Long-term dietary administration of diethyl phthalate triggers loss of insulin sensitivity in two key insulin target tissues of mice

Abstract

Over the past years, a growing body of work has linked numerous pervasive environmental chemicals with a multitude of adverse reproductive, developmental, behavioral, and metabolic changes in humans and animal models. Plasticizers include a wide variety of phthalate esters that are extensively used in a host of personal day care and cosmetic products. Many population-based studies have indicated a close association between diethyl phthalate (DEP) and diabetes albeit the mechanisms remain much unexplored. Presently, we report that long-term dietary administration of DEP to adult male Swiss albino mice at two different concentrations mirroring the recommended tolerable doses, severely impaired insulin signaling in hepatocytes and adipocytes. This was concomitant with sustained oxidative stress from the overactivation of NADPH oxidase 2, a major intracellular source of reactive oxygen species, in both the cell types. The present study provides evidences of the onset of insulin resistance in mice after chronic exposure to DEP in diet even at lower levels. This, in turn, can have serious pathological consequences with ultimate manifestations of type 2 diabetes and metabolic syndrome (MetS). Thus, by disrupting the central metabolic function of liver and adipose tissue, the key insulin target tissues, daily exposure to phthalates in plastics can potentially contribute to the alarming prevalence of MetS in recent times.

Introduction

Insulin plays a pivotal role in maintaining glucose and energy homeostasis, acting through its receptor tyrosine kinase at target tissues primarily the liver, adipose tissue, and skeletal muscle. Loss of insulin sensitivity in the target cells encompasses a pathological state, insulin resistance, which is implicated in the pathogenesis of type 2 diabetes and a plethora of diseases collectively referred to as “metabolic syndrome” (MetS). The present trends are quite alarming. According to the Diabetes Atlas of the International Diabetes Federation, the global prevalence of diabetes is expected to escalate to 10.4% (642 m) by 2040. ¹ A growing body of work over the past two decades has asserted the endocrine disrupting activities of several pervasive environmental chemicals linking them with multiple adverse reproductive, neurobehavioral, developmental changes as well as MetS and cancers in humans. ²

The diethyl ester of phthalic acid, diethyl phthalate (DEP), finds extensive usage as plasticizer in myriad personal and cosmetic day care products, insecticides, polyvinyl chloride (PVC), drugs, and pharmaceuticals. ³ The gradual leaching of phthalate from the plastic matrix into outside environment can well contaminate the DEP-coated drugs, nutrition supplements, and a host of packaged food and consumer products, thus making continuous ingestion almost unavoidable in our modern lifestyle. Higher urinary concentration of phthalate metabolites positively correlated with increased waist circumference and insulin resistance in adult men ⁴ as well as faster weight gain in women. ⁵ In addition, circulating levels of phthalate metabolites including monoethyl phthalate, the monoethyl derivative of DEP, were tightly associated with diabetes in the elderly population. ⁶ Human population-based comparative profiling of several phthalate ester metabolites in urine samples reflected higher body burden of DEP. ^7,8 Therefore, DEP-induced toxicity needs to be ascertained clearly. It is pertinent to mention that the recommended oral reference dose for DEP according to the US Environmental Protection Agency (EPA) is 800 μg kg⁻¹ bw day⁻¹ while the Concise International Chemical Assessment Document (CICAD) of the International Programme on Chemical Safety (IPCS) advised 5000 μg kg⁻¹ bw day⁻¹ as the tolerable daily intake value. ⁹ Recently, our laboratory reported the molecular mechanisms involved in testicular germ cell inflammation and sperm pathologies in mice after chronic dietary administration of DEP even at doses closer to the recommended permissible limits. ¹⁰ Higher circulatory levels of glucose in our DEP-treated mice prompted us to further investigate the regulation of blood glucose in these animals. To the best of our knowledge, the effect of DEP on insulin signaling molecules has not been evaluated. The liver, besides integrating whole body metabolism and regulating blood glucose, plays a key role in xenobiotic metabolism and detoxification as well. Adipose tissue is pivotal in energy storage and mobilization. The resultant manifestation of hyperglycemia and hyperinsulinemia, the hallmarks of insulin resistance, following long-term dietary administration of DEP adds credence to the reported close association between phthalate ester plasticizer and the alarmingly prevalent MetS in recent times.

Materials and methods

Animal care and treatment

The present work is an extension of our previous study. ¹⁰ In brief, 8-week-old adult male Swiss albino mice weighing 20–25 g were randomly assigned to three groups and fed with standard diet and drinking water ad libitum. The experimental protocol involved dietary administration of DEP (CAS No. 84-66-2, Sisco Research Laboratories Pvt. Ltd., Mumbai, Maharashtra, India) diluted in sunflower oil at two different concentrations, 1 mg kg⁻¹ bw day⁻¹ and 10 mg kg⁻¹ bw day⁻¹. Control animals were fed standard diet containing the same amount of sunflower oil vehicle. Each group had six animals and were treated for 3 months. Animal care, maintenance, and experiments were executed following the guidelines of the Institutional Animal Ethics Committee (IAEC) under the regulation of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India (Permit No. 1819/GO/Re/S/15/CPCSEA). Serum, liver, and epididymal adipose tissue were taken for biochemical and molecular analyses. All chemicals used were analytical grade.

Blood and serum analyses

Blood glucose levels were measured in mice prior to DEP exposure and also after the completion of DEP treatment using Accu-Chek glucometer (Roche, Basel, Switzerland). Insulin tolerance test (ITT) was performed in mice to determine whole-body insulin action by monitoring blood glucose levels at 0 min, 15 min, 30 min, and 60 min after insulin injection (0.75 IU/kg body weight, s.c.). Following euthanization, blood was collected and centrifuged to obtain sera. Insulin level was determined in the animal sera by commercially available kit based on enzyme linked immunosorbent assay (RayBiotech, Norcross, Georgia, USA) following the manufacturer’s instructions.

Tissue processing

The perfused liver and epididymal adipose tissue were collected in ice-cold 0.9% (w/v) normal saline solution after quick excision. The tissue was properly cleaned, weighed, and kept on ice. A 20% (w/v) homogenate was prepared in 50 mM phosphate buffer, pH 7.4 containing 0.25 M sucrose with Potter-Elvehjem motor-driven homogenizer. The homogenate was centrifuged at 10,000 × g for 20 min at 4°C to obtain cytosolic fraction which was eluted through 5 mL Sephadex G-25 column. Protein content was estimated in samples taking bovine serum albumin (BSA) as the standard. ¹¹

Isolation of hepatocytes and adipocytes

Liver and adipose tissue were immediately collected in sterile 0.9% (w/v) normal saline and thoroughly washed in Hank’s Balanced Salt Solution (HBSS) supplemented with 5.5 mM glucose. The tissues were individually digested in HBSS containing 5.5 mM glucose, 5% fatty acid free BSA, and 3.3 mg/mL type II collagenase for 30 min at 37°C. The digestion mixture was passed through a fine sieve, centrifuged at 1000 × g for 10 min and the isolated cells were suspended in phosphate-buffered saline (PBS), pH 7.4.

Intracellular ROS generation

The intracellular generation of reactive oxygen species (ROS) in the isolated hepatocytes and adipocytes was detected using cell-permeable probe, 2′,7′-dichlorofluorescein diacetate (DCFDA) (Sigma-Aldrich, St. Louis, Missouri, USA). The cells were incubated with the probe in PBS, pH 7.4 for 20 min at 37°C and then washed in PBS. Images were captured with a laser scanning confocal microscope (TCS-SP8, Leica Microsystems GmbH, Wetzlar, Germany).

Immunofluorescence of GLUT4 translocation

Isolated adipocytes were initially incubated for 30 min in incomplete media at 37°C. Cells were then treated with 100 mM insulin for 30 min, washed with PBS, and smeared on coverslip. Following the treatment, cells were incubated with 1% fatty acid free BSA, 22.52 mg mL⁻¹ glycine in PBS containing 0.1% Tween-20 for 1 h at room temperature to avoid nonspecific binding. Again, the cells were subsequently incubated with anti-GLUT4 primary antibody and FITC-conjugated anti-rabbit secondary antibody. DAPI was used as nuclear counterstain and images were captured with the help of a laser scanning confocal microscope (TCS-SP8, Leica Microsystems GmbH).

Western blotting

Equal amounts of hepatocyte and adipocyte proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore, Bedford, Massachusetts, USA). After blocking, the membranes were incubated with primary antibodies against IRβ, phosphoIRβ^Tyr1150/1151, IRS-1, phosphoIRS-1^Tyr632, phosphoPI3Kp85, PDK1, phosphoPDK1^Ser241, Akt, phosphoAkt^Thr308, GLUT2, GLUT4, NOX4, gp91^phox, and β actin (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). The membranes were well washed with Tris-buffered saline with Tween-20 and incubated with alkaline phosphatase-conjugated anti-rabbit, anti-goat, and anti-mouse antibody (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA). The bands were detected with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) (VWR Life Science AMRESCO, Radnor, Pennsylvania, USA). Densitometric analyses were performed using ImageJ software.

Statistical analyses

All data were presented as mean ± standard error of the mean (SEM) and analyzed by one-way analysis of variance followed by Duncan’s New Multiple Range test. Minimal statistical significance was considered at p < 0.05.

Results

DEP-enhanced markers of insulin resistance

There was rise in circulatory glucose level in both the treatment groups in a dose dependent manner while control animals had normal blood glucose level (Figure 1(a)). We further calculated the percent gain in blood glucose for each group in itself (blood glucose level at 3 m/blood glucose level at 0 m × 100 − 100) in order to assess the change in blood glucose concentration before DEP exposure and after completion of DEP treatment. We observed that 1 mg DEP-treated mice showed almost 45% gain in blood glucose (p < 0.01) which further escalated to 85% gain (p < 0.001) in 10 mg DEP-treated group (Figure 1(b)). This clearly indicated a severe hyperglycemic state after DEP intake. Though the serum concentration of insulin remained same as control in lower dose DEP group, a modest rise (p < 0.01) in insulin level was observed in the higher dose treatment group (Figure 1(c)). Further, insulin tolerance test (ITT) was performed to detect the sensitivity of animal body toward injected insulin. In this test, regulation of blood glucose was perfect in control mice following insulin injection as evident from the consistent decrease in circulatory glucose levels at subsequent time intervals. However, in case of both the DEP doses, blood glucose level remained significantly higher (p < 0.001) even after insulin injection indicating prevalence of hyperglycemia (Figure 1(d)). The evaluation of the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), the marker of insulin resistance, revealed significantly augmented indices in the treated groups relative to the control (Figure 1(e)).

Figure 1.

Effect of DEP on markers of insulin resistance: (a) graph showing elevated glucose level in circulation of DEP-treated animals, (b) percent gain in blood glucose of animals during the course of treatment, (c) concentration of insulin in the sera obtained from control and DEP-treated animals; (d) ITT exhibiting no significant reduction in blood glucose level after giving insulin injection to both groups of DEP-treated mice compared to control; (e) graph showing higher Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) values in both the DEP-treated mice groups compared to control. Data are presented as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, and ***p < 0.001 represent significant difference versus control; ^∧∧ p < 0.01 and ^∧∧∧ p < 0.001 represent significant difference versus lower dose DEP. DEP: diethyl phthalate; SEM: standard error of the mean; ITT: insulin tolerance test.

DEP-mediated defects in insulin receptor activation and subsequent signal transduction

Western blot analyses were performed to detect the mode of insulin signaling in two major insulin target cells, namely, hepatocytes and adipocytes isolated from control and DEP-treated mice. The protein expression of the transmembrane insulin receptor IRβ was severely reduced in both the cell lysates following administration of DEP at 10 mg kg⁻¹ bw day⁻¹ (Figure 2(a)). The receptor phosphorylation is a necessary prerequisite for subsequent activation of downstream signaling cascade. It was observed that in these two cell types, total content as well as phosphorylation of IRβ, IRS1, and PDK1 were markedly affected following DEP exposure while the phosphorylation of p85α subunit of PI3K was found to be elevated in both the DEP fed groups.

Figure 2.

Effect of DEP on insulin signaling in hepatocytes and adipocytes: immunoblot analyses of the various candidate molecules of insulin signaling pathway in the lysates of hepatocytes and adipocytes prepared from control and DEP-treated mice (n = 3). Protein expression levels were normalized to β actin. IRβ: insulin receptor β; IRS; insulin receptor substrate; PI3K: phosphoinositide-3-kinase; PDK1: phosphoinositide-dependent kinase 1; GLUT: glucose transporter; DEP: diethyl phthalate.

Cellular uptake of glucose is facilitated by Akt-mediated GLUT translocation toward the plasma membrane. Our immunoblotting revealed prominent downregulation of total Akt in both hepatocytes and adipocytes in higher dose group (Figure 2(b)). Activation of Akt by means of PDK1-mediated phosphorylation at Thr³⁰⁸ residue is essential for subsequent translocation of glucose transporters from cytosolic endosomal vesicle toward the cell membrane. Therefore, a drastic decline in Akt phosphorylation at Thr³⁰⁸ residue is indicative of a poor signal for marginal translocation of GLUT proteins. GLUT2 is predominantly expressed in hepatocytes while GLUT4 is the principal isoform in adipocytes. Marked dose-dependent decrease in GLUT4 expression was observed in adipocyte lysates prepared from DEP-treated mice. Similarly, a sharp decline in GLUT2 expression in hepatocyte lysates reflected lack of adequate uptake of the cellular energy coin glucose.

DEP reduced marginal translocation of GLUT4

Immunofluorescence depicted clear translocation of GLUT4 protein from cytosol toward the plasma membrane in adipocytes obtained from control mice as evident from the appearance of bright green fluorescence of FITC-conjugated anti-GLUT4 on the margin of the cells (Figure 3). On the other hand, drastic inhibition of GLUT4 movement was noticeable in a dose-dependent manner following the administration of DEP. It is noteworthy that less intense green fluorescence of FITC-conjugated GLUT4 antibody in both the DEP-treated groups reiterates the decreased abundance of GLUT4 protein as seen by immunoblotting.

Figure 3.

Effect of DEP on GLUT4 translocation: micrographs showing immunofluorescence of GLUT4 translocation in adipocytes isolated from control and DEP-treated mice (n = 3). Left panel: DAPI-stained, middle panel: FITC-stained, and right panel: merged images. Original magnification: ×400. White arrows are indicative of translocation of GLUT4 from cytosol to the plasma membrane of adipocytes; asterisks indicate cells showing no evidences of GLUT4 translocation. GLUT: glucose transporter; DEP: diethyl phthalate.

DEP augments generation of intracellular ROS

DCFDA assay in both hepatocytes and adipocytes displayed bright green fluorescence in phthalate-treated groups (Figure 4(a)). The presence of significantly higher percentage of fluorescent cells asserted higher ROS burden in both cell types (Figure 4(b) to (c)). Further, Western blotting was performed in cell lysates to check the expression profile of NADPH oxidase (NOX) family of enzymes, the potent contributors of intracellular ROS. Two important subunits of NOX, namely, NOX4 and NOX2 or gp91^phox showed differential trend in their expression in both the cells. The data revealed that significant NOX2 upregulation is the major ROS contributor upon DEP administration (Figure 4(d) to (e)).

Figure 4.

Effect of DEP on generation of intracellular ROS: (a) micrographs showing bright green DCFDA stained hepatocytes and adipocytes isolated from DEP-treated mice. Original magnification: ×400; (b–c) graphs showing percentage of fluorescent hepatocytes and adipocytes in control and DEP-treated groups; (d–e) immunoblots and corresponding densitometric analyses of NOX4 and gp91^phox in control and DEP-treated hepatocyte and adipocyte lysates, respectively. Data are presented as mean ± SEM (n = 3). **p < 0.01 and ***p < 0.001 represent significant difference versus control; ^p < 0.05 and ^^^p < 0.001 represent significant difference versus lower dose DEP. DEP: diethyl phthalate; DCFDA: 2′,7′-dichlorofluorescein diacetate.

Discussion

Long-term administration of DEP even at lower levels could elicit conditions of hyperglycemia and hyperinsulinemia, the hallmarks of insulin resistance, in mice. Liver and adipose tissue are the major sites for insulin-stimulated glucose uptake and excess glucose in circulation clearly indicated impediment in systemic glucose regulation. This is well corroborated by the studies of Jayashree et al. ¹² and Indumathi et al. ¹³ reporting a significant rise in circulatory insulin level in male rats after bisphenol A (BPA) treatment. Upon ligand binding, the insulin receptor (IR) autophosphorylates and becomes activated. The resultant tyrosine phosphorylation of endogenous substrates, namely, insulin receptor substrate (IRS) activates PI3K. This kinase phosphorylates phosphatidylinositol(4,5)-bisphosphate (PIP₂) into phosphatidylinositol(3,4,5)-trisphosphate (PIP₃) leading to further activation of phosphoinositide-dependent kinase 1 (PDK-1). ¹⁴ Further downstream activation of protein kinase B (PKB/Akt) leads to translocation of glucose transporter (GLUT) protein to plasma membrane which facilitates glucose uptake. ¹⁵ Since our Western blot analyses revealed significant downregulation of total IRβ and IRS-1 proteins as well as their phosphorylated forms in higher dose DEP group, it is ascertained that exposure to DEP reduced IRβ expression as well as activation. On a similar note, diethylhexyl phthalate (DEHP) diminished IR protein in cultured Chang liver cells in vitro ¹⁶ and rat gastrocnemius muscle in vivo. ¹⁷ Hyperglycemia is reported to impair IR dimerization by OGlcNAcylation. ¹⁸ The decrease in total IRS-1 protein could occur from Ser^636/639/307 phosphorylation that not only reduces tyrosine phosphorylation on IRS-1 but enhances its ubiquitin-proteasomal degradation. ¹⁹ Though the regulatory subunit p85 of PI3K was activated, exposure to DEP caused diminution of total and phosphoPDK-1 concentrations. Akt binds to PIP₃ in the plasma membrane and is phosphorylated by PDK-1 at Thr³⁰⁸ residue. ²⁰ Total Akt protein was significantly reduced in the two DEP concentrations compared to control whereas phospho-Akt^Thr308 expression was severely subdued in higher dose group affecting its activation. Akt signaling is crucial for glucose uptake into cytosol via direct regulation of GLUT expression. A sharp decline in the principal glucose transporter GLUT2 and GLUT4 protein concentration in both cell types upon DEP administration suggested inadequate cellular glucose uptake. Reduced GLUT4 translocation toward plasma membrane of DEP-treated adipocytes further confirmed the impediment in glucose uptake. This finding was similar to the effects of BPA and DEHP observed in mouse 3T3-F442A adipocytes ²¹ and in male rats, respectively. ²² Therefore, even if insulin was normal or higher than normal in DEP-treated mice, it failed to stimulate efficient cellular glucose uptake leading to persistent hyperglycemia.

Several reports have established direct relationship between systemic redox imbalance and diabetes in general human population. Kim et al. reported a panel study on an elderly Korean population reflecting oxidative damage mediated higher incidence of diabetes mellitus by DEHP. ²³ In a pilot study, Kataria et al. recorded positive correlation between BPA- and DEHP-induced oxidative stress and insulin resistance as well as endothelial dysfunction in a cohort of healthy, preadolescent children residents of New York City area. ²⁴ In fact, hyperglycemia is known to induce insulin resistance through several pathways, which are all believed to be linked with oxidative stress. ²⁵ Advanced glycation end-products (AGEs) enhanced inhibitory Ser³⁰⁷ phosphorylation of IRS-1. ²⁶ In nondiabetic rats, co-administration of antioxidants prevented insulin resistance induced by glucose infusion ²⁷ while ROS production preceded insulin resistance in cultured 3T3-L1 adipocytes. ²⁸ Previously, Rajesh et al. elaborated the protective role of antioxidant supplementation (vitamin C and E) in DEHP-induced oxidative stress-mediated insulin resistance in adipocytes of male rats. ²² Amelioration of DEHP-mediated defective insulin signal transduction by antioxidant vitamins in gastrocnemius muscle of male rats was again reported by Srinivasan et al. ¹⁷ Since oxidative stress is implicated in the development of insulin resistance, we checked the intracellular generation of ROS in both hepatocytes and adipocytes and found it substantially augmented in both DEP dose groups. NOX enzymes are one of the major cytosolic sources of ROS. Apart from the classical phagocytic NOX, ROS produced by the non-phagocytic NOX homologues have important cellular consequences. ²⁹ Earlier, activation of NOX2/gp91^phox was shown to have key role in CD95-dependent hepatocyte apoptosis. ³⁰ The robust upregulation of gp91^phox expression after DEP treatment highlights the important role of this NOX isoform in phthalate-induced toxicity. However, with regard to phthalates, it is important to consider the additive effects since the exposure involves simultaneous influence of several phthalate esters. Future investigations need to address this aspect.

Conclusion

Taken together, our results provide conclusive evidences that dietary administration of lower levels of DEP could impair insulin action in the liver and adipose tissue (Figure 5). This was accompanied with sustained oxidative stress from NOX2-mediated excess generation of ROS. The effects were prominent in mice fed DEP at 10 mg kg⁻¹ bw day⁻¹ concentration. We found that even at lower dose levels, long-term administration of DEP in mice diet could severely impair insulin signal transduction in hepatocytes and adipocytes which was concomitant with altered redox status. Considering the central metabolic function of liver and adipose tissue as key insulin target tissues, our data provide meaningful insight into the potential contribution of daily exposure to phthalates in plastics to the alarming prevalence of MetS in recent times.

Figure 5.

Schematic representation of long-term dietary DEP-induced effects in the two key insulin target cells, namely, hepatocytes and adipocytes, leading to insulin resistance in mice. DEP: diethyl phthalate.