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Abstract

The estrogenic monomer bisphenol-A (BPA) is an endocrine-disrupting chemical used in the production of epoxy resins, plastic food and beverage containers, leading to ubiquitous human exposure. Environmentally relevant doses of BPA have profound effects on mice endocrine pancreas. It increases pancreatic insulin content and favors postprandial hyperinsulinemia and insulin resistance in male mice. Skeletal muscle plays a crucial role in maintaining systemic glucose metabolism. In the present study, we investigated the possible effects of BPA on insulin-signaling molecules and glucose oxidation in skeletal muscle of male rat. Adult male Wistar albino rats were divided into three groups. Group I: control (vehicle treated) and groups II and III were administered with BPA orally (20 and 200 mg/kg bw/day, respectively). Although there was no change in the levels of insulin receptor (IR), Akt (protein kinase B) and glucose transporter-4 (GLUT4) messenger RNA, BPA significantly decreased the IR, Akt and GLUT4 protein levels (both plasma membrane and cytosolic fraction) of the gastrocnemius muscle. There was an increase in serum insulin and decrease in serum testosterone levels but fasting blood glucose level remained unaltered. In conclusion, BPA has adverse effects on phosphorylation of Akt, GLUT4 translocation and ¹⁴C-glucose oxidation.

Keywords

Bisphenol-A endocrine disruptors hyperinsulinemia skeletal muscle insulin resistance testosterone

Introduction

Glucose homeostasis is a complex process that involves intertissue communication in order to maintain blood glucose levels in a precise range throughout the day, independently of the dietary nutrient ingestion.¹ It involves a complex communication among different tissues, including the liver, skeletal muscle, adipose tissue, brain and the endocrine pancreas.^2
–4 Skeletal muscle plays a crucial role in maintaining systemic glucose metabolism. Up to 85% of whole body insulin-stimulated glucose uptake occurs in skeletal muscle by increasing the translocation of glucose transport molecules, mainly glucose transporter-4 (GLUT4). The translocation of GLUT4 vesicles occurs through intracellular molecular-signaling cascade including the consecutive phosphorylation of several cytosolic proteins, such as insulin receptor substrate (IRS) molecules, phosphatidylinositol-3-kinase and protein kinase B (Akt).⁵ In the fasting state, low glucose levels keep plasma insulin at low concentration, while increasing the levels of the counterregulatory hormones, glucagon, adrenaline and corticosteroids that promote hepatic glucose production.⁶ Insulin is the only hormone that decreases blood glucose by promoting glucose uptake by adipocytes and muscle as well as preventing the liver from producing glucose by suppressing glycogenolysis and gluconeogenesis.⁴ Therefore, these hormone concentrations serve as important regulators for glucose homeostasis and perhaps these hormone-induced metabolic changes could be modulated by the exposure of exogenous hormone-mimicking chemicals.

During the last several decades, endocrine-disrupting chemicals (EDCs) have been used in a variety of commercial products but have also emerged as environmental estrogens of great concern to human and environmental health.^7
–9 Directly or indirectly, they can act to either induce or inhibit signal transduction, hormone production, transportation and secondary messengers.¹⁰ Bisphenol-A (4,4′-dihydroxyl phenyl 2,2-diphenyl propane, BPA) was named as one of the EDCs for its weak estrogenic action. It has been proposed that EDCs may also lead to other metabolic diseases, such as metabolic syndrome and type II diabetes (T2D).¹¹ Various doses of BPA that affects body weight in Sprague-Dawley (0.1 and 1.2 mg/kg bw/day)¹² and in Swiss albino OF1 male mice profoundly disrupt the pancreatic β-cell function (1 nM BPA),^13

–16 induce and develop insulin resistance without any changes in glycemia or weight (50 μg/kg/day),¹⁷ and it causes postprandial hyperinsulinemia (50 μg/kg/day to1 mg/kg bw/day).^17,18 It rapidly changes glycemia most likely by inducing hypersecretion of insulin (1 nM BPA).^14,19 In 17β-estradiol (E2)-treated Swiss albino OF1 male mice, there was a mild insulin resistance, because of 1.7-fold higher circulating insulin levels with a decrease in blood glucose, although it was not significant. This effect is remarkably manifested with BPA, where plasma insulin levels showed 1.53-fold increase but blood glucose concentration did not vary—a clear symptom of insulin resistance.¹⁷ Insulin resistance is a crucial constituent of the metabolic syndrome, and its presence predicts T2D.²⁰ This altered blood glucose homeostasis by BPA exposure may enhance the risk of developing T2D.¹⁷

BPA, which is a monomer (228 Da) of polycarbonate plastics and a constituent of epoxy and polystyrene resins that are extensively used in the food packaging industry and in dentistry,^21

–24 has been reported to have estrogenic activity.²⁵ It is used for efficient cross linking and plastics become harder and transparent.²² Small amounts of BPA can migrate from the polymers to food or water especially upon heating.²⁶ Indeed, it has been reported that BPA is released from polycarbonate flakes during autoclaving.²⁵ It is one of the highest volume chemicals produced worldwide, over 6 billion pounds produced each year²⁷ and over 100 tons released into the atmosphere per year.²⁸ In the United States, BPA was detected in 95% of the urine samples and saliva of patients treated with dental sealants.²⁹ Higher urinary concentrations of BPA are associated with an increased prevalence of cardiovascular disease, diabetes and liver enzyme abnormalities.³⁰ Human exposures to BPA are known to be overwhelming through the oral route.³¹ FDA has never established an acceptable daily intake for BPA exposure through the use of food additive; however, the U.S. Environmental Protection Agency has published a reference dose (0.05 mg/kg/day) for BPA.³² The tolerable daily intake of BPA is 0.01 mg BPA/kg bw/day.³³

Insulin-stimulated Akt phosphorylation was reduced in the skeletal muscle and liver of pregnant mice treated with BPA.⁶ It affects glucose transport in adipocytes.³⁴ However, the specific effect of BPA on insulin-signaling molecules in skeletal muscle has received only a little attention and is largely unknown. Therefore, the present study was designed to assess the effect of BPA on insulin signal transduction and glucose oxidation in gastrocnemius muscle of adult male rat.

Materials and methods

Chemicals

All chemicals and reagents used in the present study were of molecular biology and analytical grade (AR) and they were purchased from Amersham Biosciences Ltd (Little Chalfont, UK), Sisco Research Laboratories (Mumbai, India) and Bio-Rad Laboratories Inc. (Hercules, California, USA). BPA (purity > 99%) was purchased from Sigma (St Louis, Missouri, USA).¹⁴C-glucose was purchased from Board of Radiation and Isotope Technology (Mumbai, India). Radioimmunoassay kits for the assay of insulin and testosterone were obtained from Diasorin (Saluggia, Italy). Blood Glucose Test Strips was purchased from ACON Laboratories, Inc. (San Diego, California, USA) and Life Scan Inc. (Milpitas, California, USA). Total RNA isolation reagent was purchased from Medox (Chennai, India). The RT kit was purchased from New England Biolabs, Inc. (Hitchin, UK). The polymerase chain reaction (PCR) master mix kit was purchased from Promega (Wisconsin, USA). The β-actin monoclonal antibody was purchased from Sigma Chemicals Company (St. Louis, Missouri, USA). Polyclonal antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies were obtained from GeNei (Bangalore, India).

Animals

Healthy adult male albino rats of Wistar strain (Rattus norvegicus, 90–120 days of age) used in the present study, weighing 180–210 g were obtained from Central Animal House, University of Madras (Chennai, India). Animals were housed in polypropylene cages under specific humidity (65 ± 5%) and temperature (21 ± 2°C) with constant 12-h light/dark cycle. They were fed with standard rat pelleted diet (Lipton India, Mumbai, India) and clean drinking water was made available ad libitum.

Experimental details

Rats were divided into three groups. Each group consists of six animals. Group I: control (vehicle treated): corn oil was given via oral intubation for 30 days; group II: BPA-treated group (20 mg/kg bw); group III: BPA-treated group (200 mg/kg bw). BPA was dissolved in corn oil and given via oral intubation once a day (9 AM) for 30 days. After overnight fasting, blood was collected on 29th day of treatment and blood glucose was estimated using a glucose kit (CPC Diagnostics, Montgat, Barcelona, Spain). At the end of 30 days’ treatment, rats were anesthetized with an intraperitoneal injection of thiopentone sodium (40 mg/kg bw), blood was collected, serum separated and stored at −80°C until use. And 20 ml of isotonic sodium chloride solution was perfused through the left ventricle to clear blood from the organs. Gastrocnemius muscle was dissected out and used for the measurement of various parameters.

Estimation of fasting blood glucose

Blood glucose was estimated using On-Call Plus Blood Glucose Test Strips method (ACON Laboratories, Inc. and Life Scan Inc.). After 28 days of treatment, animals were fasted overnight and water was provided ad libitum. On 29th day, fasting blood glucose was estimated in animals of all the groups. Blood sample for glucose estimation was collected from rat tail tip. In a well-restrained rat, about 1 mm at the end of the tail was cut and a drop of blood was applied directly to the end of the test strips. Result was obtained on the meter display window as milligram per deciliter.

¹⁴C-glucose oxidation

¹⁴C-glucose oxidation was estimated as per the standard method.^35,36 Briefly, 10 mg gastrocnemius muscle was weighed and placed in a 2-ml ampoule containing 170 µl Dulbecco's modified Eagle's medium (DMEM, pH 7.4), 10 IU penicillin in 10 μl DMEM and 0.5 μCi ¹⁴C-glucose. Then, the ampoules were aerated with a gas mixture (5% CO₂ and 95% air) for 30s 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, 0.1 ml of 1 N H₂SO₄was 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/10 mg tissue.

Radioimmunoassay

Serum testosterone was assayed using solid-phase RIA kit obtained from Diasorin. The limit of detection was 0.02 ng/ml at 95% confidence limit. Cross-reactivity of the testosterone antiserum to other steroids such as 5α-dihydrotestosterone and androstenedione is 6.9% and 1.1%, respectively. Intra-assay coefficient of variation (CV) was less than 8%, and interassay CV was less than 7.6%. Results are expressed as nanogram per milliliter.

Serum insulin was assayed using ¹²⁵I-labeled RIA kit obtained from Diasorin. The limit of detection is 3.0 µIU/ml at 95% confidence limit. The percentage cross-reactivity of insulin antibody to human and rat was 100%; to C-peptide was less than 0.01%. Intra-assay CV was less than 10.6%, and interassay CV was less than 10.8%. Results are expressed as micro international units per milliliter.

Gene expression analysis

Isolation of total RNA

Total RNA was isolated from control and experimental samples using TRIR kit (total RNA isolation reagent) from Medox. Briefly, 100 mg fresh gastrocnemius muscle was homogenized with 1 ml TRIR and the homogenates were transferred immediately to a microfuge tube and kept at −80°C for 60 min to permit the complete dissociation of nucleoprotein complexes. Then, 0.2 ml of chloroform was added, vortexed vigorously for1 min and placed on ice at 4°C for 5 min. The homogenates were centrifuged at 12,000g 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 12,000g for 10 min at 4°C. The supernatant was removed and RNA pellet was washed with 1 ml of 75% ethanol by vortexing and subsequent centrifugation for 5 min at 7500g (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 >1.8. The yield of RNA is expressed in microgram.

Reverse transcriptase PCR

Total RNA was used for the synthesis of complementary DNA (cDNA). Reverse transcriptase PCR (RT-PCR) purchased from New England Biolabs, Inc. was used for the generation of cDNA. Table 1 lists the primers used in the synthesis of cDNA.

Table 1.

Primers used in the study.

Gene	Sequence (5′ → 3′)
Rat IR (224 bp)
Sense primer	5′-GCC ATC CCG AAA GCG AAG ATC-3′
Antisense primer	5′-TCT GGG TCC TGA TTG CAT-3′
Rat Akt (146 bp)
Sense primer	5′-GGAAGCCTTCAGTTTGGATCCCAA-3′
Antisense primer	5′-AGTGGAAATCCAGTTCCGAGCTTG-3′
Rat GLUT4 (150 bp)
Sense primer	5′-GGG CTG TGA GTG AGT GCT TTC-3′
Antisense primer	5′-CAG CGA GGC AAG GCT AGA-3′
Rat β-actin (96 bp)
Sense primer	5′-AAG TCC CTC ACC CTC CCA AAA-3′
Antisense primer	5′-AAG CAA TGC TGT CAC CTT CCC-3′

IR: insulin receptor; Akt: protein kinase B; GLUT4: glucose transporter-4.

For the first strand cDNA synthesis, 2 μg of RNA template was added with master mix 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 RNase free water. RT-PCR was performed using the thermal cycler (Eppendorf, Hamburg, Germany) programmed as RT reaction at 42°C for 60 min, initial PCR activation at 80°C for 5 min, denaturation for 2 min at 95°C, annealing for 54–65°C for 30 s, extension for 30 s at 73°C. Thirty-five cycles were performed and final extension at 73°C for 5 min. Finally, the reaction mixture containing PCR products (5 µl) mixed with 2 µl gels loading dye were separated by 2% agarose gel electrophoresis along with 100 bp marker DNA.³⁷ The power supply was adjusted to 80 V. Then the gel containing amplified product was visualized with the help of gel doc unit (Bio-Rad). The band were normalized to that of the housekeeping gene or internal control (β-actin) which was coamplified along with the cDNA of interest using Quantity One Software (Bio-Rad).

Protein expression analysis

Preparation of sample and sucrose gradient

Plasma membrane and cytosolic fractions from skeletal muscle of control and experimental animals were prepared as described by Dombrowski et al.³⁸ and Kristiansen et al.³⁹ The gradient was prepared by layering progressively less dense sucrose solution. Briefly, sucrose solutions were added into the polyallomer tube (ultracentrifugation tube) starting with 2 ml of 35% solution, 2 ml of 32% solution and 1 ml of 25% solution. At the top, 0.5 ml of sample was loaded.

Subcellular fractionation

Gastrocnemius muscle from control and experimental animals were simultaneously processed for the preparation of different fractions. All steps were carried out on ice or at 4°C. Tissue (∼1 g) was first cleaned of all visible fat, nerve and blood vessels and minced in buffer-A. The minced tissue was homogenized (100 mg/1 ml of buffer-A) using a polytron-equipped homogenizer. Buffer-A of 100 ml contains 84 mg of sodium bicarbonate, 8557.5 mg of sucrose, 1.742 mg of phenylmethylsulfonyl fluoride and 32.5 mg of sodium azide (NaN₃). The resulting homogenate was centrifuged at 1300g for 10 min. The supernatant was centrifuged at 1,90,000g for 1 h (preparative ultracentrifuge, Hitachi, Minato-ku, Tokyo, Japan). The resultant supernatant was saved and sampled as a cytosolic fraction for protein analysis. The pellet was resuspended in buffer-A and applied on discontinuous sucrose gradients (25%, 32% and 35%, w/w) and centrifuged at 1,50,000g for 16 h. Plasma membrane at 25–32% (plasma membrane fraction) and 32–35% (total membrane (or) cytosolic fraction) interfaces were recovered, diluted with buffer-B and centrifuged at 1,90,000g for 1 h. Plasma membrane fraction (pellet) was resuspended in buffer-A and kept at −80°C until used for insulin receptor (IR) and GLUT4 protein expression analysis. Protein concentration was estimated by Lowry method⁴⁰ using BSA as a standard.

Separation of proteins

Proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) by Lammmeli.⁴¹ Equal weight (25 μg) of samples from skeletal muscle of control and experimental animals was diluted with sample buffer (1:2), heated at 95°C for 4 min and then cooled on ice for 5 min. The lysate proteins (25 µg/lane) were separated by SDS-PAGE (10% gel) and transferred by electroblotting to polyvinylidene difluoride membrane (Bio-Rad Laboratories Inc.). The membranes were blocked with 5% nonfat dry milk and probed with the primary antibodies (which were diluted to 1:1000). Following incubation, the blot was washed for three times (5 min each with Tris-buffered saline containing Tween-20 (TBS-T)). After washing with TBS-T, the membranes were incubated for 1 h with horseradish peroxidase-conjugated rabbit anti-mouse or goat anti-rabbit antibodies (which were diluted to 1:5000; GeNei). The specific signals were detected with an enhanced chemiluminescence detection system (Thermo Fisher scientific Inc., Rockford, Illinois, USA). The protein bands were captured using Chemidoc and quantified by Quantity One Image Analysis system (Bio-Rad Laboratories). Later the membranes were incubated in stripping buffer at 50°C for 30 min. After this, the membrane was reprobed using a β-actin antibody (1:5000). As the invariant control, the present study used rat β-actin.

Statistical analysis

The data were subjected to one-way analysis of variance followed by Students–Newman–Keul’s test to assess the significance between mean values of control and experimental groups. The data are expressed as mean ± SEM and p values < 0.05 were considered as significant. This was carried out using SPSS 7.5 for Windows student version.

Results

Expression of insulin-signaling molecules

Effect of BPA on IR mRNA and its protein expression

In the present study, IR messenger RNA (mRNA) was found to be unaltered but a significant decrease in the IR protein expression was recorded in the BPA-treated groups (Figure 1).

Figure 1.

Effect of BPA of IR in skeletal muscle (gastrocnemius) of adult male rats. (a) mRNA expression, (b) histogram of mRNA expression, (c) protein level of IR and (d) histogram of protein. Each bar represents mean ± SEM of three observations representing six animals. Significance at p < 0.05, ^acompared with control. Lane 1: 100 bp ladder; lane 2: control; lane 3: 20 mg BPA; lane 4: 200 mg BPA. BPA: bisphenol-A; IR: insulin receptor; mRNA: messenger RNA.

Effect of BPA on Akt mRNA and its protein

In the present study, Akt mRNA expression was found to be unaltered due to BPA treatment (Figure 2). However, a significant decrease in the Akt protein was recorded in the BPA-treated groups. Phosphorylation of Akt was also significantly decreased in both the treated groups when compared with control rats (Figure 2(f)).

Figure 2.

Effect of BPA on Akt in skeletal muscle (gastrocnemius) of adult male rats. (a) mRNA expression, (b) histogram of mRNA expression, (c) protein level of Akt, (d) histogram of protein, (e) protein level of p-Akt and (f) histogram of p-Akt protein. Each bar represents mean ± SEM of three observations representing six animals. Significance at p < 0.05, ^acompared with control and ^bcompared with 20 mg BPA. Lane 1: 100 bp ladder; lane 2: control; lane 3: 20 mg BPA; lane 4: 200 mg BPA. BPA: bisphenol-A; Akt: protein kinase B; p-Akt: phospho-Akt; mRNA: messenger RNA.

Effect of BPA on GLUT4 mRNA and protein: cytoplasmic and plasma membrane fractions

To examine whether BPA influences the expression and translocation of GLUT4, it was measured both in cytosol and in plasma membrane. In the present study, GLUT4 mRNA was found to be unaltered but a significant decrease in the protein expression was recorded in the BPA-treated groups (Figure 3), GLUT4 protein in the membrane was decreased as a result of BPA treatment (Figure 3(d)). The cytosolic GLUT4 is also reduced in both BPA-treated rats (Figure 3(f)).

Figure 3.

Effect of BPA on GLUT4 in skeletal muscle (gastrocnemius) of adult male rats. (a) mRNA expression, (b) histogram of mRNA expression, (c) protein level of plasma membrane, (d) histogram of plasma membrane protein, (e) protein level of cytosolic fraction and (f) histogram of cytosolic fraction protein. Each bar represents mean ± SEM of three observations representing six animals. Significance at p < 0.05, ^acompared with control and ^bcompared with 20 mg BPA. Lane 1: 100 bp ladder; lane 2: control; lane 3: 20 mg BPA; lane 4: 200 mg BPA. BPA: bisphenol-A; GLUT4: glucose transporter-4; mRNA: messenger RNA.

Effect of BPA on serum testosterone

In the present study, serum testosterone level was significantly decreased in BPA-treated groups when compared with control (Figure 4).

Figure 4.

Effect of BPA on serum testosterone level in adult male albino rats. Each bar represents mean ± SEM of six animals. Significance at p < 0.05, ^acompared with control and ^bcompared with 20 mg BPA. BPA: bisphenol-A.

Effect of BPA on serum insulin

In the present study, there was an elevated level of serum insulin in BPA (20 mg and 200 mg)-treated groups (Figure 5).

Figure 5.

Effect of BPA on serum insulin level in adult male albino rats. Each bar represents mean ± SEM of six animals. Significance at p < 0.05, ^acompared with control and ^bcompared with 20 mg BPA. BPA: bisphenol-A.

Effect of BPA on fasting blood glucose level

In the present study, the fasting blood glucose level was unaltered in BPA-treated groups (Figure 6) when compared with control rats.

Figure 6.

Effect of bisphenol-A on fasting blood glucose in adult male albino rats. Each bar represents mean ± SEM of six animals. Significance at p < 0.05.

Effect of BPA on glucose oxidation

This experiment was carried out to examine whether BPA influences glucose oxidation in gastrocnemius muscle. In the present study, a reduction in the oxidation of glucose was seen in both 20 and 200 mg BPA-treated rats (Figure 7) when compared with control, suggesting the adverse affect of BPA.

Figure 7.

Effect of bisphenol-A on ¹⁴C-glucose oxidation in gastrocnemius muscle of adult male rats. Each value represents mean ± SEM of six animals. Significance at p < 0.05, (a) compared with control.

Discussion

Skeletal muscle comprises a large percentage of the body mass and is the primary tissue responsible for the disposal of an oral glucose load.⁴² Insulin is essential for maintaining glucose homeostasis and regulating carbohydrate, lipid and protein metabolisms.⁴³ Insulin resistance in skeletal muscle is a hallmark feature of T2D (noninsulin dependent).⁴⁴ ^,45

The decreased IR protein levels in BPA-treated groups may be due to BPA-induced elevated levels of insulin because increased level of insulin might have caused downregulation of its receptors as evidenced by the decreased concentration of IR in the plasma membrane. Mamula et al.⁴⁶ reported that insulin is thought to downregulate its own receptor by a variety of mechanisms that can influence its synthesis and degradation. This process of downregulation occurs when there are elevated levels of insulin. When insulin binds to its receptor on the surface of a cell, the hormone receptor complex undergoes endocytosis and is subsequently attacked by intracellular lysosomal enzymes. The number of cell surface receptors for insulin is gradually reduced by the accelerated rate of receptor internalization and degradation.⁴⁷

The unaltered level of Akt mRNA suggests that BPA did not have any effect at the level of transcription. However, a significant decrease in the Akt protein and its phosphorylated form was recorded in the BPA-treated groups. In accordance with the present study, decreased levels of Akt protein and phospho-Akt (p-Akt) were also observed in 3T3-L1 adipocytes treated with BPA.⁴⁸ It has been reported that insulin-stimulated Akt phosphorylation is reduced in skeletal muscle and liver of BPA-treated pregnant mice relative to control.¹⁶ In both these studies, the exact molecular mechanism for the reduction in Akt and its phosphorylated form is not known.

GLUT4 exists in insulin-sensitive tissues mainly skeletal muscle and is the major transporter protein responsible for insulin-mediated whole body glucose uptake.⁴⁹ This may be the consequence of decreased phosphorylation of Akt which is essential for the GLUT4 translocation from cytosol to plasma membrane. Since Akt and its phosphorylation were decreased, it resulted in impaired translocation of GLUT4 to the plasma membrane. The cytosolic GLUT4 is also reduced in BPA-treated rats (Figure 3), suggesting that synthesis of GLUT4 itself is suppressed. Insulin-stimulated glucose transport from extracellular fluid into the cell is achieved by translocation of major insulin responsive glucose transporter—GLUT4 from intracellular vesicle storage site to plasma membrane in skeletal muscle.⁵⁰ This translocation of GLUT4 is mediated through insulin-signaling pathway and any abnormality in this signaling pathway results in insulin resistance which in turn leads to T2D.⁴⁹ Both cytosolic and plasma membrane GLUT4 proteins were decreased due to BPA treatment. It is therefore suggested that the increased level of insulin recorded in this study (Figure 5) might have caused internalization of its receptors as evidenced by the decreased IR in the plasma membrane (Figure 1). The decreased level of IR, Akt and its phosphorylation ultimately leads to decreased GLUT4 protein both in the cytosol and in the plasma membrane (Figure 3). The unaltered levels of IR, Akt and GLUT4 mRNA with a significant reduction in the proteins suggest a possible defect in translation of their mRNA.

Physiological level of testosterone maintains normal insulin sensitivity, whereas both excess and deficiency of testosterone promote insulin resistance.^51,52 Thus, normal circulating level of testosterone is essential to maintain optimum insulin concentration in serum. In a recent study, it was demonstrated that E2 and BPA dose dependently decrease the expressions of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes, such as cytochrome P450s, P₄₅₀17β and 17β-hydroxysteroid dehydrogenases mRNA. Therefore, the decreased expression of steroidogenic enzymes and StAR protein involved in testosterone synthesis might be primarily associated with the decreased testosterone levels as a result of BPA and E2 treatments.⁵³ BPA was found to decrease testosterone by decreasing the expression of steroidogenic enzymes such as 17α-hydroxylase/17–20 lyase.⁵⁴ It has also been reported that the serum testosterone levels were decreased in male mice following fetal exposure to BPA.^55,56 In the present study, testosterone level was decreased, and this may be due to impaired activity of steroidogenic enzymes involved in testosterone synthesis.

Whether insulin resistance precedes hyperinsulinemia or hyperinsulinemia precedes insulin resistance in the development of T2D is controversial.⁶ The only clear conclusion is that they occur in parallel.⁶ Nevertheless, it has been demonstrated that BPA increases insulin content and its release when islets of Langerhans are cultured in the presence of BPA, suggesting that BPA has a direct effect on the islets.⁶

This elevated level of serum insulin in BPA (20 mg and 200 mg)-treated groups (Figure 5) may be the direct effect of BPA on the β-cells of Langerhans to produce insulin via the estrogen receptor alpha (ERα).¹⁸ Insulin by acting through insulin-like growth factor 1 receptor stimulates the serine kinases, thereby phosphorylating the serine sites of IRS and its downstream molecules. Such an interaction could provide a mechanism for a vicious cycle of insulin-induced insulin resistance.⁵⁷ BPA-treated animals develop insulin resistance via the ERα-mediated increase in insulin content of pancreatic β-cells.¹⁸ ^,58 It has been demonstrated that BPA had some nongenomic actions on various tissues and/or cells through membrane receptors or membrane channels.⁵⁹ BPA rapidly activates cyclic adenosine monophosphate response element-binding protein in pancreatic β-cells.¹⁵ Administration of BPA in adult male mice provokes hyperinsulinemia and mild insulin resistance,¹⁷ partly due to a direct effect of BPA on the endocrine pancreas and also a possible direct effect on peripheral tissues. In the present study, BPA might have a direct or indirect action on pancreas and peripheral tissues which ultimately lead to increased insulin when compared with control.

The end product of glycolysis is pyruvate in the aerobic condition and it will enter into tricarboxylic acid cycle and the electron transport chain to produce energy. Increased free fatty acid oxidation causes elevation of the mitochondrial acetyl coenzyme A (CoA)/CoA and Nicotinamide Adenine Dinucleotide (reduced) (NADH⁺)/ Nicotinamide Adenine Dinucleotide (oxidized) (NAD⁺) ratios, which leads to inactivation of pyruvate dehydrogenase. As a result of increased acetyl-CoA, citrate concentration increases, resulting in the inhibition of phosphofructokinase and accumulation of glucose-6-phosphate. Accumulated glucose-6-phosphate inhibits hexokinase II and finally leads to decreased glucose uptake.⁶⁰ In the present study, the conversion of pyruvate into free fatty acid by BPA may be a reason for the unaltered level of glucose in BPA-treated rats. This may be a transient state, which is likely to be a forerunner for the onset of classical hyperglycemic state. Glucose might have been utilized during free fatty acid synthesis. As long as the hyperinsulinemia is adequate to overcome insulin resistance, glucose tolerance remains normal.⁶¹ Persistence of chronic physiologic euglycemic hyperinsulinemia can induce severe insulin resistance with normal glucose tolerance.⁶²

Glucose oxidation is an important process which provides energy to the cells to perform various functions. The rate of glucose oxidation in a cell depends on the rate of entry of glucose into the cell. The decreased level of glucose oxidation may be due to a defect in translocation of major insulin-stimulated glucose transporter, GLUT4, from intracellular vesicle storage site to plasma membrane. Reduced membrane GLUT4 level leads to impaired glucose uptake and this may be one of the causative factors for the decreased glucose oxidation in BPA-treated groups. Hyperinsulinemia-associated insulin resistance and low levels of testosterone appear to be responsible for impaired glucose oxidation in gastrocnemius muscle.

Conclusion

To abridge, BPA treatment significantly decreased the glucose oxidation, whereas the fasting blood glucose was unaltered. It resulted in hyperinsulinemia-associated insulin resistance and decreased the serum testosterone. BPA treatment significantly decreased the plasma membrane and cytosolic GLUT4 proteins, phosphorylated Akt, Akt and IR, whereas their mRNAs were unaltered in gastrocnemius muscle. In conclusion, this study indicates that oral gavage exposure of adult male rats to doses (20–200 mg/kg bw/day) of BPA exposure has adverse effects on insulin-signaling molecules such as IR, Akt, p-Akt and GLUT4 protein expression leading to diminished glucose uptake and oxidation in skeletal muscles of adult male rats. However, it remains to be shown whether lower doses relevant to human exposure levels would also have adverse effects on the above mentioned sites of insulin signaling and glucose uptake and oxidation.

Footnotes

Author’s Note

Animals were maintained as per the National Guidelines and Protocols, approved by the Institutional Animal Ethical Committee (IAEC No. 011/02/2011).

Funding

University Grants Commission Special Assistance Programme (UGC-SAP) and Department of Science and Technology (DST)–FIST Programme are acknowledged for the financial support.

Declaration of Conflicting Interests

The authors declared no conflicts of interest.

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Effect of bisphenol-A on insulin signal transduction and glucose oxidation in skeletal muscle of adult male albino rat

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Animals

Experimental details

Estimation of fasting blood glucose

14C-glucose oxidation

Radioimmunoassay

Gene expression analysis

Isolation of total RNA

Reverse transcriptase PCR

Protein expression analysis

Preparation of sample and sucrose gradient

Subcellular fractionation

Separation of proteins

Statistical analysis

Results

Expression of insulin-signaling molecules

Effect of BPA on IR mRNA and its protein expression

Effect of BPA on Akt mRNA and its protein

Effect of BPA on GLUT4 mRNA and protein: cytoplasmic and plasma membrane fractions

Effect of BPA on serum testosterone

Effect of BPA on serum insulin

Effect of BPA on fasting blood glucose level

Effect of BPA on glucose oxidation

Discussion

Conclusion

Footnotes

Author’s Note

Funding

Declaration of Conflicting Interests

References

¹⁴C-glucose oxidation