Cardiovasc Toxicol (2014) 14:10–20 DOI 10.1007/s12012-013-9233-z
Lactational Exposure of Phthalate Impairs Insulin Signaling in the Cardiac Muscle of F1 Female Albino Rats Viswanathan Mangala Priya • Chinnaiyan Mayilvanan Narasimhan Akilavalli • Parsanathan Rajesh • Karundevi Balasubramanian
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Published online: 3 December 2013 Ó Springer Science+Business Media New York 2013
Abstract Di-2-ethylhexyl phthalate (DEHP), a ubiquitous endocrine disruptor and plasticizer of polyvinyl chloride, is being used in the manufacture of consumer and medical products as well as in children’s toys. Fetuses and newborns are more sensitive to endocrine disruption. DEHP is a lipophilic substance, which could easily be transferred to the developing offspring through placenta or breast milk. DEHP alters the metabolism of the endocrine organs, which leads to energy imbalance associated with increased risk of insulin resistance, obesity and cardiovascular disease. The heart is an insulin-responsive organ. The effect of DEHP on the cardiac muscle insulin signaling remains obscure. Since the developmental period is more vulnerable to the adverse effect of DEHP, the present study was framed to study the impact of lactational exposure of DEHP on insulin signaling molecules in the cardiac muscle of F1 progeny female albino rat (postnatal day 60). Healthy dams were treated with DEHP orally (0, 1, 10 and 100 mg/ kg body weight/day, respectively) from the postpartum day 1–21. Both low and high doses are relevant to the human exposure, and hence, both were used in this study. At a low dose (1 mg/kg body weight/day), obvious differences were observed in the fasting blood glucose and the insulin signaling molecule when compared to control. But marked differences were observed in the cardiac tissue insulin signaling molecules of animals treated with high doses. In conclusion, the DEHP treatment significantly increased the fasting blood glucose level and decreased the insulin
V. Mangala Priya C. Mayilvanan N. Akilavalli P. Rajesh K. Balasubramanian (&) Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India e-mail: [email protected]
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receptor (IR), insulin receptor substrate (IRS-1), p-IRS1Tyr632, p-AktSer473, plasma membrane glucose transporter (GLUT4), 14C-2-deoxyglucose uptake and the 14C-glucose oxidation. Conversely, Akt and GLUT4 protein in cytosol remained unaltered compared to control. Lactational exposure of DEHP impairs insulin signal transduction and glucose oxidation in the cardiac muscle of the F1 female albino rats, suggesting its possible role in the development of type 2 diabetes. Keywords DEHP Lactational exposure Cardiac muscle Insulin resistance Type 2 diabetes Abbreviations ATP Adenosine triphosphate Atf-4 Activating transcription factor 4 Atf-6 Activating transcription factor 6 Bip Binding immunoglobulin protein CPM Counts per minute FSH Follicle-stimulating hormone GLUT4 Glucose transporter 4 IR Insulin receptor IRS-1 Insulin receptor substrate 1 IU International units JNK C-Jun N-terminal kinase MEHP Mono-(-2-ethylhexyl phthalate) MEHHP Mono-(2-ethyl-5-hydroxyhexyl) phthalate MEOHP Mono-(2-ethyl-5-oxohexyl) phthalate mM Millimolar nM Nanomolar OECD Organization for economic cooperation and development Pdx1 Pancreatic duodenal homeobox-1 PPARc Peroxisome proliferator-activated receptor c PPARa Peroxisome proliferator-activated receptor a
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Ucp2 lCi
Uncoupling protein-2 Microcurie
Introduction The incidence of type 2 diabetes, obesity and insulin resistance are rising at epidemic rates over the last decade [1]. Environmental pollutants, diet and genetic factors play an important role in the incidence of type 2 diabetes [2]. Endocrine disruptors are a heterogeneous group of chemicals found in the environment and consumer products, which are used broadly. In recent years, there has been a growing concern over the possibility of hormonal disruption even at low levels of exposure. Di-2 (ethyl hexyl phthalate) (C24H38O4), which is a ubiquitous endocrine disruptor, is used as a plasticizer in the production of polyvinyl chloride (PVC) products to impart structural flexibility [3]. Flexible material containing DEHP is used in variety of consumer products such as building materials, car products, clothing, food packaging and children’s products [4]. The general human exposure is found to be 3–30 lg/kg body weight/day. DEHP is not covalently bound to the PVC polymer, so it can easily outgas into the environment [5]. In addition to general human exposure, there is an increased risk of occupational exposure. Occupational Safety and Health Administration (OSHA) has set the permissible concentration of DEHP at workplace as 5 mg/m3 in air [6]. Also, inhalation exposure of DEHP is found as 700 lg/kg body weight/day based on workplace standards. Apart from occupational exposure, a significant exposure to DEHP occurs through medical devices. Production of DEHP amounts to approximately 2 million tons per year [7]. Evidences have accumulated that DEHP has a negative impact on the male and female reproductive functions. Additionally, DEHP has drawn considerable interest because of its contribution to energy imbalance and metabolic disorders [8]. DEHP administration resulted in impaired hepatic glycogenesis and glycogenolysis [9]. Epidemiological studies show a positive correlation between increased phthalate metabolites in urine and abdominal obesity and insulin resistance in adult males [10, 11]. Previous results confirm the adverse effects of DEHP on insulin receptor and glucose oxidation in cultured Chang liver cells [12]. Studies from our laboratory suggest that DEHP disrupts insulin signal transduction and favors glucose intolerance [13, 14]. Di-2-ethylhexyl phthalate is able to cross the placenta and also pass into the breast milk [15, 16], resulting in significant risk for the developing fetuses and newborns [17]. Neonates are more receptive to DEHP than adults as
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they are more susceptible to endocrine disruption. Even small changes in the endogenous hormone level result in structural and functional changes permanently [18]. It has been reported that developmental DEHP exposure disrupted the pancreas and altered glucose homeostasis in the whole body [19]. Although lactational exposure of DEHP results in various reproductive abnormalities, its effect on insulin signal transduction is too early to be commented on. The heart is an energy-consuming organ that needs to maintain its intracellular ATP level for the myocardial contraction/relaxation cycle. Insulin plays a key role in the regulation of cardiovascular metabolism and function, by modulating glucose transport, glycolysis and lipid metabolism. DEHP at clinically relevant concentrations impaired the mechanical and electrical behavior of cells [20]. In correlation, a study on the effect of DEHP on the cardiac muscle showed an increase in fatty acid oxidation reportedly with decreased glucose oxidation by downregulating the enzymes associated with it [21]. Despite cardiovascular disease being the leading cause of death in type 2 diabetic patients with a twofold increased risk of congestive heart failure, insulin signaling in heart has received only little attention. Therefore, the current investigation has been designed to study the effect of DEHP on insulin signaling.
Materials and Methods Chemicals and Reagents Di-2-ethylhexyl phthalate was purchased from Sigma Chemicals Company (St. Louis, USA). All other chemicals and reagents used in the present study were of analytical grade (AR), and they were purchased from Amersham Biosciences Ltd. (UK) and Sisco Research Laboratories, Mumbai. Blood glucose strips were purchased from ACON laboratories, Inc., San Diego, USA. IR, IRS-1, pIRS-1tyr632, Akt, pAktser473, GLUT4 antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. bactin antibody was purchased from Sigma Chemicals Company (St. Louis, USA). 14C-glucose and 14C-2deoxyglucose were purchased from the Board of Radiation and Isotope Technology (Mumbai, India). Animals Healthy adult female albino rats of Wistar strain (Rattus norvegicus), weighing 100–120 g, were used in the present study. The animals were housed in a clean polypropylene cage, maintained in an air-conditioned animal house (12-h light/dark and temperature 25 – 2 °C), fed with standard rat-pelleted diet (Lipton India Ltd., Mumbai, India), and clean drinking water was made available ad libitum.
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Animals were maintained as per the National Guidelines and Protocols approved by the Institutional Animal Ethical Committee. Dose Selection and Treatment Nulliparous rats were mated, and presence of sperm was taken as gestation day 0 (GD 0). After spontaneous delivery, postnatal day 1, the litters were culled to six numbers per each dam (3 dams/group) and balanced for sex to the extent possible. The litters were culled according to OECD guidelines. The dams received phthalate (DEHP) through oral gavage (vehicle—olive oil) as shown below throughout the lactational period: Group I: control (vehicle treated), Group II: 1 mg/kg body weight/day, Group III: 10 mg/kg body weight/day and Group IV: 100 mg/kg body weight/day. Dose ranges used in the present study correspond with normal to occupational human exposure. On the 58th day, the animals were kept for overnight fasting and their blood was collected for glucose estimation. All the female progeny were perfused and killed on postnatal day 60. The cardiac muscle was dissected out, washed in icecold physiological saline repeatedly and used for the assay of various parameters.
added to the ampoule 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 was 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. [14C]-2-Deoxy-D-glucose Uptake 14
C-glucose uptake in the cardiac tissue was estimated by the standard method [24]. Briefly, 100 mg of tissue was incubated in 5 ml of DMEM with 5 mM glucose for 30 min under the condition of 95 % O2, 5 % CO2. After this, the tissue was washed and incubated in glucose-free medium for 30 min with or without 100 nM of insulin. Glucose uptake was initiated by the addition of 2 ll of 0.05 lCi 14C-2-deoxyglucose. At the end of 10 min of incubation, the tissue was removed, rapidly rinsed in isotope-free medium, homogenized in 2 ml of 5 % trichloroacetic acid and placed in the scintillation vials containing 5 ml of scintillation fluid and counted in a Beta counter. Results are expressed as CPM of 14C-2-deoxyglucose/ 100 mg tissue. Protein Expression Analysis
Methods Sample Preparation Estimation of Blood Glucose Blood glucose was estimated using On-Call Plus Blood Glucose Test Strips method (ACON Laboratories, Inc., San Diego, USA.). On the 59th day, fasting blood glucose was estimated in animals of all the groups. Blood sample for glucose estimation was collected from the rat’s tail tip. Results were obtained on the meter display window as mg/ dl. Determination of 14
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C-Glucose Oxidation
C-glucose oxidation in the cardiac tissue was estimated by the standard method [22], [23]. Briefly, 10 mg of the cardiac tissue was weighed and placed in a 2-ml ampoule containing 170 ll of DMEM (pH 7.4), 10 IU penicillin in 10 ll of DMEM and 0.5 lCi of 14C-glucose. Then, the ampoules were aerated with a gas mixture (5 % CO2 and 95 % air) for 30 s and tightly closed 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 traps were replaced every 2 h. After removing the second trap, 0.1 ml of 1 N H2SO4 was
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Plasma membrane and cytosolic fractions from the cardiac tissue of the control and experimental animals were prepared by the standard methods [25], [26]. The gradient was prepared by progressively layering less dense sucrose solution upon one another. Briefly, sucrose solutions were added into the polyallomer tube (ultracentrifugation tube) slowly and steadily, starting with 35 % solution. First, 2 ml of the 35 % solution had drained into the tube, and then, 2 ml of 32 % solution could be loaded on top of the 35 % solution. This procedure was continued with 1 ml of 25 % solution. There was enough space left at the top of the tube upon which the 0.5 ml of sample was loaded. Subcellular Fractionations The cardiac tissue from the control and experimental animals was simultaneously processed for the preparation of different fractions. All the 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 (1 g/1.5 ml of buffer A) using a polytron-equipped homogenizer at a precise low setting. The resulting homogenate was centrifuged at 1,3009g for 10 min. The supernatant was centrifuged at 1,
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90,0009g for 1 h. 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, 50,0009g for 16 h. Plasma membrane from 25 to 32 interfaces was recovered, diluted with buffer B and centrifuged at 1, 90,0009g for 1 h. The plasma membrane fraction (pellet) was resuspended in buffer A and kept at -80 °C until later used for IR and GLUT4 protein expression analysis. Protein concentration in the sample was determined prior to Western blot analysis.
peroxidase-conjugated rabbit antimouse or goat antirabbit antibodies (which were diluted to 1: 5,000) (GeNei, Bangalore, India). The specific signals were detected with enhanced chemiluminescence detection system (Thermo Fisher Scientific Inc., USA). The protein bands were captured using ChemiDoc and quantified by Quantity One image analysis (Bio-Rad Laboratories, CA). Later the membranes were incubated in stripping buffer at 500c for 30 min. After this, the membranes were reprobed using a b-actin antibody (1:5,000 dilution). As the invariant control, the present study used rat b-actin.
Estimation of Protein
Statistical Analysis
Protein concentration was determined as per the method [27] with bovine serum albumin (BSA) as the standard. Ten microliters of the sample was taken in a clean test tube and made up to 1 ml with distilled water. To this, 5 ml of alkaline copper reagent was added. The contents were mixed well and allowed to stand at room temperature for 10 min. Five hundred microliters of 1 N Folin–Ciocalteu reagent was then added and immediately mixed well. After 20 min, the intensity of the blue color developed was read at 720 nm against blank. For plotting the standard graph, a set of standards (25, 50, 75, 100 and 125 lg) were taken in a series of test tubes and made up to 1 ml with distilled water and processed as that of the samples. The standard graph was drawn by plotting the concentration of standards on the x-axis and the optical density on the y-axis. The concentration of protein in the sample was calculated by referring the standard curve and expressed as lg/mg tissue.
The data were subjected to one-way analysis of variance (ANOVA), followed by Student’s Newman-Keuls (SNK) test to assess the significance between mean values of the control and experimental groups. The data were expressed as mean – standard error of mean (SEM), and p values \0.05 were considered as significant. This was carried out using SPSS package for Windows.
Results Fasting Blood Glucose Fasting blood glucose level significantly increased in treated groups compared to control (Fig. 1). DEHP treatment profoundly increased the fasting blood glucose level. This prompted us to study the action of DEHP on insulin signaling molecules.
Western Blot Analysis Separation of Proteins Proteins were separated by SDS–Polyacrylamide gel electrophoresis by standard method [28]. Equal volume (25 lg) of samples from the cardiac tissue 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. Samples were loaded to 10 or 7 % SDS-PAGE in a Bio-Rad miniature slab gel apparatus. The lysate proteins were separated by SDS-PAGE (10 % gel) and transferred by electroblotting to polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5 % nonfat dry milk and probed with primary antibodies (which were diluted to 1:1,000). Following incubation, the blot was washed 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
Fig. 1 Effect of lactational exposure of DEHP on fasting blood glucose level of female rats. After overnight, fasting blood glucose level was detected. Each bar represents mean ± SEM of 6 animals. Statistical significance at p \ 0.05, a compared with control. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
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Fig. 2 Effect of lactational exposure of DEHP on insulin receptor protein in the cardiac muscle of female rats. Total of protein was detected by Western blot analysis. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 4 Effect of lactational exposure of DEHP on phospho IRS1Tyr632 protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control, b compared with 1 mg DEHP and c compared with 10 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 3 Effect of lactational exposure of DEHP on IRS-1 protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 5 Effect of lactational exposure of DEHP on Akt protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Effect of DEHP on Insulin Signaling Molecules
IRS-1 and p-IRS-1Tyr632
Insulin Receptor
Insulin receptor substrate 1 is the major substrate involved in the cardiac muscle insulin signaling. In the present study, IRS-1 protein level was significantly decreased in 10 and 100 mg treated animals (Fig. 3) when compared to control. On the other hand, p-IRS-1Tyr632 protein level was significantly decreased in a dose-dependent manner (Fig. 4).
IR protein level was significantly decreased in the animal treated with 10 and 100 mg DEHP compared to control and 1 mg treated animals (Fig. 2). IR phosphorylation recruits IRS-1, which in turn gets phosphorylated at Tyr 632 residue.
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Fig. 6 Effect of lactational exposure of DEHP on phospho Aktser473 protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 7 Effect of lactational exposure of DEHP on cytosolic GLUT4 level in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Akt and p-AktSer473 Akt protein level (Fig. 5) was found to be unaltered, but interestingly p-AktSer473 protein level (Fig. 6) was decreased in a dose-dependent manner in the treated groups at par with the control. Cytosolic and Plasma Membrane GLUT4 Glucose transporter 4 protein, which is mandatory for the entry of glucose molecule, was found to be unaltered in the cytosol, whereas in the plasma membrane, GLUT4 was found to be significantly decreased in the 10 and 100 mg DEHP-treated groups compared to coeval control (Figs. 7, 8). 14
C-2-deoxyglucose Uptake and
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C-glucose Oxidation
It is clear from the above results that the insulin signaling molecules were drastically altered in treated groups. This tempted us to study glucose uptake and oxidation. Glucose uptake and oxidation are necessary for the maintenance of glucose homeostasis. DEHP treatment showed significant dose-dependent decrease in 14C-2-deoxyglucose uptake in treated animals compared to control (Fig. 9). DEHP treatment also showed a significant decrease in 14C-glucose oxidation in DEHP-treated animals compared to control (Fig. 10). Considering the fact that the rate of glucose oxidation in cells depends on its glucose uptake, the decrease in glucose oxidation may be attributed to reduced glucose uptake.
Fig. 8 Effect of lactational exposure of DEHP on plasma membrane GLUT4 level in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Discussion Di-2-ethylhexyl phthalate is one of the ubiquitous endocrine disruptors, which is used as a plasticizer of polyvinyl chloride in the manufacture of a wide variety of consumer products such as building and car products, clothing, food packaging and children’s products and in medical devices made of polyvinyl chloride [29, 30]. In the current study,
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Fig. 9 Effect of lactational exposure of DEHP on 14C-2-deoxyglucose uptake in the cardiac muscle of female rats. Each bar represents mean ± SEM of 6 animals. Significance at p \ 0.05; a compared with control, b compared with 1 mg DEHP and c compared with 10 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
DEHP at high doses (10 and 100 mg/kg body weight/day) exhibited a significant difference in insulin signaling molecules. Dose-dependent adverse effect was observed in this study. Previous report from our laboratory also showed a dose-dependent decrease of insulin signaling molecules in gastrocnemius muscle of adult male rats [13]. The doses used in the present study are relevant to general population, occupational and medical exposure. The highest estimated daily exposure through blood transfusion is 22.6 mg/kg of body weight for neonates and 8.5 mg/kg for adults. Dialysis patients received an average of 75 mg DEHP per treatment and average of 12 g over a period of 1 year [31]. During extracorporeal membrane oxygenation and blood transfusion, a patient would be exposed up to 42–140 mg/ kg body weight [32]. Thus, all the doses used in the present study are within the human exposure range. Current exposures to DEHP are high enough to cause serious concern as its widespread human exposure and adverse effects have triggered the interest of public and government alike [7]. Due to immaturity of the liver, neonates are unable to oxidize DEHP, and therefore, they are often more receptive to toxic chemicals [17]. Fetuses and newborns appear to be more sensitive to its endocrine disruption. Moreover, DEHP is found to be lipophilic and accumulates more in the adipose tissue, breast milk and amniotic fluid. Phthalates were also detected in pooled breast milk samples and in infant formula [15]. Sex steroids have a direct influence on glucose homeostasis and contribute to insulin resistance. DEHP and its active metabolite MEHP decrease estradiol level, ovulation [33], FSH-stimulated second messenger and progesterone production in cultured ovarian granulosa cells [34]. Moreover, adverse effects of developmental DEHP exposure on glucose homeostasis are much more severe in
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Fig. 10 Effect of lactational exposure of DEHP on glucose oxidation in the cardiac muscle of female rats. Each bar represents mean ± SEM of 6 animals. Significance at p \ 0.05; a compared with control, b compared with 1 mg DEHP and c compared with 10 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
female rats compared to male rats because females develop glucose intolerance more rapidly [19]. Further, the heart is an insulin-responsive organ, where insulin has direct effects on glucose transport, glycolysis, glucose oxidation, glycogen synthesis, protein synthesis, cardiac growth and contractility [35]. Diabetes is also associated with profound changes in cardiac metabolism, diminished glucose uptake and oxidation [36, 37]. These findings tempted us to investigate the insulin signaling pathway in the cardiac muscle of female albino rats exposed to DEHP during developmental period. In the present study, fasting blood glucose level was found to be increased in DEHP-treated animals compared to the control. In accordance with this, it was reported that administration of DEHP increased the blood glucose level by decreasing the serum insulin level [38]. DEHP exposure during developmental period led to reduction in pancreatic insulin content, loss in b-cell mass and abnormal b-cell ultrastructure during weaning [19]. Further, the DEHP exposure downregulated the expression of key genes such as Pdx1 and Ucp2, which substantially decreases pancreatic insulin content and mitochondrial function. Elevated levels of endoplasmic reticulum stress gene markers such as Atf4, Bip, Atf6 contribute to b-cell dysfunction [19]. All these changes collectively contributed to the increase in fasting blood glucose level. Further, estradiol/testosterone ratio correlated more strongly with fasting blood glucose level. It is reported that physiological estradiol level helps in maintaining normal insulin sensitivity [39]. Increase in urinary concentration of DEHP metabolites (MEHP, MEHHP and MEOHP) in Mexican women was found to be closely associated with elevated blood glucose levels through the activation of PPARc [40]. PPARc plays a major role in adipogenesis, insulin sensitivity and lipid storage [41]. PPARc interaction with peroxisome-
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proliferating chemicals perturbs physiological processes involved in adipogenesis and lipid storage [42, 43]. Type 2 diabetes mellitus is characterized by insulin resistance, which results in low levels of insulin-induced blood glucose uptake into target tissues [44]. The heart needs a steady supply of energy. In cardiomyocytes, insulin binds to extracellular part of insulin receptor and triggers the insulin signal transduction [45]. IR, expressed at the level of 10,000–100,000 receptors per cardiomyocyte, acts as a master switch for insulin signal transduction. Alteration in the insulin receptor expression and kinase activity accounts for insulin-resistant phenotypes [46]. In the present study, DEHP treatment significantly decreased IR protein level in the plasma membrane. A previous study from our laboratory also showed a reduction in IR protein in the plasma membrane of Chang liver cells exposed to DEHP in vitro [12]. Increased level of IR mRNA and a significant reduction in IR protein levels were also recorded in gastrocnemius muscle of DEHP-treated adult male rats [13]. Since DEHP induces lipid peroxidation [47], the impaired insulin receptor protein on the plasma membrane may be due to ROS-induced loss of membrane integrity. Previous studies have shown that DEHP and its active metabolite MEHP suppressed estradiol (E2) levels (inhibiting aromatase enzyme) and lead to anovulation [34]. Therefore, the present study indicates that decrease in IR protein may be due to low estradiol level. Insulin receptor gene promoter contains consensus estrogen response elements [48], and estradiol was found to increase the expression of insulin receptors in insulin-resistant HepG2 cells [49]. Insulin receptor substrate family of proteins specifically interacts with IR phosphorylating its tyrosine residues [50]. IRS-1 is the major substrate involved in cardiac insulin signaling. In the current study, DEHP mitigated the expression of IRS-1 protein. This may be due to putative generation of reactive oxygen species (ROS) and ROSinduced degradation of IRS-1 [51]. ROS lead to increased serine phosphorylation at 636/639/307, which negatively mediated insulin signaling and lead to ubiquitin proteasomal degradation [52]. It was observed in 3T3L1 adipocytes that increased serine phosphorylation inhibits IRS tyrosine phosphorylation [53]. This may be one of the reasons for the depletion of IRS-1 tyrosine phosphorylation in the present study. Tyrosine-phosphorylated IRS-1 activates Akt (serine/ threonine kinase), which is present downstream of PI3kinase pathway. Akt is known to play a key role in insulin signaling and glucose transport [50]. In the present study, Akt protein expression was unaltered, but Akt (ser473) phosphorylation was significantly diminished in the cardiac muscle. Decrease in phosphorylation of Akt may be due to oxidative stress. Phthalates induce free radical production
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in vivo by activating NADPH oxidase complex, which generates superoxide anion and hydroxyl radical [54]. Chronic oxidative stress induces stress signaling pathways such as JNK and IKappaB kinase b (IKK) [51]. Tumor necrosis factor [55], an inflammatory cytokine produced upon oxidative stress, inhibits insulin signaling through dephosphorylation of Akt by stimulating ceramide to produce protein phosphatase in adipocytes [56]. One of the terminal events in insulin signal transduction is the translocation of GLUT4 to the plasma membrane. GLUT4 is responsible for maintaining postprandial glucose disposal, and any alteration in insulin signaling leads to insulin resistance and eventually type 2 diabetes mellitus. GLUT4 is an insulin-regulated glucose transporter found in the cardiac muscle, skeletal muscle and adipose tissue. It is completely sequestered in intracellular vesicular structures, and when stimulated with insulin, GLUT4 is translocated to the plasma membrane [57, 58, 59]. Phosphorylation of Akt activates AS160, which in turn translocates GLUT4 to the plasma membrane. In the current study, significant decrease in GLUT4 was found in the plasma membrane, whereas no significant change was observed in the cytosol. DEHP acts as a PPARa agonist. Chronic activation of PPARa is associated with downregulation of GLUT4 protein [60]. GLUT4 promoter contains critical myocyte-responsive element 2A (MEF) in the proximal region. PPARa downregulated MEF and GLUT4 promoter activity by abolishing MEF [60]. Further studies regarding the role of PPARa in the cardiac muscle would help to resolve the molecular mechanisms involved. The reduction in phosphorylation of IRS-1 and Akt in the present study may contribute to the defective translocation of GLUT4 vesicles to the plasma membrane. It has also been recorded that intact microtubule network is essential for the translocation of GLUT4 to the plasma membrane [61]. It is worth to recall the previous reports [20, 21], which have shown disruption of microtubule kinesin, connexin and tubulin in DEHP-treated cardiomyocytes. It is therefore suggested that the DEHP-induced loss of microtubule proteins may be responsible for the impaired GLUT4 translocation. Di-2-ethylhexyl phthalate treatment has lowered the glucose uptake significantly in the cardiac muscle of 100 mg treated offspring, which may be due to defective insulin signaling. This clearly shows that DEHP treatment significantly reduces GLUT4 translocation, which eventually leads to reduced glucose uptake by the cardiac muscle. The decrease in glucose uptake may partly be due to chronic activation of PPARa by DEHP [62]. PPAR is the family of nuclear hormone receptors that control genes involved in glucose and lipid metabolism. Cardiac-specific overexpression of PPARa and PGC-1a (coactivator of PPARa and PPARc) results in increased fatty acid
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oxidation with the reciprocal inhibition of glucose uptake and utilization as observed in both in vivo and in vitro studies [21, 63]. 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. In this study, the lactational exposure of DEHP reduced the oxidation of glucose compared to control in a dose-dependent manner. The dose-dependent decrease in glucose oxidation may be the consequence of impaired glucose uptake. In support of this finding, enhanced glycogenolysis and reduction in glucose-6-phosphate, fructose-6-phosphate and pyruvate, activity of pyruvate dehydrogenase complex and pyruvate kinase were observed in the liver of the DEHP-treated animal [9].
Conclusion It is concluded from this study that DEHP exposure during lactational period elevates fasting blood glucose and impairs insulin signal transduction, glucose uptake and oxidation in the cardiac muscle of F1 female progeny rat. Acknowledgments Financial assistance from UGC-SAP-DRS, UGC-ASIST and DST-FIST programs are gratefully acknowledged.
References 1. Eckardt, K., Taube, A., & Eckel, J. (2011). Obesity-associated insulin resistance in skeletal muscle: Role of lipid accumulation and physical inactivity. Reviews in Endocrine and Metabolic Disorders, 12, 163–172. 2. Hectors, T. L., Vanparys, C., van der Ven, K., Martens, G. A., Jorens, P. G., Van Gaal, L. F., et al. (2011). Environmental pollutants and type 2 diabetes: A review of mechanisms that can disrupt beta cell function. Diabetologia, 54, 1273–1290. 3. Kobayashi, K., Miyagawa, M., Wang, R. S., Suda, M., Sekiguchi, S., & Honma, T. (2006). Effects of in utero and lactational exposure to di(2-ethylhexyl)phthalate on somatic and physical development in rat offspring. Industrial Health, 44, 652–660. 4. Fay, M., Donohue, J. M., & De Rosa, C. (1999). ATSDR evaluation of health effects of chemicals. VI. Di(2-ethylhexyl)phthalate. Agency for toxic substances and disease registry. Toxicology and Industrial Health, 15, 651–746. 5. FDA. (2002). Safety assessment of Di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices, (Administration, F.a.D., ed). 6. ATSDR. (2002). Toxicological Profile for Di(2-ethylhexyl)phthalate (DEHP). Atlanta, GA: Agency for toxic substances and disease registry. Available: http://www.atsdr.cdc.gov/ toxprofiles/tp9.html (Accessed 11 August 2003). 7. NTP-CERHR. (2006). NTP-CERHR monograph on the potential human reproductive and developmental effects of Di(2-Ethylhexyl) Phthalate (DEHP), U.S. Department of Health and Human Services.
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Cardiovasc Toxicol (2014) 14:10–20 8. Desvergne, B., Feige, J. N., & Casals-Casas, C. (2009). PPARmediated activity of phthalates: A link to the obesity epidemic? Molecular and Cellular Endocrinology, 304, 43–48. 9. Martinelli, M. I., Mocchiutti, N. O., & Bernal, C. A. (2006). Dietary di(2-ethylhexyl)phthalate-impaired glucose metabolism in experimental animals. Human and Experimental Toxicology, 25, 531–538. 10. Stahlhut, R. W., van Wijngaarden, E., Dye, T. D., Cook, S., & Swan, S. H. (2007). Concentrations of urinary phthalate metabolites are associated with increased waist circumference and insulin resistance in adult U.S. males. Environmental Health Perspectives, 115, 876–882. 11. Hatch, E. E., Nelson, J. W., Qureshi, M. M., Weinberg, J., Moore, L. L., Singer, M., et al. (2008). Association of urinary phthalate metabolite concentrations with body mass index and waist circumference: A cross-sectional study of NHANES data, 1999–2002. Environmental Health: A Global Access Science Source, 7, 27. 12. Rengarajan, S., Parthasarathy, C., Anitha, M., & Balasubramanian, K. (2007). Diethylhexyl phthalate impairs insulin binding and glucose oxidation in Chang liver cells. Toxicology In Vitro: An International Journal Published in Association With BIBRA, 21, 99–102. 13. Srinivasan, C., Khan, A. I., Balaji, V., Selvaraj, J., & Balasubramanian, K. (2011). Diethyl hexyl phthalate-induced changes in insulin signaling molecules and the protective role of antioxidant vitamins in gastrocnemius muscle of adult male rat. Toxicology and Applied Pharmacology, 257, 155–164. 14. Rajesh, P., Sathish, S., Srinivasan, C., Selvaraj, J., & Balasubramanian, K. (2013). Phthalate is associated with insulin resistance in adipose tissue of male rat: Role of antioxidant vitamins. Journal of Cellular Biochemistry, 114, 558–569. 15. Calafat, A. M., Slakman, A. R., Silva, M. J., Herbert, A. R., & Needham, L. L. (2004). Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 805, 49–56. 16. Latini, G., De Felice, C., Presta, G., Del Vecchio, A., Paris, I., Ruggieri, F., et al. (2003). In utero exposure to di-(2-ethylhexyl)phthalate and duration of human pregnancy. Environmental Health Perspectives, 111, 1783–1785. 17. Latini, G. (2000). Potential hazards of exposure to di-(2-ethylhexyl)-phthalate in babies. A Review. Biology of the Neonate, 78, 269–276. 18. WHO. (2002). Global assessment of the state-of-the-science of endocrine disruptors. 19. Lin, Y., Wei, J., Li, Y., Chen, J., Zhou, Z., Song, L., et al. (2011). Developmental exposure to di(2-ethylhexyl) phthalate impairs endocrine pancreas and leads to long-term adverse effects on glucose homeostasis in the rat. American Journal of Physiology. Endocrinology and Metabolism, 301, E527–E538. 20. Gillum, N., Karabekian, Z., Swift, L. M., Brown, R. P., Kay, M. W., & Sarvazyan, N. (2009). Clinically relevant concentrations of di (2-ethylhexyl) phthalate (DEHP) uncouple cardiac syncytium. Toxicology and Applied Pharmacology, 236, 25–38. 21. Posnack, N. G., Lee, N. H., Brown, R., & Sarvazyan, N. (2011). Gene expression profiling of DEHP-treated cardiomyocytes reveals potential causes of phthalate arrhythmogenicity. Toxicology, 279, 54–64. 22. Johnson, A. D., & Turner, P. C. (1971). Epididymal carbohydrate metabolism. I. Glucose-1-14-C and glucose-6-14-C metabolism by mouse, rat and rabbit tissues. Comparative Biochemistry and Physiology A, Comparative Physiology, 39, 599–604. 23. Kraft, L. A., & Johnson, A. D. (1972). Epididymal carbohydrate metabolism. II. Substrates and pathway utilization of caput and cauda epididymal tissue from the rabbit, rat and mouse.
Cardiovasc Toxicol (2014) 14:10–20
24.
25.
26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39. 40.
41.
Comparative Biochemistry and Physiology B, Comparative Biochemistry, 42, 451–461. Ishizuka, T., Kajita, K., Miura, A., Ishizawa, M., Kanoh, Y., Itaya, S., et al. (1999). DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase. The American Journal of Physiology, 276, E196–E204. Dombrowski, L., Roy, D., Marcotte, B., & Marette, A. (1996). A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle. The American journal of physiology, 270, E667–E676. Kristiansen, S., Nielsen, J. N., Bourgoin, S., Klip, A., Franco, M., & Richter, E. A. (2001). GLUT-4 translocation in skeletal muscle studied with a cell-free assay: Involvement of phospholipase D. American Journal of Physiology. Endocrinology and Metabolism, 281, E608–E618. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Latini, G., De Felice, C., & Verrotti, A. (2004). Plasticizers, infant nutrition and reproductive health. Reproductive Toxicology, 19, 27–33. Hauser, R., & Calafat, A. M. (2005). Phthalates and human health. Occupational and Environmental Medicine, 62, 806–818. Faouzi, M. A., Dine, T., Gressier, B., Kambia, K., Luyckx, M., Pagniez, D., et al. (1999). Exposure of hemodialysis patients to di-2-ethylhexyl phthalate. International Journal of Pharmaceutics, 180, 113–121. Shneider, B., Schena, J., Truog, R., Jacobson, M., & Kevy, S. (1989). Exposure to di(2-ethylhexyl)phthalate in infants receiving extracorporeal membrane oxygenation. The New England Journal of Medicine, 320, 1563. Davis, B. J., Maronpot, R. R., & Heindel, J. J. (1994). Di-(2ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicology and Applied Pharmacology, 128, 216–223. Treinen, K. A., Dodson, W. C., & Heindel, J. J. (1990). Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulosa cell cultures. Toxicology and Applied Pharmacology, 106, 334–340. Young, L. H. (2010). Diet-induced obesity obstructs insulin signaling in the heart. American Journal of Physiology. Heart and Circulatory Physiology, 298, H306–H307. Belke, D. D., Betuing, S., Tuttle, M. J., Graveleau, C., Young, M. E., Pham, M., et al. (2002). Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression. The Journal of Clinical Investigation, 109, 629–639. Muniyappa, R., Montagnani, M., Koh, K. K., & Quon, M. J. (2007). Cardiovascular actions of insulin. Endocrine Reviews, 28, 463–491. Gayathri, N. S., Dhanya, C. R., Indu, A. R., & Kurup, P. A. (2004). Changes in some hormones by low doses of di (2-ethyl hexyl) phthalate (DEHP), a commonly used plasticizer in PVC blood storage bags & medical tubing. The Indian Journal of Medical Research, 119, 139–144. Livingstone, C., & Collison, M. (2002). Sex steroids and insulin resistance. Clinical Science, 102, 151–166. Svensson, K., Hernandez-Ramirez, R. U., Burguete-Garcia, A., Cebrian, M. E., Calafat, A. M., Needham, L. L., et al. (2011). Phthalate exposure associated with self-reported diabetes among Mexican women. Environmental Research, 111, 792–796. Ferre, P. (2004). The biology of peroxisome proliferator-activated receptors: Relationship with lipid metabolism and insulin sensitivity. Diabetes, 53(Suppl 1), S43–S50.
19 42. Brun, R. P., Kim, J. B., Hu, E., & Spiegelman, B. M. (1997). Peroxisome proliferator-activated receptor gamma and the control of adipogenesis. Current Opinion in Lipidology, 8, 212–218. 43. Tontonoz, P., Nagy, L., Alvarez, J. G., Thomazy, V. A., & Evans, R. M. (1998). PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell, 93, 241–252. 44. Batista, T. M., Alonso-Magdalena, P., Vieira, E., Amaral, M. E., Cederroth, C. R., Nef, S., et al. (2012). Short-term treatment with bisphenol-A leads to metabolic abnormalities in adult male mice. PLoS One, 7, e33814. 45. Desrois, M., Sidell, R. J., Gauguier, D., King, L. M., Radda, G. K., & Clarke, K. (2004). Initial steps of insulin signaling and glucose transport are defective in the type 2 diabetic rat heart. Cardiovascular Research, 61, 288–296. 46. Pessin, J. E., & Saltiel, A. R. (2000). Signaling pathways in insulin action: Molecular targets of insulin resistance. The Journal of Clinical Investigation, 106, 165–169. 47. Santhosh, A., Nair, K. G., Arun, P., Deepadevi, K. V., Manojkumar, V., Lakshmi, L. R., et al. (1998). Effect of DEHP [di-(2ethyl hexyl) phthalate] on lipid peroxidation in liver in rats and in primary cultures of rat hepatocytes. The Indian Journal of Medical Research, 108, 17–23. 48. Garcia-Arencibia, M., Davila, N., Campion, J., Carmen Carranza, M., & Calle, C. (2005). Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter. The Journal of Steroid Biochemistry and Molecular Biology, 94, 1–14. 49. Xie, P., Liu, M. L., Gu, Y. P., Lu, J., Xu, X., Zeng, W. M., et al. (2003). Oestrogen improves glucose metabolism and insulin signal transduction in HepG2 cells. Clinical and Experimental Pharmacology and Physiology, 30, 643–648. 50. Khan, A. H., & Pessin, J. E. (2002). Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways. Diabetologia, 45, 1475–1483. 51. Kaneto, H., Katakami, N., Matsuhisa, M., & Matsuoka, T. A. (2010). Role of reactive oxygen species in the progression of type 2 diabetes and atherosclerosis. Mediators of Inflammation, 2010, 453892. 52. Potashnik, R., Bloch-Damti, A., Bashan, N., & Rudich, A. (2003). IRS1 degradation and increased serine phosphorylation cannot predict the degree of metabolic insulin resistance induced by oxidative stress. Diabetologia, 46, 639–648. 53. Pirola, L., Johnston, A. M., & Van Obberghen, E. (2004). Modulation of insulin action. Diabetologia, 47, 170–184. 54. Rusyn, I., Kadiiska, M. B., Dikalova, A., Kono, H., Yin, M., Tsuchiya, K., et al. (2001). Phthalates rapidly increase production of reactive oxygen species in vivo: Role of Kupffer cells. Molecular Pharmacology, 59, 744–750. 55. Peraldi, P., Xu, M., & Spiegelman, B. M. (1997). Thiazolidinediones block tumor necrosis factor-alpha-induced inhibition of insulin signaling. The Journal of Clinical Investigation, 100, 1863–1869. 56. Teruel, T., Hernandez, R., & Lorenzo, M. (2001). Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes, 50, 2563–2571. 57. Slot, J. W., Geuze, H. J., Gigengack, S., James, D. E., & Lienhard, G. E. (1991). Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proceedings of the National Academy of Sciences of the United States of America, 88, 7815–7819. 58. Uphues, I., Kolter, T., Goud, B., & Eckel, J. (1995). Failure of insulin-regulated recruitment of the glucose transporter GLUT4 in cardiac muscle of obese Zucker rats is associated with alterations of small-molecular-mass GTP-binding proteins. The Biochemical Journal, 311(Pt 1), 161–166.
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20 59. Russell, R. R, 3rd, Bergeron, R., Shulman, G. I., & Young, L. H. (1999). Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. The American Journal of Physiology, 277, H643–H649. 60. Finck, B. N., Bernal-Mizrachi, C., Han, D. H., Coleman, T., Sambandam, N., LaRiviere, L. L., et al. (2005). A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes. Cell Metabolism, 1, 133–144. 61. Lizunov, V. A., Matsumoto, H., Zimmerberg, J., Cushman, S. W., & Frolov, V. A. (2005). Insulin stimulates the halting, tethering,
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Cardiovasc Toxicol (2014) 14:10–20 and fusion of mobile GLUT4 vesicles in rat adipose cells. The Journal of Cell Biology, 169, 481–489. 62. Lapinskas, P. J., Brown, S., Leesnitzer, L. M., Blanchard, S., Swanson, C., Cattley, R. C., et al. (2005). Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver. Toxicology, 207, 149–163. 63. Park, S. Y., Cho, Y. R., Kim, H. J., Higashimori, T., Danton, C., Lee, M. K., et al. (2005). Unraveling the temporal pattern of dietinduced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice. Diabetes, 54, 3530–3540.
Lactational Exposure of Phthalate Impairs Insulin Signaling in the Cardiac Muscle of F1 Female Albino Rats Viswanathan Mangala Priya • Chinnaiyan Mayilvanan Narasimhan Akilavalli • Parsanathan Rajesh • Karundevi Balasubramanian
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Published online: 3 December 2013 Ó Springer Science+Business Media New York 2013
Abstract Di-2-ethylhexyl phthalate (DEHP), a ubiquitous endocrine disruptor and plasticizer of polyvinyl chloride, is being used in the manufacture of consumer and medical products as well as in children’s toys. Fetuses and newborns are more sensitive to endocrine disruption. DEHP is a lipophilic substance, which could easily be transferred to the developing offspring through placenta or breast milk. DEHP alters the metabolism of the endocrine organs, which leads to energy imbalance associated with increased risk of insulin resistance, obesity and cardiovascular disease. The heart is an insulin-responsive organ. The effect of DEHP on the cardiac muscle insulin signaling remains obscure. Since the developmental period is more vulnerable to the adverse effect of DEHP, the present study was framed to study the impact of lactational exposure of DEHP on insulin signaling molecules in the cardiac muscle of F1 progeny female albino rat (postnatal day 60). Healthy dams were treated with DEHP orally (0, 1, 10 and 100 mg/ kg body weight/day, respectively) from the postpartum day 1–21. Both low and high doses are relevant to the human exposure, and hence, both were used in this study. At a low dose (1 mg/kg body weight/day), obvious differences were observed in the fasting blood glucose and the insulin signaling molecule when compared to control. But marked differences were observed in the cardiac tissue insulin signaling molecules of animals treated with high doses. In conclusion, the DEHP treatment significantly increased the fasting blood glucose level and decreased the insulin
V. Mangala Priya C. Mayilvanan N. Akilavalli P. Rajesh K. Balasubramanian (&) Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India e-mail: [email protected]
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receptor (IR), insulin receptor substrate (IRS-1), p-IRS1Tyr632, p-AktSer473, plasma membrane glucose transporter (GLUT4), 14C-2-deoxyglucose uptake and the 14C-glucose oxidation. Conversely, Akt and GLUT4 protein in cytosol remained unaltered compared to control. Lactational exposure of DEHP impairs insulin signal transduction and glucose oxidation in the cardiac muscle of the F1 female albino rats, suggesting its possible role in the development of type 2 diabetes. Keywords DEHP Lactational exposure Cardiac muscle Insulin resistance Type 2 diabetes Abbreviations ATP Adenosine triphosphate Atf-4 Activating transcription factor 4 Atf-6 Activating transcription factor 6 Bip Binding immunoglobulin protein CPM Counts per minute FSH Follicle-stimulating hormone GLUT4 Glucose transporter 4 IR Insulin receptor IRS-1 Insulin receptor substrate 1 IU International units JNK C-Jun N-terminal kinase MEHP Mono-(-2-ethylhexyl phthalate) MEHHP Mono-(2-ethyl-5-hydroxyhexyl) phthalate MEOHP Mono-(2-ethyl-5-oxohexyl) phthalate mM Millimolar nM Nanomolar OECD Organization for economic cooperation and development Pdx1 Pancreatic duodenal homeobox-1 PPARc Peroxisome proliferator-activated receptor c PPARa Peroxisome proliferator-activated receptor a
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Ucp2 lCi
Uncoupling protein-2 Microcurie
Introduction The incidence of type 2 diabetes, obesity and insulin resistance are rising at epidemic rates over the last decade [1]. Environmental pollutants, diet and genetic factors play an important role in the incidence of type 2 diabetes [2]. Endocrine disruptors are a heterogeneous group of chemicals found in the environment and consumer products, which are used broadly. In recent years, there has been a growing concern over the possibility of hormonal disruption even at low levels of exposure. Di-2 (ethyl hexyl phthalate) (C24H38O4), which is a ubiquitous endocrine disruptor, is used as a plasticizer in the production of polyvinyl chloride (PVC) products to impart structural flexibility [3]. Flexible material containing DEHP is used in variety of consumer products such as building materials, car products, clothing, food packaging and children’s products [4]. The general human exposure is found to be 3–30 lg/kg body weight/day. DEHP is not covalently bound to the PVC polymer, so it can easily outgas into the environment [5]. In addition to general human exposure, there is an increased risk of occupational exposure. Occupational Safety and Health Administration (OSHA) has set the permissible concentration of DEHP at workplace as 5 mg/m3 in air [6]. Also, inhalation exposure of DEHP is found as 700 lg/kg body weight/day based on workplace standards. Apart from occupational exposure, a significant exposure to DEHP occurs through medical devices. Production of DEHP amounts to approximately 2 million tons per year [7]. Evidences have accumulated that DEHP has a negative impact on the male and female reproductive functions. Additionally, DEHP has drawn considerable interest because of its contribution to energy imbalance and metabolic disorders [8]. DEHP administration resulted in impaired hepatic glycogenesis and glycogenolysis [9]. Epidemiological studies show a positive correlation between increased phthalate metabolites in urine and abdominal obesity and insulin resistance in adult males [10, 11]. Previous results confirm the adverse effects of DEHP on insulin receptor and glucose oxidation in cultured Chang liver cells [12]. Studies from our laboratory suggest that DEHP disrupts insulin signal transduction and favors glucose intolerance [13, 14]. Di-2-ethylhexyl phthalate is able to cross the placenta and also pass into the breast milk [15, 16], resulting in significant risk for the developing fetuses and newborns [17]. Neonates are more receptive to DEHP than adults as
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they are more susceptible to endocrine disruption. Even small changes in the endogenous hormone level result in structural and functional changes permanently [18]. It has been reported that developmental DEHP exposure disrupted the pancreas and altered glucose homeostasis in the whole body [19]. Although lactational exposure of DEHP results in various reproductive abnormalities, its effect on insulin signal transduction is too early to be commented on. The heart is an energy-consuming organ that needs to maintain its intracellular ATP level for the myocardial contraction/relaxation cycle. Insulin plays a key role in the regulation of cardiovascular metabolism and function, by modulating glucose transport, glycolysis and lipid metabolism. DEHP at clinically relevant concentrations impaired the mechanical and electrical behavior of cells [20]. In correlation, a study on the effect of DEHP on the cardiac muscle showed an increase in fatty acid oxidation reportedly with decreased glucose oxidation by downregulating the enzymes associated with it [21]. Despite cardiovascular disease being the leading cause of death in type 2 diabetic patients with a twofold increased risk of congestive heart failure, insulin signaling in heart has received only little attention. Therefore, the current investigation has been designed to study the effect of DEHP on insulin signaling.
Materials and Methods Chemicals and Reagents Di-2-ethylhexyl phthalate was purchased from Sigma Chemicals Company (St. Louis, USA). All other chemicals and reagents used in the present study were of analytical grade (AR), and they were purchased from Amersham Biosciences Ltd. (UK) and Sisco Research Laboratories, Mumbai. Blood glucose strips were purchased from ACON laboratories, Inc., San Diego, USA. IR, IRS-1, pIRS-1tyr632, Akt, pAktser473, GLUT4 antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. bactin antibody was purchased from Sigma Chemicals Company (St. Louis, USA). 14C-glucose and 14C-2deoxyglucose were purchased from the Board of Radiation and Isotope Technology (Mumbai, India). Animals Healthy adult female albino rats of Wistar strain (Rattus norvegicus), weighing 100–120 g, were used in the present study. The animals were housed in a clean polypropylene cage, maintained in an air-conditioned animal house (12-h light/dark and temperature 25 – 2 °C), fed with standard rat-pelleted diet (Lipton India Ltd., Mumbai, India), and clean drinking water was made available ad libitum.
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Animals were maintained as per the National Guidelines and Protocols approved by the Institutional Animal Ethical Committee. Dose Selection and Treatment Nulliparous rats were mated, and presence of sperm was taken as gestation day 0 (GD 0). After spontaneous delivery, postnatal day 1, the litters were culled to six numbers per each dam (3 dams/group) and balanced for sex to the extent possible. The litters were culled according to OECD guidelines. The dams received phthalate (DEHP) through oral gavage (vehicle—olive oil) as shown below throughout the lactational period: Group I: control (vehicle treated), Group II: 1 mg/kg body weight/day, Group III: 10 mg/kg body weight/day and Group IV: 100 mg/kg body weight/day. Dose ranges used in the present study correspond with normal to occupational human exposure. On the 58th day, the animals were kept for overnight fasting and their blood was collected for glucose estimation. All the female progeny were perfused and killed on postnatal day 60. The cardiac muscle was dissected out, washed in icecold physiological saline repeatedly and used for the assay of various parameters.
added to the ampoule 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 was 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. [14C]-2-Deoxy-D-glucose Uptake 14
C-glucose uptake in the cardiac tissue was estimated by the standard method [24]. Briefly, 100 mg of tissue was incubated in 5 ml of DMEM with 5 mM glucose for 30 min under the condition of 95 % O2, 5 % CO2. After this, the tissue was washed and incubated in glucose-free medium for 30 min with or without 100 nM of insulin. Glucose uptake was initiated by the addition of 2 ll of 0.05 lCi 14C-2-deoxyglucose. At the end of 10 min of incubation, the tissue was removed, rapidly rinsed in isotope-free medium, homogenized in 2 ml of 5 % trichloroacetic acid and placed in the scintillation vials containing 5 ml of scintillation fluid and counted in a Beta counter. Results are expressed as CPM of 14C-2-deoxyglucose/ 100 mg tissue. Protein Expression Analysis
Methods Sample Preparation Estimation of Blood Glucose Blood glucose was estimated using On-Call Plus Blood Glucose Test Strips method (ACON Laboratories, Inc., San Diego, USA.). On the 59th day, fasting blood glucose was estimated in animals of all the groups. Blood sample for glucose estimation was collected from the rat’s tail tip. Results were obtained on the meter display window as mg/ dl. Determination of 14
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C-Glucose Oxidation
C-glucose oxidation in the cardiac tissue was estimated by the standard method [22], [23]. Briefly, 10 mg of the cardiac tissue was weighed and placed in a 2-ml ampoule containing 170 ll of DMEM (pH 7.4), 10 IU penicillin in 10 ll of DMEM and 0.5 lCi of 14C-glucose. Then, the ampoules were aerated with a gas mixture (5 % CO2 and 95 % air) for 30 s and tightly closed 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 traps were replaced every 2 h. After removing the second trap, 0.1 ml of 1 N H2SO4 was
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Plasma membrane and cytosolic fractions from the cardiac tissue of the control and experimental animals were prepared by the standard methods [25], [26]. The gradient was prepared by progressively layering less dense sucrose solution upon one another. Briefly, sucrose solutions were added into the polyallomer tube (ultracentrifugation tube) slowly and steadily, starting with 35 % solution. First, 2 ml of the 35 % solution had drained into the tube, and then, 2 ml of 32 % solution could be loaded on top of the 35 % solution. This procedure was continued with 1 ml of 25 % solution. There was enough space left at the top of the tube upon which the 0.5 ml of sample was loaded. Subcellular Fractionations The cardiac tissue from the control and experimental animals was simultaneously processed for the preparation of different fractions. All the 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 (1 g/1.5 ml of buffer A) using a polytron-equipped homogenizer at a precise low setting. The resulting homogenate was centrifuged at 1,3009g for 10 min. The supernatant was centrifuged at 1,
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90,0009g for 1 h. 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, 50,0009g for 16 h. Plasma membrane from 25 to 32 interfaces was recovered, diluted with buffer B and centrifuged at 1, 90,0009g for 1 h. The plasma membrane fraction (pellet) was resuspended in buffer A and kept at -80 °C until later used for IR and GLUT4 protein expression analysis. Protein concentration in the sample was determined prior to Western blot analysis.
peroxidase-conjugated rabbit antimouse or goat antirabbit antibodies (which were diluted to 1: 5,000) (GeNei, Bangalore, India). The specific signals were detected with enhanced chemiluminescence detection system (Thermo Fisher Scientific Inc., USA). The protein bands were captured using ChemiDoc and quantified by Quantity One image analysis (Bio-Rad Laboratories, CA). Later the membranes were incubated in stripping buffer at 500c for 30 min. After this, the membranes were reprobed using a b-actin antibody (1:5,000 dilution). As the invariant control, the present study used rat b-actin.
Estimation of Protein
Statistical Analysis
Protein concentration was determined as per the method [27] with bovine serum albumin (BSA) as the standard. Ten microliters of the sample was taken in a clean test tube and made up to 1 ml with distilled water. To this, 5 ml of alkaline copper reagent was added. The contents were mixed well and allowed to stand at room temperature for 10 min. Five hundred microliters of 1 N Folin–Ciocalteu reagent was then added and immediately mixed well. After 20 min, the intensity of the blue color developed was read at 720 nm against blank. For plotting the standard graph, a set of standards (25, 50, 75, 100 and 125 lg) were taken in a series of test tubes and made up to 1 ml with distilled water and processed as that of the samples. The standard graph was drawn by plotting the concentration of standards on the x-axis and the optical density on the y-axis. The concentration of protein in the sample was calculated by referring the standard curve and expressed as lg/mg tissue.
The data were subjected to one-way analysis of variance (ANOVA), followed by Student’s Newman-Keuls (SNK) test to assess the significance between mean values of the control and experimental groups. The data were expressed as mean – standard error of mean (SEM), and p values \0.05 were considered as significant. This was carried out using SPSS package for Windows.
Results Fasting Blood Glucose Fasting blood glucose level significantly increased in treated groups compared to control (Fig. 1). DEHP treatment profoundly increased the fasting blood glucose level. This prompted us to study the action of DEHP on insulin signaling molecules.
Western Blot Analysis Separation of Proteins Proteins were separated by SDS–Polyacrylamide gel electrophoresis by standard method [28]. Equal volume (25 lg) of samples from the cardiac tissue 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. Samples were loaded to 10 or 7 % SDS-PAGE in a Bio-Rad miniature slab gel apparatus. The lysate proteins were separated by SDS-PAGE (10 % gel) and transferred by electroblotting to polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked with 5 % nonfat dry milk and probed with primary antibodies (which were diluted to 1:1,000). Following incubation, the blot was washed 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
Fig. 1 Effect of lactational exposure of DEHP on fasting blood glucose level of female rats. After overnight, fasting blood glucose level was detected. Each bar represents mean ± SEM of 6 animals. Statistical significance at p \ 0.05, a compared with control. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
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Fig. 2 Effect of lactational exposure of DEHP on insulin receptor protein in the cardiac muscle of female rats. Total of protein was detected by Western blot analysis. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 4 Effect of lactational exposure of DEHP on phospho IRS1Tyr632 protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control, b compared with 1 mg DEHP and c compared with 10 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 3 Effect of lactational exposure of DEHP on IRS-1 protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 5 Effect of lactational exposure of DEHP on Akt protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Effect of DEHP on Insulin Signaling Molecules
IRS-1 and p-IRS-1Tyr632
Insulin Receptor
Insulin receptor substrate 1 is the major substrate involved in the cardiac muscle insulin signaling. In the present study, IRS-1 protein level was significantly decreased in 10 and 100 mg treated animals (Fig. 3) when compared to control. On the other hand, p-IRS-1Tyr632 protein level was significantly decreased in a dose-dependent manner (Fig. 4).
IR protein level was significantly decreased in the animal treated with 10 and 100 mg DEHP compared to control and 1 mg treated animals (Fig. 2). IR phosphorylation recruits IRS-1, which in turn gets phosphorylated at Tyr 632 residue.
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Fig. 6 Effect of lactational exposure of DEHP on phospho Aktser473 protein in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Fig. 7 Effect of lactational exposure of DEHP on cytosolic GLUT4 level in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Akt and p-AktSer473 Akt protein level (Fig. 5) was found to be unaltered, but interestingly p-AktSer473 protein level (Fig. 6) was decreased in a dose-dependent manner in the treated groups at par with the control. Cytosolic and Plasma Membrane GLUT4 Glucose transporter 4 protein, which is mandatory for the entry of glucose molecule, was found to be unaltered in the cytosol, whereas in the plasma membrane, GLUT4 was found to be significantly decreased in the 10 and 100 mg DEHP-treated groups compared to coeval control (Figs. 7, 8). 14
C-2-deoxyglucose Uptake and
14
C-glucose Oxidation
It is clear from the above results that the insulin signaling molecules were drastically altered in treated groups. This tempted us to study glucose uptake and oxidation. Glucose uptake and oxidation are necessary for the maintenance of glucose homeostasis. DEHP treatment showed significant dose-dependent decrease in 14C-2-deoxyglucose uptake in treated animals compared to control (Fig. 9). DEHP treatment also showed a significant decrease in 14C-glucose oxidation in DEHP-treated animals compared to control (Fig. 10). Considering the fact that the rate of glucose oxidation in cells depends on its glucose uptake, the decrease in glucose oxidation may be attributed to reduced glucose uptake.
Fig. 8 Effect of lactational exposure of DEHP on plasma membrane GLUT4 level in the cardiac muscle of female rats. Each bar represents mean ± SEM of 3 observations representing six animals. Significance at p \ 0.05; a compared with control and b compared with 1 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
Discussion Di-2-ethylhexyl phthalate is one of the ubiquitous endocrine disruptors, which is used as a plasticizer of polyvinyl chloride in the manufacture of a wide variety of consumer products such as building and car products, clothing, food packaging and children’s products and in medical devices made of polyvinyl chloride [29, 30]. In the current study,
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Fig. 9 Effect of lactational exposure of DEHP on 14C-2-deoxyglucose uptake in the cardiac muscle of female rats. Each bar represents mean ± SEM of 6 animals. Significance at p \ 0.05; a compared with control, b compared with 1 mg DEHP and c compared with 10 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
DEHP at high doses (10 and 100 mg/kg body weight/day) exhibited a significant difference in insulin signaling molecules. Dose-dependent adverse effect was observed in this study. Previous report from our laboratory also showed a dose-dependent decrease of insulin signaling molecules in gastrocnemius muscle of adult male rats [13]. The doses used in the present study are relevant to general population, occupational and medical exposure. The highest estimated daily exposure through blood transfusion is 22.6 mg/kg of body weight for neonates and 8.5 mg/kg for adults. Dialysis patients received an average of 75 mg DEHP per treatment and average of 12 g over a period of 1 year [31]. During extracorporeal membrane oxygenation and blood transfusion, a patient would be exposed up to 42–140 mg/ kg body weight [32]. Thus, all the doses used in the present study are within the human exposure range. Current exposures to DEHP are high enough to cause serious concern as its widespread human exposure and adverse effects have triggered the interest of public and government alike [7]. Due to immaturity of the liver, neonates are unable to oxidize DEHP, and therefore, they are often more receptive to toxic chemicals [17]. Fetuses and newborns appear to be more sensitive to its endocrine disruption. Moreover, DEHP is found to be lipophilic and accumulates more in the adipose tissue, breast milk and amniotic fluid. Phthalates were also detected in pooled breast milk samples and in infant formula [15]. Sex steroids have a direct influence on glucose homeostasis and contribute to insulin resistance. DEHP and its active metabolite MEHP decrease estradiol level, ovulation [33], FSH-stimulated second messenger and progesterone production in cultured ovarian granulosa cells [34]. Moreover, adverse effects of developmental DEHP exposure on glucose homeostasis are much more severe in
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Fig. 10 Effect of lactational exposure of DEHP on glucose oxidation in the cardiac muscle of female rats. Each bar represents mean ± SEM of 6 animals. Significance at p \ 0.05; a compared with control, b compared with 1 mg DEHP and c compared with 10 mg DEHP. The data were subjected to one-way analysis of variance, followed by Student’s Newman–Keuls test
female rats compared to male rats because females develop glucose intolerance more rapidly [19]. Further, the heart is an insulin-responsive organ, where insulin has direct effects on glucose transport, glycolysis, glucose oxidation, glycogen synthesis, protein synthesis, cardiac growth and contractility [35]. Diabetes is also associated with profound changes in cardiac metabolism, diminished glucose uptake and oxidation [36, 37]. These findings tempted us to investigate the insulin signaling pathway in the cardiac muscle of female albino rats exposed to DEHP during developmental period. In the present study, fasting blood glucose level was found to be increased in DEHP-treated animals compared to the control. In accordance with this, it was reported that administration of DEHP increased the blood glucose level by decreasing the serum insulin level [38]. DEHP exposure during developmental period led to reduction in pancreatic insulin content, loss in b-cell mass and abnormal b-cell ultrastructure during weaning [19]. Further, the DEHP exposure downregulated the expression of key genes such as Pdx1 and Ucp2, which substantially decreases pancreatic insulin content and mitochondrial function. Elevated levels of endoplasmic reticulum stress gene markers such as Atf4, Bip, Atf6 contribute to b-cell dysfunction [19]. All these changes collectively contributed to the increase in fasting blood glucose level. Further, estradiol/testosterone ratio correlated more strongly with fasting blood glucose level. It is reported that physiological estradiol level helps in maintaining normal insulin sensitivity [39]. Increase in urinary concentration of DEHP metabolites (MEHP, MEHHP and MEOHP) in Mexican women was found to be closely associated with elevated blood glucose levels through the activation of PPARc [40]. PPARc plays a major role in adipogenesis, insulin sensitivity and lipid storage [41]. PPARc interaction with peroxisome-
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proliferating chemicals perturbs physiological processes involved in adipogenesis and lipid storage [42, 43]. Type 2 diabetes mellitus is characterized by insulin resistance, which results in low levels of insulin-induced blood glucose uptake into target tissues [44]. The heart needs a steady supply of energy. In cardiomyocytes, insulin binds to extracellular part of insulin receptor and triggers the insulin signal transduction [45]. IR, expressed at the level of 10,000–100,000 receptors per cardiomyocyte, acts as a master switch for insulin signal transduction. Alteration in the insulin receptor expression and kinase activity accounts for insulin-resistant phenotypes [46]. In the present study, DEHP treatment significantly decreased IR protein level in the plasma membrane. A previous study from our laboratory also showed a reduction in IR protein in the plasma membrane of Chang liver cells exposed to DEHP in vitro [12]. Increased level of IR mRNA and a significant reduction in IR protein levels were also recorded in gastrocnemius muscle of DEHP-treated adult male rats [13]. Since DEHP induces lipid peroxidation [47], the impaired insulin receptor protein on the plasma membrane may be due to ROS-induced loss of membrane integrity. Previous studies have shown that DEHP and its active metabolite MEHP suppressed estradiol (E2) levels (inhibiting aromatase enzyme) and lead to anovulation [34]. Therefore, the present study indicates that decrease in IR protein may be due to low estradiol level. Insulin receptor gene promoter contains consensus estrogen response elements [48], and estradiol was found to increase the expression of insulin receptors in insulin-resistant HepG2 cells [49]. Insulin receptor substrate family of proteins specifically interacts with IR phosphorylating its tyrosine residues [50]. IRS-1 is the major substrate involved in cardiac insulin signaling. In the current study, DEHP mitigated the expression of IRS-1 protein. This may be due to putative generation of reactive oxygen species (ROS) and ROSinduced degradation of IRS-1 [51]. ROS lead to increased serine phosphorylation at 636/639/307, which negatively mediated insulin signaling and lead to ubiquitin proteasomal degradation [52]. It was observed in 3T3L1 adipocytes that increased serine phosphorylation inhibits IRS tyrosine phosphorylation [53]. This may be one of the reasons for the depletion of IRS-1 tyrosine phosphorylation in the present study. Tyrosine-phosphorylated IRS-1 activates Akt (serine/ threonine kinase), which is present downstream of PI3kinase pathway. Akt is known to play a key role in insulin signaling and glucose transport [50]. In the present study, Akt protein expression was unaltered, but Akt (ser473) phosphorylation was significantly diminished in the cardiac muscle. Decrease in phosphorylation of Akt may be due to oxidative stress. Phthalates induce free radical production
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in vivo by activating NADPH oxidase complex, which generates superoxide anion and hydroxyl radical [54]. Chronic oxidative stress induces stress signaling pathways such as JNK and IKappaB kinase b (IKK) [51]. Tumor necrosis factor [55], an inflammatory cytokine produced upon oxidative stress, inhibits insulin signaling through dephosphorylation of Akt by stimulating ceramide to produce protein phosphatase in adipocytes [56]. One of the terminal events in insulin signal transduction is the translocation of GLUT4 to the plasma membrane. GLUT4 is responsible for maintaining postprandial glucose disposal, and any alteration in insulin signaling leads to insulin resistance and eventually type 2 diabetes mellitus. GLUT4 is an insulin-regulated glucose transporter found in the cardiac muscle, skeletal muscle and adipose tissue. It is completely sequestered in intracellular vesicular structures, and when stimulated with insulin, GLUT4 is translocated to the plasma membrane [57, 58, 59]. Phosphorylation of Akt activates AS160, which in turn translocates GLUT4 to the plasma membrane. In the current study, significant decrease in GLUT4 was found in the plasma membrane, whereas no significant change was observed in the cytosol. DEHP acts as a PPARa agonist. Chronic activation of PPARa is associated with downregulation of GLUT4 protein [60]. GLUT4 promoter contains critical myocyte-responsive element 2A (MEF) in the proximal region. PPARa downregulated MEF and GLUT4 promoter activity by abolishing MEF [60]. Further studies regarding the role of PPARa in the cardiac muscle would help to resolve the molecular mechanisms involved. The reduction in phosphorylation of IRS-1 and Akt in the present study may contribute to the defective translocation of GLUT4 vesicles to the plasma membrane. It has also been recorded that intact microtubule network is essential for the translocation of GLUT4 to the plasma membrane [61]. It is worth to recall the previous reports [20, 21], which have shown disruption of microtubule kinesin, connexin and tubulin in DEHP-treated cardiomyocytes. It is therefore suggested that the DEHP-induced loss of microtubule proteins may be responsible for the impaired GLUT4 translocation. Di-2-ethylhexyl phthalate treatment has lowered the glucose uptake significantly in the cardiac muscle of 100 mg treated offspring, which may be due to defective insulin signaling. This clearly shows that DEHP treatment significantly reduces GLUT4 translocation, which eventually leads to reduced glucose uptake by the cardiac muscle. The decrease in glucose uptake may partly be due to chronic activation of PPARa by DEHP [62]. PPAR is the family of nuclear hormone receptors that control genes involved in glucose and lipid metabolism. Cardiac-specific overexpression of PPARa and PGC-1a (coactivator of PPARa and PPARc) results in increased fatty acid
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oxidation with the reciprocal inhibition of glucose uptake and utilization as observed in both in vivo and in vitro studies [21, 63]. 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. In this study, the lactational exposure of DEHP reduced the oxidation of glucose compared to control in a dose-dependent manner. The dose-dependent decrease in glucose oxidation may be the consequence of impaired glucose uptake. In support of this finding, enhanced glycogenolysis and reduction in glucose-6-phosphate, fructose-6-phosphate and pyruvate, activity of pyruvate dehydrogenase complex and pyruvate kinase were observed in the liver of the DEHP-treated animal [9].
Conclusion It is concluded from this study that DEHP exposure during lactational period elevates fasting blood glucose and impairs insulin signal transduction, glucose uptake and oxidation in the cardiac muscle of F1 female progeny rat. Acknowledgments Financial assistance from UGC-SAP-DRS, UGC-ASIST and DST-FIST programs are gratefully acknowledged.
References 1. Eckardt, K., Taube, A., & Eckel, J. (2011). Obesity-associated insulin resistance in skeletal muscle: Role of lipid accumulation and physical inactivity. Reviews in Endocrine and Metabolic Disorders, 12, 163–172. 2. Hectors, T. L., Vanparys, C., van der Ven, K., Martens, G. A., Jorens, P. G., Van Gaal, L. F., et al. (2011). Environmental pollutants and type 2 diabetes: A review of mechanisms that can disrupt beta cell function. Diabetologia, 54, 1273–1290. 3. Kobayashi, K., Miyagawa, M., Wang, R. S., Suda, M., Sekiguchi, S., & Honma, T. (2006). Effects of in utero and lactational exposure to di(2-ethylhexyl)phthalate on somatic and physical development in rat offspring. Industrial Health, 44, 652–660. 4. Fay, M., Donohue, J. M., & De Rosa, C. (1999). ATSDR evaluation of health effects of chemicals. VI. Di(2-ethylhexyl)phthalate. Agency for toxic substances and disease registry. Toxicology and Industrial Health, 15, 651–746. 5. FDA. (2002). Safety assessment of Di(2-ethylhexyl)phthalate (DEHP) released from PVC medical devices, (Administration, F.a.D., ed). 6. ATSDR. (2002). Toxicological Profile for Di(2-ethylhexyl)phthalate (DEHP). Atlanta, GA: Agency for toxic substances and disease registry. Available: http://www.atsdr.cdc.gov/ toxprofiles/tp9.html (Accessed 11 August 2003). 7. NTP-CERHR. (2006). NTP-CERHR monograph on the potential human reproductive and developmental effects of Di(2-Ethylhexyl) Phthalate (DEHP), U.S. Department of Health and Human Services.
123
Cardiovasc Toxicol (2014) 14:10–20 8. Desvergne, B., Feige, J. N., & Casals-Casas, C. (2009). PPARmediated activity of phthalates: A link to the obesity epidemic? Molecular and Cellular Endocrinology, 304, 43–48. 9. Martinelli, M. I., Mocchiutti, N. O., & Bernal, C. A. (2006). Dietary di(2-ethylhexyl)phthalate-impaired glucose metabolism in experimental animals. Human and Experimental Toxicology, 25, 531–538. 10. Stahlhut, R. W., van Wijngaarden, E., Dye, T. D., Cook, S., & Swan, S. H. (2007). Concentrations of urinary phthalate metabolites are associated with increased waist circumference and insulin resistance in adult U.S. males. Environmental Health Perspectives, 115, 876–882. 11. Hatch, E. E., Nelson, J. W., Qureshi, M. M., Weinberg, J., Moore, L. L., Singer, M., et al. (2008). Association of urinary phthalate metabolite concentrations with body mass index and waist circumference: A cross-sectional study of NHANES data, 1999–2002. Environmental Health: A Global Access Science Source, 7, 27. 12. Rengarajan, S., Parthasarathy, C., Anitha, M., & Balasubramanian, K. (2007). Diethylhexyl phthalate impairs insulin binding and glucose oxidation in Chang liver cells. Toxicology In Vitro: An International Journal Published in Association With BIBRA, 21, 99–102. 13. Srinivasan, C., Khan, A. I., Balaji, V., Selvaraj, J., & Balasubramanian, K. (2011). Diethyl hexyl phthalate-induced changes in insulin signaling molecules and the protective role of antioxidant vitamins in gastrocnemius muscle of adult male rat. Toxicology and Applied Pharmacology, 257, 155–164. 14. Rajesh, P., Sathish, S., Srinivasan, C., Selvaraj, J., & Balasubramanian, K. (2013). Phthalate is associated with insulin resistance in adipose tissue of male rat: Role of antioxidant vitamins. Journal of Cellular Biochemistry, 114, 558–569. 15. Calafat, A. M., Slakman, A. R., Silva, M. J., Herbert, A. R., & Needham, L. L. (2004). Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 805, 49–56. 16. Latini, G., De Felice, C., Presta, G., Del Vecchio, A., Paris, I., Ruggieri, F., et al. (2003). In utero exposure to di-(2-ethylhexyl)phthalate and duration of human pregnancy. Environmental Health Perspectives, 111, 1783–1785. 17. Latini, G. (2000). Potential hazards of exposure to di-(2-ethylhexyl)-phthalate in babies. A Review. Biology of the Neonate, 78, 269–276. 18. WHO. (2002). Global assessment of the state-of-the-science of endocrine disruptors. 19. Lin, Y., Wei, J., Li, Y., Chen, J., Zhou, Z., Song, L., et al. (2011). Developmental exposure to di(2-ethylhexyl) phthalate impairs endocrine pancreas and leads to long-term adverse effects on glucose homeostasis in the rat. American Journal of Physiology. Endocrinology and Metabolism, 301, E527–E538. 20. Gillum, N., Karabekian, Z., Swift, L. M., Brown, R. P., Kay, M. W., & Sarvazyan, N. (2009). Clinically relevant concentrations of di (2-ethylhexyl) phthalate (DEHP) uncouple cardiac syncytium. Toxicology and Applied Pharmacology, 236, 25–38. 21. Posnack, N. G., Lee, N. H., Brown, R., & Sarvazyan, N. (2011). Gene expression profiling of DEHP-treated cardiomyocytes reveals potential causes of phthalate arrhythmogenicity. Toxicology, 279, 54–64. 22. Johnson, A. D., & Turner, P. C. (1971). Epididymal carbohydrate metabolism. I. Glucose-1-14-C and glucose-6-14-C metabolism by mouse, rat and rabbit tissues. Comparative Biochemistry and Physiology A, Comparative Physiology, 39, 599–604. 23. Kraft, L. A., & Johnson, A. D. (1972). Epididymal carbohydrate metabolism. II. Substrates and pathway utilization of caput and cauda epididymal tissue from the rabbit, rat and mouse.
Cardiovasc Toxicol (2014) 14:10–20
24.
25.
26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39. 40.
41.
Comparative Biochemistry and Physiology B, Comparative Biochemistry, 42, 451–461. Ishizuka, T., Kajita, K., Miura, A., Ishizawa, M., Kanoh, Y., Itaya, S., et al. (1999). DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase. The American Journal of Physiology, 276, E196–E204. Dombrowski, L., Roy, D., Marcotte, B., & Marette, A. (1996). A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle. The American journal of physiology, 270, E667–E676. Kristiansen, S., Nielsen, J. N., Bourgoin, S., Klip, A., Franco, M., & Richter, E. A. (2001). GLUT-4 translocation in skeletal muscle studied with a cell-free assay: Involvement of phospholipase D. American Journal of Physiology. Endocrinology and Metabolism, 281, E608–E618. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Latini, G., De Felice, C., & Verrotti, A. (2004). Plasticizers, infant nutrition and reproductive health. Reproductive Toxicology, 19, 27–33. Hauser, R., & Calafat, A. M. (2005). Phthalates and human health. Occupational and Environmental Medicine, 62, 806–818. Faouzi, M. A., Dine, T., Gressier, B., Kambia, K., Luyckx, M., Pagniez, D., et al. (1999). Exposure of hemodialysis patients to di-2-ethylhexyl phthalate. International Journal of Pharmaceutics, 180, 113–121. Shneider, B., Schena, J., Truog, R., Jacobson, M., & Kevy, S. (1989). Exposure to di(2-ethylhexyl)phthalate in infants receiving extracorporeal membrane oxygenation. The New England Journal of Medicine, 320, 1563. Davis, B. J., Maronpot, R. R., & Heindel, J. J. (1994). Di-(2ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicology and Applied Pharmacology, 128, 216–223. Treinen, K. A., Dodson, W. C., & Heindel, J. J. (1990). Inhibition of FSH-stimulated cAMP accumulation and progesterone production by mono(2-ethylhexyl) phthalate in rat granulosa cell cultures. Toxicology and Applied Pharmacology, 106, 334–340. Young, L. H. (2010). Diet-induced obesity obstructs insulin signaling in the heart. American Journal of Physiology. Heart and Circulatory Physiology, 298, H306–H307. Belke, D. D., Betuing, S., Tuttle, M. J., Graveleau, C., Young, M. E., Pham, M., et al. (2002). Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression. The Journal of Clinical Investigation, 109, 629–639. Muniyappa, R., Montagnani, M., Koh, K. K., & Quon, M. J. (2007). Cardiovascular actions of insulin. Endocrine Reviews, 28, 463–491. Gayathri, N. S., Dhanya, C. R., Indu, A. R., & Kurup, P. A. (2004). Changes in some hormones by low doses of di (2-ethyl hexyl) phthalate (DEHP), a commonly used plasticizer in PVC blood storage bags & medical tubing. The Indian Journal of Medical Research, 119, 139–144. Livingstone, C., & Collison, M. (2002). Sex steroids and insulin resistance. Clinical Science, 102, 151–166. Svensson, K., Hernandez-Ramirez, R. U., Burguete-Garcia, A., Cebrian, M. E., Calafat, A. M., Needham, L. L., et al. (2011). Phthalate exposure associated with self-reported diabetes among Mexican women. Environmental Research, 111, 792–796. Ferre, P. (2004). The biology of peroxisome proliferator-activated receptors: Relationship with lipid metabolism and insulin sensitivity. Diabetes, 53(Suppl 1), S43–S50.
19 42. Brun, R. P., Kim, J. B., Hu, E., & Spiegelman, B. M. (1997). Peroxisome proliferator-activated receptor gamma and the control of adipogenesis. Current Opinion in Lipidology, 8, 212–218. 43. Tontonoz, P., Nagy, L., Alvarez, J. G., Thomazy, V. A., & Evans, R. M. (1998). PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell, 93, 241–252. 44. Batista, T. M., Alonso-Magdalena, P., Vieira, E., Amaral, M. E., Cederroth, C. R., Nef, S., et al. (2012). Short-term treatment with bisphenol-A leads to metabolic abnormalities in adult male mice. PLoS One, 7, e33814. 45. Desrois, M., Sidell, R. J., Gauguier, D., King, L. M., Radda, G. K., & Clarke, K. (2004). Initial steps of insulin signaling and glucose transport are defective in the type 2 diabetic rat heart. Cardiovascular Research, 61, 288–296. 46. Pessin, J. E., & Saltiel, A. R. (2000). Signaling pathways in insulin action: Molecular targets of insulin resistance. The Journal of Clinical Investigation, 106, 165–169. 47. Santhosh, A., Nair, K. G., Arun, P., Deepadevi, K. V., Manojkumar, V., Lakshmi, L. R., et al. (1998). Effect of DEHP [di-(2ethyl hexyl) phthalate] on lipid peroxidation in liver in rats and in primary cultures of rat hepatocytes. The Indian Journal of Medical Research, 108, 17–23. 48. Garcia-Arencibia, M., Davila, N., Campion, J., Carmen Carranza, M., & Calle, C. (2005). Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter. The Journal of Steroid Biochemistry and Molecular Biology, 94, 1–14. 49. Xie, P., Liu, M. L., Gu, Y. P., Lu, J., Xu, X., Zeng, W. M., et al. (2003). Oestrogen improves glucose metabolism and insulin signal transduction in HepG2 cells. Clinical and Experimental Pharmacology and Physiology, 30, 643–648. 50. Khan, A. H., & Pessin, J. E. (2002). Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways. Diabetologia, 45, 1475–1483. 51. Kaneto, H., Katakami, N., Matsuhisa, M., & Matsuoka, T. A. (2010). Role of reactive oxygen species in the progression of type 2 diabetes and atherosclerosis. Mediators of Inflammation, 2010, 453892. 52. Potashnik, R., Bloch-Damti, A., Bashan, N., & Rudich, A. (2003). IRS1 degradation and increased serine phosphorylation cannot predict the degree of metabolic insulin resistance induced by oxidative stress. Diabetologia, 46, 639–648. 53. Pirola, L., Johnston, A. M., & Van Obberghen, E. (2004). Modulation of insulin action. Diabetologia, 47, 170–184. 54. Rusyn, I., Kadiiska, M. B., Dikalova, A., Kono, H., Yin, M., Tsuchiya, K., et al. (2001). Phthalates rapidly increase production of reactive oxygen species in vivo: Role of Kupffer cells. Molecular Pharmacology, 59, 744–750. 55. Peraldi, P., Xu, M., & Spiegelman, B. M. (1997). Thiazolidinediones block tumor necrosis factor-alpha-induced inhibition of insulin signaling. The Journal of Clinical Investigation, 100, 1863–1869. 56. Teruel, T., Hernandez, R., & Lorenzo, M. (2001). Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes, 50, 2563–2571. 57. Slot, J. W., Geuze, H. J., Gigengack, S., James, D. E., & Lienhard, G. E. (1991). Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proceedings of the National Academy of Sciences of the United States of America, 88, 7815–7819. 58. Uphues, I., Kolter, T., Goud, B., & Eckel, J. (1995). Failure of insulin-regulated recruitment of the glucose transporter GLUT4 in cardiac muscle of obese Zucker rats is associated with alterations of small-molecular-mass GTP-binding proteins. The Biochemical Journal, 311(Pt 1), 161–166.
123
20 59. Russell, R. R, 3rd, Bergeron, R., Shulman, G. I., & Young, L. H. (1999). Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. The American Journal of Physiology, 277, H643–H649. 60. Finck, B. N., Bernal-Mizrachi, C., Han, D. H., Coleman, T., Sambandam, N., LaRiviere, L. L., et al. (2005). A potential link between muscle peroxisome proliferator- activated receptor-alpha signaling and obesity-related diabetes. Cell Metabolism, 1, 133–144. 61. Lizunov, V. A., Matsumoto, H., Zimmerberg, J., Cushman, S. W., & Frolov, V. A. (2005). Insulin stimulates the halting, tethering,
123
Cardiovasc Toxicol (2014) 14:10–20 and fusion of mobile GLUT4 vesicles in rat adipose cells. The Journal of Cell Biology, 169, 481–489. 62. Lapinskas, P. J., Brown, S., Leesnitzer, L. M., Blanchard, S., Swanson, C., Cattley, R. C., et al. (2005). Role of PPARalpha in mediating the effects of phthalates and metabolites in the liver. Toxicology, 207, 149–163. 63. Park, S. Y., Cho, Y. R., Kim, H. J., Higashimori, T., Danton, C., Lee, M. K., et al. (2005). Unraveling the temporal pattern of dietinduced insulin resistance in individual organs and cardiac dysfunction in C57BL/6 mice. Diabetes, 54, 3530–3540.
