Ligand Activated Transcription Factors Biology Essay

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Estrogens exert their physiological effects through two ER subtypes, ER and ER, which belong to the nuclear receptor family of ligand-activated transcription factors. The major physiological estrogen is 17b-estradiol (E2), which has a similar affinity for both ERs. Like other steroid hormone receptors, ERs act as dimers to regulate transcriptional activation. In the classical model of ER action, ligand-activated ER binds specifically to DNA at estrogen-responsive elements (EREs) through its DNA binding domain (1). Estrogen also modulates gene expression by a second mechanism, in which ER interacts with other transcription factors, such as AP-1, through a process referred to as transcription factor cross-talk (2). Both ERa and ERb are able to modulate gene expression by either classical ERE-mediated signaling or by interacting with other transcription factors.

Estrogen signaling and central regulation of metabolism

Estrogen is a major player in regulation of appetite, energy expenditure, body weight and fat deposition/distribution in females. Food intake varies across the menstrual cycle with daily food intake lowest during the peri-ovulatory period when estrogen levels are at maximum (4). Menopausal women tend to gain body fat, which appears to be a consequence of the decline in endogenous estrogens (5-7). In animal models, ovariectomy induces an increase in food intake and decreases ambulatory and wheel running activities, which is reversed with estrogen replacement (8-12). Therefore, hypo-estrogenic states are associated with decreased activity and an increase in body weight in females. The anorectic effects of estrogen are thought to be mediated through the CNS actions based on the findings that direct injections of estradiol into the paraventricular nucleus or arcuate/ventromedial nucleus are the most effective to reduce food intake, body weight and increase wheel running activity especially in females (8,9,13).

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In rodents and primates, energy homeostasis (defined by food intake, body weight, metabolic rates, etc.) is altered by the phasic changes in estrogen levels during the estrous cycle. A strong link between the reproductive cycle in females and the central control of energy homeostasis and feeding behaviour in the hypothalamus has been previously reviewed (4,14,15). Briefly, during the estrous cycle, female guinea pigs, which have a true luteal phase unlike rats and mice, eat less during and immediately after the pro-estrous phase where estrogen levels are at their peak (13) and during the estrous cycle in rats, energy expenditure and respiratory quotients, all measures of metabolic rates, decrease during the estrous phase of the cycle (16). Female rodents may eat about 25% less during a significant portion of the estrous cycle when estrogen is at peak levels (4). The decrease in food intake is mostly due to a decrease in meal size (meal frequency may actually increase) (11). In rats, estrogen inhibits meal size during the light cycle through a constant or tonic effect while having a phasic effect on both meal size and meal number during the dark cycle (17). The role of estrogen in energy homeostasis has been further substantiated through studies of mutant mice with targeted disruption of the aromatase (ArKO) gene, the product of which converts androgens into estrogens. In the ArKO genotype, females are severely obese and the obesity is reversed with estrogen treatment (18).

The greater tendency for post-menopausal women towards obesity and the alterations of weight gain and feeding behaviour in rodent models clearly indicate that gonadal steroids, especially estrogen, have a significant role in the CNS control of energy homeostasis. The cellular mechanisms and hypothalamic circuits involved in the CNS effects of estrogen on energy homeostasis are only partially understood. Estrogen is known to attenuate weight gain post-ovariectomy in multiple rodent models primarily through an ER-dependent mechanism although there is evidence suggesting a role for another receptor-mediated mechanism (12,19). Furthermore, the multiple hypothalamic neuronal circuits that control feeding and metabolism are known to express ERs (20-24). This review will discuss the potential transcriptional and rapid response mechanisms estrogen has to control energy homeostasis and feeding including classical ERs and novel membrane receptors. While the nucleus of the solitary tract (NTS) in the brain stem is also involved in the control of feeding behaviour, this discussion is limited to the hypothalamic control of energy homeostasis and feeding because of the importance of estradiol in the control of multiple hypothalamic and homeostatic functions.

Over the past decade since the production of ER knockout mice strains, there has been conflicting evidence as to which ER subtype is involved in the effects of estrogen on energy homeostasis.

ERa

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Among the isoforms, ERalpha seems to be the major player in the central control of body weight by estrogens. Whether ERalpha mediates these effects via the regulation of food intake or actions on energy expenditure has been discussed controversially. Targeted deletion of ERalpha in mice resulted in an obese phenotype with increased fat accumulation but absence of marked differences in food intake between wild-type and knockout mice. In contrast, ovariectomy of mice or rats leads to a weight gain of 10-25% which has been shown to be associated with an increase in food intake [99,100]. These data suggest that estrogens reduce food intake which has been attributed to augment cholecystokinin-signalling and reduction in meal size [100]. It can be speculated that differences between genetic disruption of estrogen signalling during embryogenesis and ligand deficiency after birth account for this discrepancies. This controversy might be resolved by a recent study in which ERalpha was directly silenced in the VMN of adult mice by adeno-associated viral vector based injection of small hairpin (sh) RNA [101]. ERalpha silencing in the VMN resulted in an increase of food consumption as well as reduced energy expenditure caused by diminished physical activity and impaired thermogenic responses to feeding [101]. Importantly recently work published by Thammacharoen et al. [102] done on OVX-rats and -mice treated with E2 and PPT indicated a strong ERalpha-dependent inhibitory effect on eating behaviour observed in those animals, when compared to vehicle-treated littermates and ERalpha-deficient mice. Other hypothalamic regions might also be involved in the anorexigenic actions of estrogens. In summary, among the ER isoforms ERalpha is the major regulator of central energy homeostasis and its activation results in a reduction of food intake and increased energy expenditure contributing to the overall reduction of body weight by estrogens.

ERa knockouts exhibited an obesity phenotype while the ER² knockout mice did not (52,53). In general, studies in ±ERKO animals have found that females gain fat deposits at the expense of muscle mass, although there are some inconsistencies depending on the KO mouse model (52,54,55). ER± was involved in the oestrogenic decrease in white adipose accumulation (52). ER± was also deemed important for the attenuation of weight gain and food intake and potentiation of cholecystokinin (CCK) function by oestrogen (19) and is involved in the extrahypothalamic (NTS) control of food intake (56,56).

ERb

Although ER is expressed in the arcuate nucleus, the DMH and the lateral hypothalamus, the highest expression of ER in the hypothalamus is found in PVN primarily in the magnocellular division of the PVN (20-22). The expression of ER in the PVN is also differentially regulated during pregnancy and postnatal development (45) indicating that sex steroids regulate ER expression in the PVN. This is in accordance with data from ERbeta mice in which food consumption is similar with wild-type mice when fed a high fat diet [11].

Conversely, ER may have a central role in the control of energy homeostasis because co-administration of oestradiol with ER anti-sense oligodeoxynucleotides (ODN) into the third ventricle attenuated the inhibitory effects of oestradiol on food intake in ovariectomised rats while infusion of ER anti-sense ODN did not (57). Another study suggested a role for ER in adipose tissue accumulation based on an increase in weight gain and fat accumulation during oestrogen treatment in ER knockout mice (53).

Estrogen signaling in lipogenesis

Interestingly, fat accumulation is sexually dimorphic. Females have a higher percentage of body fat and tend to accumulate more SC fat than males, whereas males have a lower percentage of body fat and accumulate more visceral fat. Despite the higher level of body fat, female humans and rodents are more insulin sensitive than males. Thus, women have improved glucose tolerance and increased insulin sensitivity compared with men (17,18) and are more resistant to fatty acid-induced insulin resistance (8,19,20). Prevalence of early abnormalities of glucose metabolism is three times higher in men compared with women (7). Likewise, female mice are less prone to diet-induced insulin resistance (10,21,22), and many genetically induced forms of insulin resistance have a milder phenotype in females compared with males (11,21,22). Indeed, we find that the increased insulin sensitivity of female versus male mice can be detected on simple intraperitoneal glucose and insulin tolerance tests.

We find that adipocytes of female mice have increased lipogenic capacity compared with adipocytes from male mice and are also more insulin sensitive, especially those from the intra-abdominal (PG) depot, which are more insulin sensitive than female adipocytes from the SC depot and male adipocytes from both depots. This increased lipogenic capacity in female adipocytes is the result of at least two factors. One is enhanced insulin sensitivity at intermediate steps in the insulin signaling network such as stimulation of Akt and ERK. Thus, PG adipocytes from female mice show increased phosphorylation/activation of Akt at lower doses of insulin than male adipocytes, and Akt has been shown to be a key intermediate in insulin stimulation of glucose transport (23). The other factor is increased expression of glucose and lipid metabolism genes such as GLUT1, GLUT4, FAS, and ACC. Interestingly, despite the increased lipogenic rates, female adipocytes are smaller than male adipocytes, especially those from the PG depot, consistent with studies showing that females adipocytes have increased lipolytic rates compared with those from males (24,25). As a result, in humans, females have higher FFA serum levels than males (20) but appear to be protected against insulin resistance induced by elevated FFA (8). However, in mice, we did not detect any significant differences in the FFA serum levels between males and females.

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Our results, as well as previous studies (26), demonstrate that female adipocytes have increased insulin sensitivity compared with male adipocytes. This is particularly true for female PG adipocytes, which have a lower EC50 (0.1-0.2 nmol/l insulin) for insulin stimulation of lipogenesis than female SC adipocytes (0.5-0.6 nmol/l) and male adipocytes from either depot (0.4-0.7 nmol/l). Female PG adipocytes have a higher lipogenic rate than male adipocytes. Because PG fat in females also has a higher lipolytic capacity than in males (25), this would suggest a higher metabolic turnover of PG fat in females leading to decreased fat accumulation in visceral depots in females compared with males.

Sex steroids are known to play a role in the regulation of adipose tissue development and function as well as whole-body insulin sensitivity. Ovariectomy reduced lipogenic capacity in female adipocytes from both PG and SC depots and reduced insulin sensitivity in female PG adipocytes, indicating a positive role of estrogen in insulin sensitivity and lipogenesis in females. The exact mechanism of this effect is confounded by the fact that castration increases food intake and fat mass in females, and this could contribute indirectly to a reduction in insulin sensitivity.

Thus, an increase in circulating estrogen above the physiological levels preferentially alters PG adipose tissue metabolism, reducing lipogenic rates and depot size. These findings are in agreement with previous reports (27) and consistent with evidence that white adipocytes in different fat depots have fundamentally different properties and may have different developmental lineage (28). Which estrogen receptor (ER) is mediating these effects is unclear. In females, ER expression is similar between depots, whereas we and others found that the levels of ER are higher in the SC depot (data not shown) (29). Studies with knockout mice of ER or ER suggest that the effects of estrogen in the adipose tissue are mediated mainly through ER (30-32). The preferential effect of estrogen on adipocyte metabolism in the PG depot in females might also involve differences in coactivators such as the steroid receptor coactivator p160 family of proteins (33).

ERa

ERalpha-deficient mice exhibit increased adipose tissue mass in the absence of differences in energy intake suggesting a role of ERalpha in adipose tissue biology [8]. This notion is corroborated by data in 3T3-L1 pre-/adipocytes in which cells stably transfected with ERalpha showed attenuated triglyceride accumulation and reduced LPL expression [75].

ERb

We could recently show that ERbeta-deficient female mice have a higher body weight under high fat diet feeding than their wild type littermates. Higher body weight in ERbeta−/− mice resulted from enhanced adipogenesis and subsequent increased adipose tissue mass. Lipogenesis was not investigated in this study. However, we could demonstrate that PPARgamma, a key adipogenic and lipogenic factor, is negatively regulated by ERbeta suggesting also anti-lipogenic actions of this isoform.

Together, it appears that both ER isoforms participate in the antilipogenic actions of estrogens.

Estrogen signaling in glucose homeostasis and insulin tolerance

Studies in humans and rodents link estrogen to the maintenance of glucose homeostasis. Treatment of healthy postmenopausal women with estrogen has been shown to improve insulin sensitivity and to lower blood glucose. Additional observations in rodents support a notion that estrogen mediates anti-diabetic effects. For example, female rodents are protected against hyperglycemia unless they are ovariectomized in spontaneous rodent models of T2D. Studies in knock-out mouse models have shed light on the role of estrogen and its receptors in rodent obesity and glucose tolerance. Mice with functional knock-out of aromatase (ArKO mice) are unable to synthesize endogenous estrogen and display an obese and insulin resistant phenotype. A similar phenotype was observed in mice lacking ER (ERKO) but not in mice lacking ER (ERKO), indicating that ER is the major mediator for the estrogenic effects on insulin sensitivity and body weight.

The evolution of T2D requires the presence of defects in both insulin secretion and insulin action. Both defects have a recognized genetic background as well as an environmental component, where the lack of exercise and obesity play important roles. During the prediabetic phase with developed IR, the -cell hypersecretes insulin to maintain normal blood glucose levels provoking hyperinsulinemia. This hyperinsulinemia may produce an excess of insulin signaling in the liver, kidneys and ovaries, leading to hypertriglyceridemia.

At physiological levels, E2 is thought to be involved in maintaining normal insulin sensitivity and to be beneficial for -cell function. However, E2 levels above or below the physiological range may promote IR and type II diabetes.

Evidence that high E2 concentrations are important for blood glucose homeostasis has existed for a long time. In humans, the most consistent effects of oral contraceptives and estrogen replacement therapy (HRT) are decreased levels of fasting plasma glucose and impairedglucose tolerance.

Low levels of estrogen due to ovariectomy/menopause are associated with impaired glucose tolerance and IR and can be counteracted by HRT. Deficiency of estrogen signaling in men due to absence of ER or the key enzyme involved in estrogen production, aromatase, results in impaired glucose metabolism. Individuals with aromatase deficiency, due to mutations in the aromatase gene, have glucose metabolism impairment and IR. Similar abnormalities were found in aromatase-deficient (ArKO) mice. In humans it has been shown that polymorphisms in the ER gene are associated with T2D and metabolic syndrome.

ER and ER have both been suggested to be involved in energy balance and blood glucose homeostasis with ER being the main mediator.

ERa

ER knockout mice (ERKO) are obese and insulin resistant and ER has been shown to be involved in regulation of glucose metabolism by acting in different tissues including liver, skeletal muscle, adipose tissue, endocrine pancreas and the central nervous system (CNS).

The maintenance of glucose homeostasis is depending on whole body glucose uptake and glucose production by glycogenolysis and gluconeogenesis in the liver. Estrogens were shown to regulate liver glucose homeostasis and hepatic cholesterol output, mediated mainly via ER. Studies in ER-/-deficient mice indicate that ER plays the predominant role in the regulation of hepatic glucose homeostasis. The endogenous glucose production, assessed by euglycaemic-hyperinsulinaemic clamp analysis, revealed that ER deficiency was associated with a pronounced hepatic IR. Furthermore, microarray analysis of hepatic tissue isolated from ER-deficient and control mice revealed ER-dependent upregulation of key genes involved in hepatic lipid biosynthesis, and successive downregulation of the genes regulating lipid transport. Those findings are in consonance with studies in diabetic ob/ob mice showing that a major anti-diabetic effect of long E2-treatment is associated with decreased expression of lipogenic genes in the liver.

Glucose clearance in response to postprandial insulin secretion is mainly mediated by skeletal muscle. The insulin signaling pathways inducing sufficient glucose uptake in skeletal muscle are well studied and involve insulin receptor, insulin receptor substrate (IRS), phosphatidylinositol-3 kinase (PI3-K) and AKT kinase leading to subsequent translocation of Glut-4 to the cell membrane. ER and ER receptors seem to have opposing effects on the expression of Glut-4 transporters. ER was shown to induce and ER seems to inhibit-Glut-4 expression in skeletal muscle. Recent data indicates that tamoxifen-treated ER-deficient mice showed increased Glut-4 expression in skeletal muscle, which indicates pro-diabetogenic effects of ER. It appears that both ER isoforms determine metabolic estrogen actions in skeletal muscle where, in accordance with other tissues, ER mediates protective actions and ER deleterious.

Adipose tissue plays a major role in the regulation of glucose homeostasis and insulin sensitivity. There are well-documented sex differences in the pathophysiology of obesity and metabolic disorders. Women tend to accumulate more subcutaneous fat whereas men accumulate more visceral fat. The prevalence of early IR and impaired glucose tolerance seems to be higher in men than in women. Furthermore, increased abdominal obesity observed in postmenopausal women associated with IR can be improved by HRT. Together, these data implicate a central role of estrogens in adipose tissue biology.

In summary, it seems that the majority of previous reports point towards a direct anti-lipogenic and pro-lipolytic action of estrogens in adipose tissue. Whether these actions are mediated through ERa or ERb is currently unknown. ERa-deficient mice exhibit increased adipose tissue mass in the absence of differences in energy intake suggesting a role of ERa in adipose tissue metabolism.

Estrogens are known regulators of pancreatic b-cell function. A recently published study in mice suggested that long-term exposure to E2 increased insulin content, insulin gene expression, and insulin release without changing b-cell mass. ER± has been identified as the functional predominant receptor isoform in the murine pancreas. E2-dependent insulin release in cultured pancreatic islets was reduced in ER±-deficient mice, when compared to islets derived from either ER²-deficient or wt mice. However, ER²-deficient mice show mild islet hyperplasia and delayed first phase IR.

ERb

Estrogen signaling in cholesterol homeostasis

Accumulated evidence has revealed that estrogen increases the risk for the formation of cholesterol gallstones by promoting hepatic secretion of biliary cholesterol that induces an increase in cholesterol saturation of bile in humans and in several animal models of cholesterol gallstones [29-31,101-113]. In addition, observations from human and animal studies [101,105,108,114] have shown that high levels of estrogen significantly enhance the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in hepatic cholesterol biosynthesis, even under high dietary cholesterol loads. These findings suggest that there could be an increased delivery of cholesterol to bile from de novo synthesis in the liver. Furthermore, some studies in humans and animals observe that estrogen could augment the capacity of dietary cholesterol to induce cholesterol supersaturation of bile [100-103]. It is also found that high doses of estrogen augment intestinal cholesterol absorption [102], contributable in part to an up-regulated expression of intestinal sterol influx transporter Niemann-Pick C1 like 1 protein (NPC1L1) via the intestinal ESR1 pathway [115]. Because of species differences, NPC1L1 is expressed in the livers of humans and hamsters [116,117,118], but not in mouse livers [117,119,120]. Together with hepatic lipid transporters such as ABCG5/G8 on cholesterol secretion, ABCB11 on bile salt secretion and ABCB4 on phospholipid secretion [1], NPC1L1 may participate in the regulation of biliary cholesterol secretion. It has been found that ezetimibe treatment can prevent the formation of cholesterol gallstones in mice [117,119] and reduce cholesterol concentrations in bile in humans and hamsters [118,119]. In a preliminary, it is observed that E2 could promote biliary cholesterol secretion mostly through upregulating expression levels of ABCG5/G8 and ABCB11 in the liver. However, it needs to further investigate the effect of NPC1L1 on biliary cholesterol secretion under E2 treatment conditions. Despite these observations, little information is available on the metabolic abnormalities underlying major sources of the excess cholesterol leading to the supersaturation of bile and the formation of cholesterol gallstones induced by estrogen.

To investigate whether the high hepatic output of biliary cholesterol observed in E2-treated mice is the result of a higher rate of hepatic cholesterogenesis, the contribution of newly synthesized hepatic cholesterol to biliary output are measured [105]. Also, biliary secretion rates of total and newly synthesized cholesterol are observed over 4 hours in mice administered with [3H]water for 6 hours before the commencement of total biliary diversion. As a consequence of the surgery that ensures complete interruption of the enterohepatic circulation of bile salts, external biliary drainage in each mouse gave a "washout curve" as evidenced by measurement of bile salt secretion rate. It is observed that during the 4-hour period of interrupted enterohepatic circulation, biliary bile salt output gradually decreases with time. In contrast, cholesterol secretion rate is unaltered during the first 4 hours of biliary washout [121]. Hence, this 4 hour analysis provides biliary cholesterol output during simulation of an intact enterohepatic circulation. In addition, it has been reported that the proportion of biliary cholesterol derived from newly synthesized sources is not influenced by the amount of bile salts available for biliary secretion, and the rate of biliary output of both total and labeled cholesterol is constant during the first 4 hours of biliary washout [121]. These experiments further observe hepatic outputs of biliary total and newly synthesized cholesterol in female AKR mice with intact ovaries (i.e., control mice) and in ovariectomized mice treated with E2 or E2 plus ICI 182,780, and on chow or fed the high (1%) cholesterol diet for 14 days. In the chow-fed state, hepatic outputs of biliary total and newly synthesized cholesterol are ~5.0 µmol/h/kg and ~0.7 µmol/h/kg, respectively, and the relative contribution of newly synthesized cholesterol to biliary output is ~15% in control mice. E2 treatment induces a significant increase in hepatic outputs of biliary total (~8.0 µmol/h/kg) and newly synthesized cholesterol (~3.0 µmol/h/kg) and the relative contribution of newly synthesized cholesterol to biliary output is increased to ~45%. In addition, the high (1%) cholesterol diet slightly increases biliary total cholesterol output to ~7.0 µmol/h/kg in control mice, because the AKR mouse is a gallstone-resistant strain. However, the relative contribution of newly synthesized cholesterol to biliary total cholesterol output is reduced to ~6%. In contrast, the secreted newly synthesized cholesterol is 5-fold higher in E2-treated mice on the high cholesterol diet than those in control mice. Also, E2 treatment results in a significant increase in biliary total cholesterol outputs (~18.0 µmol/h/kg). Under these circumstances, the origin of biliary cholesterol possibly comes mostly from the high cholesterol diet and partly from lipoproteins such as HDL carrying cholesterol from extrahepatic tissues via a reverse cholesterol transport pathway. Furthermore, the biological actions of E2 are blocked by the antiestrogenic agent ICI 182,780. As a result, hepatic outputs of biliary total and newly synthesized cholesterol are essentially similar between ovariectomized mice treated with E2 plus ICI 182,780 and control mice, regardless of whether chow or the high cholesterol diet is fed.

To explore whether there is an "estrogen-ESR1-SREBP-2" pathway for the regulation of hepatic cholesterol biosynthesis, expression levels of the sterol regulatory element-binding protein-2 (Srebp-2) gene and five major SREBP-2-responsive genes in the liver are investigated by quantitative real-time PCR methods [105]. On the chow diet, high doses of E2 significantly increase the relative mRNA levels of the Srebp-2 gene. Furthermore, high dietary cholesterol significantly reduces expression levels of Srebp-2 by approximately 50% compared with the chow diet in control mice. These observations show that cholesterol biosynthesis may be inhibited by a negative feedback regulation possibly through the SREBP-2 pathway [122,123]. In contrast, E2-treated mice still display significantly higher expression levels of Srebp-2, even under high dietary cholesterol loads. These results indicate that under conditions of high levels of E2, mice continue to synthesize cholesterol in the liver because the negative feedback regulation of cholesterol synthesis by the SREBP-2 pathway may be inhibited by E2 through the hepatic ESR1. Again, these biological actions of E2 are abolished by the antiestrogenic ICI 182,780, regardless of whether chow or high dietary cholesterol is fed.

When expression levels of five major SREBP-2-responsive genes including HMG-CoA synthase (isoforms 1 and 2), HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, and lathosterol synthase in the liver are further investigated under the same experimental conditions as described above, it is observed that in control mice, expression levels of these SREBP-2-responsive genes are significantly reduced by the high cholesterol diet compared with the chow diet. Furthermore, high doses of E2 significantly up-regulate the relative mRNA levels for the SREBP-2-responsive genes in the liver, regardless of whether chow or high dietary cholesterol is fed. Again, these biological effects of E2 on expression levels of the SREBP-2-responsive genes are significantly attenuated by ICI 182,780. These findings support the notion that under the normal physiological conditions, there is a negative feedback regulation of cholesterol biosynthesis by cholesterol; however, under conditions of high levels of E2, these important regulatory effects are attenuated possibly by E2 via the hepatic ESR1 pathway. Obviously, these findings suggest a possible "estrogen-ESR1-SREBP-2" pathway that regulates hepatic cholesterol biosynthesis [105]. Furthermore, these results show that during estrogen treatment, mice continue to synthesize cholesterol in the face of its excess availability from the high cholesterol diet. It suggests that there is a loss in the negative feedback regulation of cholesterol biosynthesis, which results in excess secretion of newly synthesized cholesterol and supersaturation of bile that predisposes to cholesterol precipitation and gallstone formation [105].

In addition, estrogen could decrease plasma low-density lipoprotein (LDL) cholesterol and increase plasma high-density lipoprotein (HDL) cholesterol because high doses of E2 amplify expression levels of HDL receptor SR-BI and LDL receptor [124-127]. The decrease in plasma LDL is a result of increased hepatic LDL receptor expression, which increases the clearance of plasma LDL. Therefore, the increased uptake of LDL by the liver may result in increased secretion of cholesterol into the bile. These alterations could induce an apparent increase in hepatic output of biliary cholesterol derived from circulating lipoproteins such as HDL and LDL, although LDL cholesterol could have a less effect on biliary secretion.

Although estrogen has been proposed to be one important risk factor for cholesterol gallstones, the predominant mechanisms of lithogenic action of E2 depend on the ESR subtypes and the dose of hormone administered. Obviously, these observations suggest a critical role for ESR1 in E2-induced cholesterol gallstones. To explore whether deletion of the Esr1 gene decreases susceptibility to estrogen-induced cholesterol gallstones, ESR1 deficient mice are challenged to the lithogenic diet and treated with high doses of estrogen [128]. The ESR1 knockout mice in an AKR genetic background of gallstone-resistant strain are generated by targeting deletion of the Esr1 gene. At 4 weeks of age, ESR1 knockout and wild-type mice are gonadectomized. At 8 weeks, these mice are implanted subcutaneously with pellets designed to release E2 at 6 µg/day for 8 weeks. Under conditions of the lithogenic diet feeding, cholesterol crystallization and gallstone formation are greatly accelerated in wild-type mice when challenged to high levels of E2, mainly through up-regulating expression levels of the Esr1 gene in the liver. Compared with ESR1 knockout mice, E2-treated wild-type mice display significantly increased expression levels of mRNAs of SREBP-2 and other four major genes for cholesterol biosynthesis pathway, no matter if the chow or the lithogenic diet is fed. The increase in hepatic cholesterol synthesis could be associated with a significant increase in hepatic secretion of biliary cholesterol. However, the E2 effects on increasing cholesterol biosynthesis and promoting cholesterol gallstone formation are partially blocked by deletion of the Esr1 gene. The marked reduction in cholesterol synthesis correlates with the significant decrease in the amount of mRNAs of SREBP-2 and multiple genes for cholesterol biosynthesis and biliary cholesterol secretion in ESR1 knockout mice.

ERa

ERb

Early postnatal events can predispose to metabolic and endocrine disease in adulthood. In this study, we evaluated the programming effects of a single early postnatal oestradiol injection on insulin sensitivity in adult female rats. We also assessed the expression of genes involved in inflammation and glucose metabolism in skeletal muscle and adipose tissue and analysed circulating inflammation markers as possible mediators of insulin resistance. Neonatal oestradiol exposure reduced insulin sensitivity and increased plasma levels of monocyte chemoattractant protein-1 (MCP-1) and soluble intercellular adhesion molecule-1. In skeletal muscle, oestradiol increased the expression of genes encoding complement component 3 (C3), Mcp-1, retinol binding protein-4 (Rbp4) and transforming growth factor 1 (Tgf1). C3 and MCP-1 are both related to insulin resistance, and C3, MCP-1 and TGF1 are also involved in inflammation. Expression of genes encoding glucose transporter-4 (Glut 4), carnitine-palmitoyl transferase 1b (Cpt1b), peroxisome proliferator-activated receptor {delta} (Ppard) and uncoupling protein 3 (Ucp3), which are connected to glucose uptake, lipid oxidation, and energy uncoupling, was down regulated. Expression of several inflammatory genes in skeletal muscle correlated negatively with whole-body insulin sensitivity. In s.c. inguinal adipose tissue, expression of Tgf1, Ppard and C3 was decreased, while expression of Rbp4 and Cpt1b was increased. Inguinal adipose tissue weight was increased but adipocyte size was unaltered, suggesting an increased number of adipocytes. We suggest that early neonatal oestrogen exposure may reduce insulin sensitivity by inducing chronic, low-grade systemic and skeletal muscle inflammation and disturbances of glucose and lipid metabolism in skeletal muscle in adulthood.

Estrogen signaling in metabolic inflammatory processes

This hypothesis is supported by the fact that impaired glucose tolerance and type 2 diabetes are characterized by a low-grade inflammatory state (2). Several cytokines, mostly derived from the adipose tissue, have been demonstrated to directly impair the insulin signaling pathway in experimental models and hence play a role in the regulation of insulin resistance (19, 20). Along the same line of investigation, the deleterious metabolic effect of high-fat diet in mice, leading to the initiation of insulin resistance and glucose intolerance, has been recently demonstrated to be mainly mediated through the induction of inflammatory cytokine expression in adipose tissues and the liver (3). However, although estrogens are well recognized to exert regulatory functions on the immune system and the expression of cytokines (18), the hypothesis that these hormones could interfere with the early inflammatory response observed in animal models of insulin resistance and type 2 diabetes has not been addressed to date.

Our data clearly demonstrate that a chronic in vivo exposure to estrogens reduced the occurrence of HFD-induced insulin resistance in the mouse, leading to an improved adipose tissue and muscle glucose use rate and insulin signaling. Furthermore, we show that this beneficial metabolic effect occurs despite an increased inflammatory tone.

To our knowledge, the present study was the first to investigate the therapeutic effect of exogenous estrogens on diabetes, insulin resistance, and glucose intolerance in NCD- and HFD-fed wild-type mice. To date, all the animal studies that examined the influence of estrogens on glucose metabolism have been conducted in monkeys, rats, or transgenic mice fed with a normal chow or a moderately fat-enriched diet (10, 11, 25, 26, 27). Accordingly to these previous works, we demonstrate here that the beneficial effect of estrogens on insulin sensitivity and glucose tolerance in HFD-fed mice are mediated through ER{alpha} activation (15, 17). Conversely, ER appeared dispensable because ER-deficient mice demonstrated a normal glucose tolerance (28), although no complete metabolic study has been reported to date in this model. Interestingly, although our results indicate that the activation of the estrogen pathway prevents the occurrence and the course of insulin resistance and glucose intolerance resulting from high-fat feeding in wild-type mice, we did not observe any effect of either endogenous estrogens or chronic E2 administration on insulin sensitivity, and only a weak effect of E2 treatment on glucose tolerance, in our normal chow-diet experimental condition. This latter observation suggests that, despite a modest contribution to glucose homeostasis in a normal metabolic state, estrogens reveal their early and strong preventive or protective effect when the animals are exposed to a diabetogenic stress such as a HFD.

The second main finding of our study is that estrogens, especially chronic E2 administration to ovariectomized mice, enhance the expression of inflammatory factors induced by a HFD in tissues directly involved in insulin action and glucose metabolism, finally leading to the elevation of plasma cytokine concentrations. Furthermore, our observations in chimeric mice strongly suggest that the proinflammatory effect of E2, at least in visceral adipose tissues, results from the ER{alpha}-dependent activation of bone marrow-derived cells. These data fit nicely with the conclusion from recent animal studies reporting the proinflammatory influence of estrogens in vivo. Indeed, despite some in vitro studies suggesting that estrogens could exert antiinflammatory properties on several immune cells, in vivo E2 administration was clearly demonstrated to enhance the production of numerous proinflammatory cytokines by conventional antigen-specific CD4+ T lymphocytes, and natural killer T cells, as well as antigen-presenting cells (24, 29, 30).

Interestingly, in HFD-fed mice, the early inflammatory response was recently shown to result from an increase in plasma lipopolysaccharide concentrations, which lead to the activation of CD14-Toll-like receptor 4-expressing cellular targets (3, 31). Among them, macrophages are known to infiltrate both the liver and adipose tissues and thus to largely contribute to the secretion of inflammatory mediators (19). Interestingly, in our model, macrophage density in adipose tissues was significantly increased by E2 treatment. Thus, it is tempting to speculate that a direct effect of estrogens on these pivotal cells of the innate immune system could explain the increase in cytokine expression induced by both endogenous estrogens and E2 substitution in ovariectomized mice. Supporting this hypothesis, monocytes/macrophages, as lymphocytes and dendritic cells, express ERs, especially ER{alpha} (18). Furthermore, contrasting with previous in vitro studies suggesting that E2 exerts antiinflammatory properties on lipopolysaccharide-activated monocyte/macrophage cell lines, we recently demonstrated that in vivo E2 administration increases the capacity of murine peritoneal macrophages to produce proinflammatory cytokines (IL-1, IL-6, and IL-12) on Toll-like receptor 4 stimulation (32), as previously reported in microglial cells, the resident macrophages of the brain (30). This proinflammatory effect of in vivo exposure to estrogens is mediated through ER{alpha} activation and results from an increase in the activity of the nuclear factor-{kappa}B pathway (32).

However, because the low-grade inflammatory tone induced by high-fat feeding initiates insulin resistance and glucose intolerance (2, 3) and because inflammatory cytokines are known to impair insulin signaling, the proinflammatory effect of estrogens should be considered as controversial. However, bone marrow graft experiments indicated that the main part of this proinflammatory effect results from a direct hormonal role on hematopoietic cells. This allow us to conclude that the beneficial metabolic influence of estrogens cannot be explained by the modulation of cytokine expression but results from a direct targeting of nonhematopoietic cells that overcome the deleterious effect of HFD-induced inflammation. In this line, despite their proinflammatory potential, estrogens have been demonstrated to exert protective effects in several inflammatory disease models, preventing target organs or tissues (24, 33). Thus, the inflammatory potential of the actors of the inflammatory-immune system can be balanced by the action of E2 on extramedullar cell targets, which should be now investigated.

Although ER{alpha} signaling has been demonstrated to regulate weight gain and adiposity, as confirmed by our observations in mice fed with the HFD for 3 months, we found that, after 1 month of high-fat feeding, E2 administration improved insulin sensitivity and glucose tolerance without any significant effect on body weight. Alternatively, previous studies reported that ER{alpha} signaling improves insulin sensitivity through the modulation of lipogenic genes and signal transducer and activator of transcription 3 in the liver (26, 28) and through the regulation of IRS-1 phosphorylation or intra- vs. extracellular resistin distribution in 3T3-L1 adipocytes (34, 35), but the cellular and molecular mechanisms underlying the effect of estrogens on insulin action remain poorly understood. In the present study, E2 administration did not influence plasma triglyceride and FFA concentrations as well as triglyceride content and phosphoenolpyruvate carboxykinase mRNA abundance in the liver (data not shown), and circulating adiponectin levels were significantly decreased in E2-treated mice, as recently reported (27). Furthermore, as described above, our experiments ruled out the hypothesis that estrogens could exert their beneficial metabolic effect through the downmodulation of the inflammatory response to high-fat feeding. In contrast, the demonstration that E2 enhances IRS-1 expression and Akt phosphorylation in insulin-stimulated muscles suggests that the estrogens pathway could directly interfere with insulin signaling in peripheral insulin-sensitive tissues. However, the mechanisms leading to the regulation of IRS-1 expression remains to be clarified because the effect of E2 was observed only after insulin stimulation. Interestingly, AMPK{alpha} phosphorylation in muscles was also increased by E2 treatment, suggesting that alternative pathways could be responsible for the improved metabolic status.