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The estrogenic effect of bisphenol a disrupts pancreatic [beta]-cell function in vivo and induces insulin resistance.



The function of the pancreatic [beta]-cell is the storage and release of insulin, the main hormone involved in blood glucose blood glucose Diabetology The principal sugar produced by the body from food–especially carbohydrates, but also from proteins and fats; glucose is the body's major source of energy, is transported to cells via the circulation and used by cells in the presence  homeostasis homeostasis

Any self-regulating process by which a biological or mechanical system maintains stability while adjusting to changing conditions. Systems in dynamic equilibrium reach a balance in which internal change continuously compensates for external change in a feedback
. The results in this article show that the widespread environmental contaminant contaminant /con·tam·i·nant/ (kon-tam´in-int) something that causes contamination.

contaminant

something that causes contamination.
 bisphenol-A (BPA BPA British Paediatric Association. ) imitates 17[beta]-estradiol ([E.sub.2]) effects in vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body.

in vi·vo
adj.
Within a living organism.



in vivo adv.
 on blood glucose homeostasis through genomic and nongenomic pathways. The exposure of adult mice to a single low dose (10 [micro]g/kg) of either [E.sub.2] or BPA induces a rapid decrease in glycemia glycemia /gly·ce·mia/ (gli-se´me-ah) the presence of glucose in the blood.

gly·ce·mi·a
n.
The presence of glucose in the blood.
 that correlates with a rise of plasma insulin. Longer exposures to [E.sub.2] and BPA induce an increase in pancreatic [beta]-cell insulin content in an estrogen-receptor--dependent manner. This effect is visible after 2 days of treatment and starting at doses as low as 10 [micro]g/kg/day. After 4 days of treatment with either [E.sub.2] or BPA, these mice developed chronic hyperinsulinemia, and their glucose and insulin tolerance tests were altered. These experiments unveil the link between environmental estrogens Estrogens
Hormones produced by the ovaries, the female sex glands.

Mentioned in: Acne, Polycystic Ovary Syndrome

estrogens (es´trōjenz),
n.
 and insulin resistance Insulin Resistance Definition

Insulin resistance is not a disease as such but rather a state or condition in which a person's body tissues have a lowered level of response to insulin, a hormone secreted by the pancreas that helps to regulate the level
. Therefore, either abnormal levels of endogenous estrogens or environmental estrogen exposure enhances the risk of developing type 2 diabetes mellitus Type 2 diabetes mellitus
One of the two major types of diabetes mellitus, characterized by late age of onset (30 years or older), insulin resistance, high levels of blood sugar, and little or no need for supple-mental insulin.
, hypertension, and dyslipidemia. Key words: bisphenol A, diabetes, endocrine disruptors, estradiol, estrogen receptor estrogen receptor A protein of a superfamily of nuclear receptors for small hydrophilic ligands–eg, steroid hormones, thyroid hormone, vitamin D, retinoids; the presence of ERs in breast CA generally is associated with a better prognosis, as they respond to , insulin, islet islet /is·let/ (-lit) an island.

islets of Langerhans  irregular microscopic structures scattered throughout the pancreas and comprising its endocrine portion.
 of Langerhans, nongenomic, xenoestrogens. Environ Health Perspect 114:106-112 (2006). doi:10.1289/ehp.8451 available via http://dx.doi.org/[Online 20 September 2005]

**********

Insulin resistance is a crucial constituent of the metabolic syndrome metabolic syndrome
n.
See syndrome X.


Metabolic syndrome
A group of risk factors for heart disease, diabetes, and stroke.
, and its presence predicts type 2 diabetes type 2 diabetes
n.
See diabetes mellitus.
 and atherosclerotic cardiovascular disease Cardiovascular disease
Disease that affects the heart and blood vessels.

Mentioned in: Lipoproteins Test

cardiovascular disease 
 (DeFronzo and Ferrannini 1991). In addition to insulin resistance, type 2 diabetes mellitus is also characterized by a progressive [beta]-cell dysfunction. In most patients, both symptoms are present several years before the onset of hyperglycemia hyperglycemia: see diabetes. .

The incidence of diabetes has increased in the last decades, and at present it is reaching epidemic levels (177 million persons with diabetes in the world; World Health Organization 2005). The cornerstones of its development are related to modern lifestyle, principally, a lack of exercise and an unhealthy diet. Other pathologies whose incidence rose dramatically after World War II, such as cancer, reproductive impairment, and neurodegenerative diseases neurodegenerative diseases

diseases characterized by neurodegeneration. Lesions are microscopic only but in chronic disease with massive involvement there may be grossly visible atrophy of affected nervous tissue.
, are probably related to the increase of endocrine-disrupting chemicals (EDCs) in the environment (Colborn et al. 1996). However, an experimental link between EDCs and diabetes has not yet been established, although a connection at the epidemiologic level in humans has been recently proposed for dioxin, an environmental contaminant that acts through other than estrogen receptors (ERs) as an endocrine disruptor (Bertazzi et al. 2001; Rene and Bunce n. 1. a sudden unexpected piece of good fortune.

Noun 1. bunce - a sudden happening that brings good fortune (as a sudden opportunity to make money); "the demand for testing has created a boom for those unregulated laboratories where boxes of
 2002).

A large number of EDCs act by mimicking the action of the sex hormone 17[beta]-estradiol ([E.sub.2]) (Colborn et al. 1993). In most cases they bind to the classic ERs, ER-[alpha] and ER-[beta] (McLachlan 2001; Newbold 2004), but they can also act through novel estrogen targets (Nadal et al. 2005). At physiologic levels, [E.sub.2] is thought to be involved in maintaining normal insulin sensitivity and to be beneficial for [beta]-cell function (Livingstone and Collison 2002; Louet et al. 2004). However, abnormal levels of [E.sub.2] may promote insulin resistance (Livingstone and Collison 2002), similar to what occurs in normal puberty and pregnancy (Amiel et al. 1991; Hollingsworth 1983). Therefore, the exposure to an exogenous chemical acting as the natural hormone [E.sub.2], but at an inappropriate concentration and during an improper time window, may enhance the risk of developing insulin resistance.

In spite of the many clinical studies that link sex steroids and actions of insulin, few studies have investigated the molecular basis of the interaction between [E.sub.2], the pancreatic [beta]-cell function, blood glucose homeostasis, and the development of diabetes. Pancreatic [beta]-cells contain both types of ERs, ER-[alpha] and ER-[beta] (Nadal et al. 2000). Although their functions are still greatly undetermined, ER-[alpha] and ER-[beta] are involved in important aspects of the [beta]-cell physiology (Nadal et al. 2004; Sutter-Dub 2002). These include protection against [beta]-cell death caused by cytokines Cytokines
Chemicals made by the cells that act on other cells to stimulate or inhibit their function. Cytokines that stimulate growth are called "growth factors.
 (Contreras et al. 2002) and, after a prolonged application, a beneficial effect on diabetes in mice expressing human islet amyloid amyloid /am·y·loid/ (am´i-loid)
1. starchlike; amylaceous.

2. the pathologic, extracellular, waxy, amorphous substance deposited in amyloidosis, being composed of fibrils in bundles or in a meshwork of polypeptide
 peptide (Geisler et al. 2002). The involvement of ERs in lipid and glucose metabolism has been demonstrated in ER-[alpha] knockout mice that display increased adiposity adiposity /ad·i·pos·i·ty/ (ad?i-pos´i-te) obesity.

cerebral adiposity  fatness due to cerebral disease, especially of the hypothalamus.


adiposity

obesity.
, insulin resistance, and glucose intolerance (Heine et al. 2000). In addition, [beta]-cells have the nonclassical membrane ER (ncmER) that triggers rapid effects (Nadal et al. 1998, 2000; Quesada et al. 2002). Recently, a similar receptor has been found in Drosophila Drosophila: see fruit fly.
drosophila

Any member of about 1,000 species in the dipteran genus Drosophila, commonly known as fruit flies but also called vinegar flies. Some species, particularly D.
 (Srivastava et al. 2005). [E.sub.2] rapidly potentiates [beta]-cell signaling systems and insulin release via this ncmER, an effect that is mimicked by EDCs, including bisphenol A (BPA) (Nadal et al. 2004). BPA is one of the most common chemicals that behaves as an endocrine disruptor. It was the first synthetic estrogen without a steroid structure (Dodds and Lawson 1936), but because of its properties as a cross-linking chemical, BPA was widely chosen by the chemical industry to produce plastic polymers, mainly polycarbonates. Nowadays, it is used in the manufacture of barrier coatings for the inner surfaces of food and beverage F&B is a common abbreviation in the United States and Commonwealth countries, including Hong Kong. F&B is typically the widely accepted abbreviation for "Food and Beverage," which is the sector/industry that specializes in the conceptualization, the making of, and delivery of foods.  cans. High concentrations of BPA have been detected in food and water extracted from autoclaved cans (Brotons et al. 1995). BPA is one of the highest-volume chemicals produced in the world, and its exposure is widespread: it has been found in 95% of the urine samples from people in the United States and to a similar extent is found in human blood, as well (Ikezuki et al. 2002; vom Saal and Hughes 2005). The European Commission's Scientific Committee on Food (ECSCF 2002) reported a tolerable daily intake (TDI TDI - Transport Driver Interface ) of 10[micro]g/kg/day. However, the U.S. Environmental Protection Agency Environmental Protection Agency (EPA), independent agency of the U.S. government, with headquarters in Washington, D.C. It was established in 1970 to reduce and control air and water pollution, noise pollution, and radiation and to ensure the safe handling and  (U.S. EPA EPA eicosapentaenoic acid.

EPA
abbr.
eicosapentaenoic acid


EPA,
n.pr See acid, eicosapentaenoic.

EPA,
n.
) considers 50 [micro]g/kg/day the reference dose based on the lowest observed adverse effect level (LOAEL LOAEL Lowest Observed Adverse Effect Level ) of 50 mg/kg/day, according to studies performed in the 1980s (vom Saal and Hughes 2005). In vivo studies using much lower doses of BPA than the LOAEL have shown that it affects sexual maturation (Howdeshell et al. 1999), induces a decrease in

daily sperm count and fertility (vom Saal et al. 1998), disrupts chromosome alignment (Hunt et al. 2003), and affects synaptogenesis (McLusky et al. 2005). In spite of this evidence, there is an ongoing debate with the chemical industry that is still skeptical about the risk of low doses of BPA (Bisphenol A Global Industry Group 2005).

In this article we show that the exposure of adult mice to BPA, at doses about 1,000-fold less than the LOAEL established by the U.S. EPA, alters blood glucose homeostasis in vivo. It rapidly increases plasma insulin, altering blood glucose concentration through a nonclassical estrogen pathway. Longer exposure increases [beta]-cell insulin content, an effect that involves classic ERs. These longer exposures generate chronic hyperinsulinemia in the fed state and peripheral insulin resistance.

Materials and Methods

Materials. We obtained ICI (language) ICI - An extensible, interpretated language by Tim Long with syntax similar to C. ICI adds high-level garbage-collected associative data structures, exception handling, sets, regular expressions, and dynamic arrays. 182,780 (ICI) from Tocris Cookson Ltd. (Avonmouth, UK), tocopherol-stripped corn oil from MP Biomedicals, LLC (Logical Link Control) See "LANs" under data link protocol.

LLC - Logical Link Control
 (Solon Solon, Athenian statesman
Solon (sō`lən), c.639–c.559 B.C., Athenian statesman, lawgiver, and reformer. He was also a poet, and some of his patriotic verse in the Ionic dialect is extant. At some time (perhaps c.600 B.C.
, OH, USA), and soluble insulin from Humulina Regular (Lilly, Madrid, Spain). Other substances were obtained from Sigma (Madrid, Spain).

Animals. We used Swiss albino albino (ălbī`nō) [Port.,=white], animal or plant lacking normal pigmentation. The absence of pigment is observed in the body covering (skin, hair, and feathers) and in the iris of the eye.  OF1 male mice (8-10 weeks of age) throughout this study. All animals were kept under standard housing conditions and were treated humanely and with regard for alleviation of suffering. An internal animal care and use committee reviewed and approved the method used.

Treatment. Stimuli ([E.sub.2] and BPA) were dissolved in tocopherol-stripped corn oil and administered subcutaneously at various concentrations. The amount of vehicle was kept constant at 100 [micro]L. In long-term experiments, animals were injected twice per day, at 0900 hr and 2000 hr, with 50 [micro]g/kg or 5 [micro]g/kg of test compound. Two injections of 50 [micro]g/kg/day during 4 days gave a plasma concentration of [E.sub.2] similar to that found in late pregnancy (Song et al. 2001). In ICI experiments, animals were injected intraperitoneally with a single dose of 500 [micro]g/kg/day for 3-4 days, always at 0800 hr.

Glycemia determination. We determined glucose in blood obtained from the tail vein using an Accu-check compact glucometer (Roche Diagnostic GmbH, Mannheim, Germany).

Insulin secretion and content. To measure plasma insulin, mice were anesthetized a·nes·the·tize also a·naes·the·tize  
tr.v. a·nes·the·tized, a·nes·the·tiz·ing, a·nes·the·tiz·es
To induce anesthesia in.



a·nes
 with 50 mg/kg body weight sodium pentobarbital pentobarbital /pen·to·bar·bi·tal/ (pen?to-bahr´bi-tal) a short- to intermediate-acting barbiturate; the sodium salt is used as a hypnotic and sedative, usually presurgery, and as an anticonvulsant. . Blood (~ 1 mL) was obtained by cardiac puncture with a syringe containing 24 mM EDTA EDTA: see chelating agents. . We determined the levels of plasma insulin by enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay
n.
ELISA.


Enzyme-linked immunosorbent assay (ELISA)
A diagnostic blood test used to screen patients for AIDS or other viruses.
 (ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.

ELISA
n.
) using a mouse insulin assay kit from Mercodia AB (Uppsala, Sweden).

To measure insulin release from isolated islets, mice were killed by cervical dislocation, and pancreatic islets of Langerhans were isolated by collagenase collagenase /col·la·ge·nase/ (kah-laj´e-nas) an enzyme that catalyzes the hydrolysis of peptide bonds in triple helical regions of collagen.

col·lag·e·nase
n.
 digestion as described previously (Morimoto et al. 2001). Islets were washed twice with a buffer solution containing 20 mM HEPES HEPES N-2-Hydroxyethylpiperazine-N'-2-Ethanesulfonic Acid , 115 mM NaCI, 5 mM NaHC[O.sub.3], 5 mM KCI KCI Kansas City International (airport)
KCI Kennel Club of India
KCI Key Club International
KCI Korea Concrete Institute
KCI Kitchener Collegiate Institute
KCI Kids Central, Inc.
KCI The Kitchen Collection, Inc.
KCI Kodak Canada Inc.
, 2.6 mM Ca[Cl.sub.2], 1.2 mM KH2P[O.sub.4], 1.2 mM MgS[O.sub.4], 3 mM D-glucose, and 1% bovine serum albumin (pH 7.4). Groups of 10 islets were then incubated in 1 mL of this buffer in the presence of 3, 7, and 16 mM glucose. After 1 hr, the medium was collected, and insulin was measured in duplicate samples by radioimmunoassay using a Coat-a-Count kit (DPC DPC Department of Premier and Cabinet (Victoria, Australia)
DPC Dutch Power Cows
DPC Deferred Procedure Calls (Microsoft Windows NT 4.
, Los Angeles, CA, USA).

The insulin content was determined in islets isolated as described above. Islets were grouped in batches of 10 and incubated overnight in an ethanol/HC1 buffer at 4[degrees]C. At the end of the incubation period, the buffer was removed and studied for insulin content using radioimmunoassay with a Coat-a-Count kit (DPC). Protein determination was performed by the Bradford dye method.

Immunocytochemistry im·mu·no·cy·to·chem·is·try
n.
The study of cell constituents by immunologic methods, such as the use of fluorescent antibodies.



immunocytochemistry
 and insulin content. Islets isolated as above were dispersed into single cells as previously described (Nadal et al. 1998). Briefly, islets were disaggregated Broken up into parts.  into single cells with trypsin trypsin, enzyme that acts to degrade protein; it is often referred to as a proteolytic enzyme, or proteinase. Trypsin is one of the three principal digestive proteinases, the other two being pepsin and chymotrypsin. . Cells were then centrifuged and resuspended in Hank's modified medium supplemented with 200 U/mL penicillin, 0.2 mg/mL streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other , 5 mM glucose, and 1% fatty acid--free albumin, pH 7.4. They were then plated on 24-well tissue culture plates. After 30 min, cells were washed with phosphate-buffered saline (PBS PBS
 in full Public Broadcasting Service

Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural,
) and fixed with Bouin's solution for 5 min. Then, they were dehydrated de·hy·drate  
v. de·hy·drat·ed, de·hy·drat·ing, de·hy·drates

v.tr.
1. To remove water from; make anhydrous.

2. To preserve by removing water from (vegetables, for example).
 with 30, 50, and 70% ethanol, 3 min each, and then washed with PBS. After washing, these cells were first incubated with a monoclonal anti-insulin antibody (1:200 dilution; Sigma, Madrid, Spain) for 2 hr and then for 1 hr with a secondary anti-body, anti-mouse IgG-conjugated fluorescein isothiocyanate (IgG-FITC; 1:200 dilution; Sigma), both at room temperature. Cells were washed with PBS overnight. Images were acquired with a confocal confocal

see confocal microscopy.
 microscope Zeiss Pascal 5 using a Zeiss 20x objective (numerical aperture = 0.5) and analyzed using LSM LSM Linux Software Map
LSM Louisiana State Museum
LSM Linux Security Module
LSM Living Stream Ministry
LSM Laser Scanning Microscopy
LSM Legato Storage Manager
LSM Land-Surface Model
LSM Lutheran Student Movement
LSM Logical Storage Manager
 Zeiss software (Zeiss, Jena, Germany). We measured immunofluorescence Immunofluorescence

A technique that uses a fluorochrome to indicate the occurrence of a specific antigen-antibody reaction. The fluorochrome labels either an antigen or an antibody.
 intensity in random fields. The results were expressed as average pixel intensity and normalized with respect to vehicle-treated animals. Pixel intensity was normalized with respect to the mean value of the pixel intensity of vehicle-treated animals at the corresponding days.

Glucose and insulin tolerance tests. For glucose tolerance tests, animals were fasted overnight for 12 hr, and blood samples were obtained from the tail vein. Animals were then injected intraperitoneally with 2 g/kg body weight of glucose, and blood samples were taken at the indicated intervals.

For insulin tolerance tests, fed animals were used. Animals were injected intraperitoneally with 0.75 IU/kg body weight of soluble insulin. Blood glucose was measured in each sample using an Accu-check compact glucometer (Roche).

Statistical analysis. Data are expressed as mean [+ or -] SE. Pairwise comparisons were made using Student's t-test. A probability level < 0.05 was considered statistically significant.

Results

[E.sub.2] and BPA rapidly alter glycemia and insulinemia. In vitro, both [E.sub.2] and BPA alter the function of pancreatic [beta]-cells through the ncmER (Nadal et al. 1998, 2000; Quesada et al. 2002; Ropero et al. 2002). In vivo, the administration of 10 [micro]g/kg [E.sub.2] to adult male mice resulted in a significant decrease of glycemia measured at 30, 60, and 120 min after the injection, compared to the increase of blood glucose produced by the fatty acids contained in the vehicle injection (tocopherol-stripped corn oil) (Figure 1A). The administration of 1, 10, and 100[micro]g/kg [E.sub.2] evoked a clear dose-dependent decrease in the rise of glycemia 30 min after the [E.sub.2] injection (Figure 1B). This effect is mimicked by equal doses of the environmental estrogen BPA (Figure 1C). Thirty minutes after injection, this decrease in blood glucose is parallel to an increase in plasma insulin (Figure 1D) of 3.20 [+ or -] 0.45-fold for [E.sub.2]-treated mice and 2.76 [+ or -] 0.5-fold for BPA-treated mice. Rapid effects elicited by [E.sub.2] and BPA in islets of Langerhans islets of Langerhans: see pancreas.  in vitro are initiated after they bind at the ncmER that is insensitive to the pure antiestrogen ICI (Nadal et al. 2004). This anti-estrogen was described as blocking classic ER-mediated actions in vivo (Johnson et al. 2003; Perez-Martin et al. 2003). To evaluate whether the [E.sub.2] and BPA actions described above require an ER with a sensitivity to the pure antiestrogen similar to a classical ER, we undertook experiments using mice treated with 500 [micro]g/kg/day ICI administered intraperitoneally for 3 days (Johnson et al. 2003). As shown in Figure 2A, ICI had no effect on the [E.sub.2] and BPA-dependent blood glucose decrease nor on the [E.sub.2]- and BPA-dependent increase of plasma insulin (Figure 2B).

Therefore, both [E.sub.2] and BPA rapidly change glycemia most likely by inducing an hypersecretion of insulin through a non-classical ER-mediated mechanism that may involve the ncmER previously described in these cells (Nadal et al. 2000, 2004).

[E.sub.2] and BPA increase [beta]-cell insulin content. Adult male mice were injected twice a day with the vehicle, [E.sub.2] or BPA for 4 days.

Afterward, insulin content was measured in individual cells by immunocytochemistry. Figure 3A shows confocal images of [beta]-cells obtained from mice treated 4 days with vehicle, 100[micro]g/kg/day [E.sub.2], or 100[micro]g/kg/day BPA. Figure 3B illustrates a three-dimensional reconstruction of images in Figure 3A showing a pixel intensity scale from 0 to 256 pixels. The images and the three-dimensional graphs illustrate that [beta]-cells from animals treated with [E.sub.2] and BPA presented higher staining than those treated with the vehicle and thus higher insulin content in every cell. After 4 days of treatment, the insulin increase was already manifested at doses of 10[micro]g/kg/day of either [E.sub.2] or BPA (Figure 3C). Nonetheless, this effect was small and 100 [micro]g/kg/day was needed to produce a potent increase in insulin content (Figure 3C). A treatment of 100 [micro]g/kg/day [E.sub.2] for 4 days gives an [E.sub.2] plasma concentration similar to that found in late pregnancy (Song et al. 2001). Therefore, the chronic action of [E.sub.2] and BPA was manifested at higher concentrations than was required for the acute effect. On the basis of this response, we use 100 [micro]g/kg/day as the paradigmatic See paradigm.  concentration in long-term experiments.

Time-course experiments demonstrated that the onset of the increase in insulin content occurs after 2448 hr of treatment with either [E.sub.2] or BPA (Figure 3D). The experiment in Figure 3D shows that most single [beta]-cells increased their insulin content. The experiment in Figure 3D shows that after 4 days of treatment, the insulin content is higher in [E.sub.2] and BPA-treated mice. The radioimmunoassay analysis of insulin content performed 4 days after the treatment with 100 [micro]g/kg/day of either [E.sub.2] or BPA produced similar results as those presented with immunocytochemistry (Figure 3E). These results demonstrate that the insulin content was increased by both the natural hormone and the environmental estrogen to a similar extent.

To study the involvement of classical ERs in the regulation of [E.sub.2]- and BPA-induced insulin expression, we used mice treated with the antiestrogen ICI, as described for the experiment presented in Figure 2. Animals treated with 100 [micro]g/kg/day [E.sub.2] for 4 days presented higher levels of insulin immunoreactivity than the control (Figure 4A). In animals treated with ICI, [E.sub.2] had no effect (Figure 4A). The action of the pure anti-estrogen is manifested to the same extent with BPA, as demonstrated by immunocyto-chemistry (Figure 4B) and radioimmunoassay (Figure 4C). The ability of the pure anti-estrogen to completely block the effect of both [E.sub.2] and BPA indicates that the action performed on insulin content is mediated by a classical ER.

[E.sub.2] and BPA administration provokes hyperinsulinemia and insulin resistance. An increment in the insulin content of every [beta]-cell within an islet causes it to release a higher amount of insulin every time it is stimulated (Marban et al. 1989). This occurs, for instance, during pregnancy, when islets adapt to deal with peripheral insulin resistance (Costrini and Kalkhoff 1971; Faure et al. 1985). In our system (Figure 5A), we show that, at high glucose concentrations (16 mM), islets from mice treated with 100 [micro]g/kg/day [E.sub.2] for 4 days secrete 1.54 [+ or -] 0.12-fold more insulin than those from mice treated with the vehicle, and the same occurs in islets from mice treated with BPA. In the latter case, differences are significant even at lower glucose concentrations (7 mM). This effect has been recently described in vitro (Adachi et al. 2005). Therefore, if [E.sub.2] and BPA-treated mice have higher insulin content, they should release more insulin than untreated mice, and accordingly, these animals should be hyperinsulinemic in the fed state. This is shown in Figure 5B: 4 days after the [E.sub.2] treatment, fed mice had blood insulin levels 1.7-fold higher than those of vehicle-treated mice. Blood glucose levels were 186 [+ or -] 5 mg/dL in vehicletreated mice and 170 [+ or -] 6 mg/dL in [E.sub.2]-treated mice (p = 0.065, not significant). Plasma insulin levels for the group treated with BPA were 1.5-fold higher than those for the vehicle-treated mice; however, their blood glucose levels were 153 [+ or -] 4 mg/dL for the vehicle (n = 5) and 154 [+ or -] 7 mg/dL for BPA (n = 7; p = 0.93). This experiment indicates that, in the [E.sub.2]-treated animals, there was mild insulin resistance, because there are 1.7-fold higher circulating insulin levels but a decrease of blood glucose, although it is not significant. This effect is remarkably manifested with BPA; in this case, plasma insulin levels are 1.53-fold higher and blood glucose concentration does not vary, a clear symptom of insulin resistance.

To prove that glucose tolerance is altered, we performed an intraperitoneal glucose tolerance test in fasted (12 hr) mice. Contrary to vehicle-treated mice, blood glucose increased to a higher level in [E.sub.2]-treated (Figure 6A) and BPA-treated mice (Figure 6B). The higher increase in blood glucose manifested 15 and 30 rain after a glucose challenge. We found that the area under the curve (milligrams per deciliter-minute) was 162 [+ or -] 6 (vehicle, n = 24), 181 [+ or -] 5 ([E.sub.2], n = 16, p = 0.025 vs. vehicle), and 190 [+ or -] 8 (BPA, n = 8, p = 0.028 vs. vehicle). These results indicate that an impaired glucose tolerance Impaired Glucose Tolerance (IGT) is a pre-diabetic state of dysglycemia, that is associated with insulin resistance and increased risk of cardiovascular pathology. IGT may precede type 2 diabetes mellitus by many years. IGT is also a risk factor for mortality.  was present in [E.sub.2] treated mice and in those treated with BPA. When an insulin tolerance test (intraperitoneal injection of 0.75 IU/kg body weight soluble insulin) was performed in fed mice, a significantly reduced hypoglycemic hypoglycemic /hy·po·gly·ce·mic/ (-gli-sem´ik)
1. pertaining to, characterized by, or causing hypoglycemia.

2. an agent that lowers blood glucose levels.
 response was observed in both [E.sub.2] and BPA-treated mice (Figure 6C). This insulin intolerance also manifested in animals given an oral dose of 100 [micro]g/kg/day BPA for 4 days. In this case, the BPA was dissolved in tocopherol-stripped corn oil and delivered through a pipette pipette /pi·pette/ (pi-pet´) [Fr.]
1. a glass or transparent plastic tube used in measuring or transferring small quantities of liquid or gas.

2. to dispense by means of a pipette.
 placed into the animal's mouth (Howdeshell et al. 1999) (Figure 6D). All these results indicate that [E.sub.2]-treated animals and, in a remarkably similar manner, BPA-treated animals develop insulin resistance without any changes in glycemia or weight (35-40 g, with no significant differences between the diverse treatments applied).

Discussion

The results presented in this article demonstrate a link between estrogenic endocrine disruptors and insulin resistance. We have shown that BPA mimics [E.sub.2] effects on blood glucose homeostasis through two different pathways. A nonclassical pathway produces a rapid increase in plasma insulin and a decrease in blood glucose. This is unaffected by the anti-estrogen ICI and is most likely initiated by the ncmER already described in islet cells (Alonso-Magdalena et al. 2005; Nadal et al. 2000; Quesada et al. 2002; Ropero et al. 2002). Nonetheless, other estrogen and xenoestrogen actions initiated at the plasma membrane are mediated via classical ERs (Losel lo·sel  
n.
One that is worthless.



[Middle English, from lsen, past participle of l
 et al. 2003; Wozniak et al. 2005). The activation of this alternative pathway occurs at low doses of both [E.sub.2] and BPA, becoming significant at 10 [micro]g/kg. When animals were treated with either the natural hormone or BPA for a longer period of time, there was an increase in the pancreatic insulin content. This effect was completely blocked by ICI, suggesting that a classical ER is involved. This implies that the action described here for [E.sub.2] and BPA can be extrapolated to other estrogenic EDCs. Any EDC EDC

See: Export Development Corp.
 having an estrogenic effect through classical ERs can be a candidate to induce insulin overexpression. This chronic treatment induced insulin resistance.

Type 2 diabetes mellitus is characterized by insulin resistance, which results in lower levels of blood glucose uptake into target tissues. Consequently, blood glucose levels increase and more insulin is released, producing hyperinsulinemia, which manifests early in type 2 diabetes. In addition, several studies have demonstrated that the hypersecretion of insulin is a primary defect of type 2 diabetes and that insulin resistance develops secondarily to the chronic hyperinsulinemia (Charollais et al. 2000; DeFronzo 1997; Devedjian et al. 2000; McGurry 1992). Indeed, the persistence of chronic physiologic euglycemic hyperinsulinemia for 3-5 days can induce severe insulin resistance in healthy subjects with normal glucose tolerance (Del Prato et al. 1994). Furthermore, patients with insulinoma display a correlation between hyperinsulinemia and insulin resistance (Pontiroli et al. 1992; Skrha et al. 1996). When insulin is overexpressed, a chronic hyperinsulinemia manifests, as shown in transgenic mice that overexpressed the insulin gene (Marban et al. 1989). Remarkably, these transgenic mice developed insulin resistance. Here we have shown that 4 days of treatment with either [E.sub.2] or BPA induces an increase in [beta]-cell insulin content; these mice were hyperinsulinemic and presented altered glucose and insulin tolerance tests. These alterations may result from a direct effect of [E.sub.2] and BPA on [beta]-cell insulin content, or a compensatory response resulting from the insulin resistance noted in these animals, or both.

We have demonstrated that after 4 days of treatment, isolated islets respond more vigorously to glucose (Figure 5A), likely because their insulin content is higher (Marban et al. 1989). This phenomenon may be responsible for the chronic hyperinsulinemia that manifested in the fed state (Figure 5B). In addition, the altered glucose and insulin tolerance test results were consistent with the fact that these mice had developed insulin resistance. Therefore, our results suggest that the sustained hyperinsulinemia produced by [E.sub.2] and BPA affects peripheral tissues, producing insulin resistance, most likely by down-regulation of insulin receptor number and function. However, the extent of the insulin resistance with this treatment is not enough to induce hyperglycemia in the fasted state.

The assumption described above does not rule out a direct effect of both [E.sub.2] and BPA on peripheral tissue. BPA produces down-regulation of glucose transporters in adipocytes (Sakurai et al. 2004), an action that may induce insulin resistance. Moreover, BPA combined with insulin favors the conversion of fibroblasts Fibroblasts
A type of cell found in connective tissue; produces collagen.

Mentioned in: Skin Grafting
 to adipocytes (Masuno et al. 2002), enhancing the risk of obesity, a metabolic disorder that has been related to endocrine disruptors in the last years (Heindel 2003; Mead 2004). Hence, the direct effect of BPA on peripheral tissue might also be of importance to developing insulin resistance.

At present, there is a debate about determining the safe levels of BPA exposure and whether there is a need for a new risk assessment (vom Saal and Hughes 2005). The U.S. EPA considers 50 [micro]g/kg/day as the reference dose based on a LOAEL of 50 mg/kg/day, according to studies performed in the 1980s (vom Saal and Hughes 2005). The ECSCF (2002) reported a TDI of 10 [micro]g/kg/day. The results presented here show a rapid nongenomic effect at a dose of 10 [micro]g/kg/day, five times lower than the reference dose established by the U.S. EPA and equal to the TDI reported by the ECSCF. This dose of BPA results in levels of parent (unconjugated) BPA in blood of 3-4 nM after 30 min and maintained 24 hr later (Zalko et al. 2003), below the level reported in blood from human fetuses at parturition parturition
 or birth or childbirth or labour or delivery

Process of bringing forth a child from the uterus, ending pregnancy. It has three stages.
 (Schonfelder et al. 2002). This low dose of BPA produces a 2.5-fold increase in plasma insulin and a 20% decrease in blood glucose levels, 30 min after its application. Remarkably, a low dose of 10 [micro]g/kg/day was able to slightly change insulin content as well. A higher dose of 100 [micro]g/kg/day dramatically increased pancreatic insulin content after only 4 days of exposure. Moreover, this treatment, delivering BPA either via injection or through oral intake, induced insulin resistance. This dose is only twice the reference dose recommended by the U.S. EPA and 10 times higher than the TDI recommended by the ECSCF.

The present study demonstrates a connection between BPA and insulin resistance at doses much lower than the LOAEL used up to now (50 mg/kg/day), and therefore it is strong evidence supporting a review of the risk assessment concerning BPA.

We thank B. Fernandez for excellent technical assistance and I. Quesada, A. Gomis, and A.B. Ropero for critical reading of the manuscript.

This study was supported by the Spanish Ministry of Education and Science (grant BFI BFI - brute force and ignorance 2002-01469 and BFU BFU Bundesstelle für Flugunfalluntersuchung (German: federal agency for flight accident investigation)
BFU Battlefield Unit (gaming)
BFU Blinding Fast Upgrade
2005-01052) and Instituto de Salud Carlos III (grants RCMN C03/08 and 03/0178). P.A.M. has a fellowship from the Ministry of Education and Science. S.M. was a postdoctoral fellow from CONACYT CONACYT Consejo Nacional de Ciencia y Tecnología (National Board of Science and Technology; Mexico, Bolivia, Paraguay) , Mexico.

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Paloma Alonso-Magdalena, (1) Sumiko Morimoto, (1,2) Cristina Ripoll, (1) Esther Fuentes, (1) and Angel Nadal (1)

(1) lnstituto de Bioingenieria, Universidad Miguel Hernandez de Elche, Alicante, Spain; (2) Departamento de Biologia de la Reproduccion,

Instituto Nacional de Ciencias Medicas y Nutricion "Salvador Zubiran," Mexico City, Mexico

The authors declare they have no competing financial interests.

Address correspondence to A. Nadal, Instituto de Bioingenieria, Universidad Miguel Hernandez de Elche, Carretera Alicante-Valencia Km 87, Sant Joan d'Alacant Sant Joan d'Alacant (San Juan de Alicante in Spanish) is a municipality belonging to Alicante metropolitan area, in the southeast of Land of Valencia, Spain. It is located 8 km north-east of the provincial capital, Alicante, and 3 km west of the 7-km sandy beach called , 03550 Alicante, Spain. Telephone: 34-96-5919535. Fax: 34-96-5919547. E-mail: nadal@umh.es

Received 30 June 2005; accepted 19 September 2005.
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