1978; Buren et al., 2002). Likewise,
low levels of estradiol were shown to be associated with insulin resistance in men (Dunajska et al., 2004). In view of these findings, it is proposed that castration-induced changes in corticosterone and estradiol could have contributed for the changes recorded in the present study. It is concluded from the present study that testosterone deficiency-induced defective glucose oxidation in skeletal muscles, liver and adipose tissue is mediated through impaired serum insulin level and its receptor gene expression. Acknowledgement Financial assistance from the Indian Council of Medical Research (ICMR), New Delhi in the form of Senior Research Fellowship (SRF) to T. Muthusamy (Award No. 3/1/2/15/05RHN dated 9-8-2005), DST-FIST, UGC-SAP-DRS-II and UGC-ASIST programmes are gratefully acknowledged. References Aarnisalo, P., Palvimo, J.J., Janne, O.A., 1998. CREB-binding protein in androgen receptor-mediated signaling. Proceedings of the National Academy of Sciences of the United States of America 95 (5), 2122–2127. Andersson, B., Marin, P., Lissner, L., Vermeulen, A., Bjorntorp, P., 1994. Testosterone concentrations in women and men with NIDDM. Diabetes Care 17 (5), 405–411. Ashcroft, F.M., Rorsman, P., 2004. Molecular defects in insulin secretion in type-2 diabetes. Reviews in Endocrine and Metabolic Disorders 5 (2), 135–142. Bajaj, M., Defronzo, R.A., 2003. Metabolic and molecular basis of insulin resistance. Journal of Nuclear Cardiology 10 (3), 311–323. Banu, S.K., Govindarajulu, P., Aruldhas, M.M., 2002. Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo. Steroids 67 (13–14), 1007–1014. Barrett-Connor, E., 1992. Lower endogenous androgen levels and dyslipidemia in men with non insulin-dependent diabetes mellitus. Annals of Internal Medicine 117 (10), 807–811. Boyanov, M.A., Boneva, Z., Christov, V.G., 2003. Testosterone supplementation in men with type-2 diabetes, visceral obesity and partial androgen deficiency. Aging Male 6 (1), 1–7. Buren, J., Liu, H.X., Jensen, J., Eriksson, J.W., 2002. Dexamethasone impairs insulin signalling and glucose transport by depletion of insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase B in primary cultured rat adipocytes. European Journal of Endocrinology 146 (3), 419–429. Carpentier, J.L., 1994. Insulin receptor internalization: molecular mechanisms and physiopathological implications. Diabetologia 37 (Suppl 2), S117–S124. Carson, J.A., Lee, W.J., Mc Clung, J., Hand, G.A., 2002. Steroid receptor concentration in aged rat hindlimb muscle: effect of anabolic steroid administration. Journal of Applied Physiology 93 (1), 242–250. Cato, A.C.B., Peterziel, H., 1998. The androgen receptor as mediator of gene expression and signal transduction pathways. Trends in Endocrinology and Metabolism 9 (4), 150–154. Chen, X., Li, X., Huang, H.Y., Li, X., Lin, J.F., 2006. Effects of testosterone on insulin receptor substrate-1 and glucose transporter 4 expression in cells sensitive to insulin. Zhonghua Yi Xue Za Zhi 86 (21), 1474–1477. Cohen, P.G., 1999. The hypogonadal-obesity cycle: role of aromatase in modulating the testosterone-estradiol shunt-a major factor in the genesis of morbid obesity. Medical Hypotheses 52 (1), 49–51. Collins, S., Ahima, R.S., Kahn, B.B., 2005. Biology of adipose tissue. In: Kahn, C.R., Weir, G.C., King, G.L., Jacobson, A.M., Moses, A.C., Smith, R.J. (Eds.), Joslin's diabetes Mellitus. A Wolters Klunwer Company, Indian edition by BI publication, India, pp. 207–226. Defronzo, R.A., Mandarino, L.J., 2003. Insulin action. In: Henry, H.L., Norman, A.W. (Eds.), Encyclopedia of hormones. Elseivier Inc, California, pp. 333–347.
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