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Metabolic diseases affect more than 230 million people worldwide with an expected increase to around 350 million in 25 years, which makes it the fourth leading cause of death by disease. Type 2 diabetes (T2D) is a heterogeneous metabolic disorder diagnosed by imbalance in glucose homeostasis, which stems from impaired insulin secretion, defects in insulin action or from both. Hyperglycemia is often associated with other serious health complications, such as liver and intestine toxicities, renal dysfunction, dyslipidemia and cardiovascular disorders.
Estrogen and the estrogen receptors (ERs) are well-known regulators of glucose homeostasis. Premenopausal women are more sensitive to insulin and have improved glucose tolerance, as well as are less prone to develop insulin resistance (IR) compared to men (Kuhl, et al. 2005; Macotela et al. 2009). In healthy postmenopausal women, hormone replacement therapy (HRT) has been shown to lower circulating glucose levels and to improve insulin sensitivity. The development of T2D in postmenopausal women with coronary heart diseases is also reduced upon HRT administration.
Carbohydrate catabolism starts with digestion in the small intestine where monosaccharides are absorbed into circulation. The most important carbohydrate is glucose, which is metabolized by most organisms. Circulating levels of glucose are controlled by three hormones; insulin, glucagon and epinephrine. When circulating levels of glucose is raised, insulin is secreted by the pancreatic ï¢ cells to stimulate glucose uptake into liver and muscles, where excess glucose is stored as glycogen by the process of glycogenesis. When blood glucose levels decrease, glucagon is secreted to stimulate the breakdown of glycogen to glucose through glycogenolysis.
The ï¢ cells in the pancreatic islets of Langerhans release insulin in two phases. The first is a rapid response to increased blood glucose levels and the second phase is a slow release that is triggered independently of glucose. There are several substances apart from glucose known to stimulate insulin release, including amino acids from dietary proteins, acetylcholine released from vagus nerve endings, gastrointestinal hormones and glucose-dependent insulinotropic peptides.
The hormone glucagon is secreted from the ï¡ cells in the pancreatic isets of Langerhans and probes conversion of hepatic glycogen into glucose, which is subsequently released into the blood. Muscle cells lack the ability to release glucose into circulation. The output of glucagon is triggered by low levels of circulating glucose. Other factors, for example growth hormone, cortisol and epinephrine also display glucose regulatory actions similar to glucagon.
IR is a physiological disease condition where insulin is less effective at lowering circulating glucose. IR in muscle and adipocytes reduces glucose uptake whereas hepatic IR results in reduced glycogen synthesis and storage and a failure to suppress glucose production and subsequent release into the blood. IR commonly refers to the reduced glucose lowering effects of insulin described above, however, other functions of insulin are also affected. For example, IR in adipocytes results in reduced uptake of circulating lipids and increased hydrolysis of stored triglycerides, which leads to elevated levels of circulating free fatty acids. High plasma levels of insulin, glucose and lipids due to IR are a major component of the metabolic syndrome, which could develop into T2D.
1.1 Estrogen signaling and estrogen receptors
Estrogens exert their physiological effects through two estrogen receptor (ER) subtypes, ERα and ERβ, which are members of the steroid receptor gene superfamily of the nuclear receptors. ERï¡ is mainly expressed in reproductive tissues, kidney, bone, white adipose tissue and liver, while ERï¢ is expressed in the ovary, prostate, lung, gastrointestinal tract, bladder and hematopoietic and the central nervous system (CNS) (Matthews and Gustafsson 2003).
ERs share a common structure with the other members of the nuclear receptor family. The N-terminal A/B domain is the most variable region with less than 20% amino acid identity between the two ERs, and could confer subtype specific actions on target genes. This region harbors the activation function-1 (AF-1) that is ligand-independent and shows promoter- and cell-specific activity. The centrally located C-domain harbors the DNA binding domain (DBD), which is involved in DNA binding and receptor dimerization. This domain is highly conserved between ERï¡ and ERï¢ with 95% amino acid identity. The D-domain is referred to as the hinge domain and shows low conservation between ERï¡ and ERï¢ (30%). This domain has been shown to contain a nuclear localization signal. The C-terminal E-domain is the ligand-binding domain (LBD) and the two subtypes display 59% conservation in this region. The LBD contains a hormone-dependent activation function (AF-2) and is responsible for ligand binding and receptor dimerization. The F-domain has less than 20% amino acid identity between the two ER subtypes and the functions of this domain remain undefined (Zhao, et al. 2008).
Fig. 1. Structure and homology degree between ERï¡ and ERï¢. The A/B domain is referring the ligand independent transcription activation function-1 (AF-1). The C domain is mediating DNA binding and D domain represents a hinge and harbors nuclear localization signals. The E domain contains the ligand dependent AF-2 function, which is involved in ligand binding.
Estrogens are sex steroids, which stem from the common pre-cursor cholesterol. The last step in the synthesis of estrogen from androgens is catalyzed by P450 aromatase. The three major estrogens include 17ï¢-estradiol (E2), estrone and estriol. The major physiological estrogen is E2, which has a similar affinity for both ERs. In addition, ERs are activated by a range of ligands including selective estrogen receptor modulators (SERMs) such as raloxifen and tamoxifen, the ERα selective agonist propyl-pyrazole-triol (PPT) and the ERβ-selective agonist diarylpropionitrile (DPN), as well as many other compounds (Heldring, et al. 2007).
Like other nuclear receptors, ligand-bound ERs act as dimers to regulate transcriptional activation. Full transcriptional activity of the ERs is mediated through a synergistic action between the two activation domains, AF-1 and AF-2. Both ERï¡ and ERï¢ contain a potent AF-2 function, but unlike ERï¡, ERï¢ seems to have a weaker corresponding AF-1 function and depends more on the ligand-dependent AF-2 for its transcriptional activation function (Dahlman-Wright, et al. 2006). In their unliganded state, ERs are associated with protein complexes of heat shock proteins, which inhibit their functions. The classical estrogen signaling occurs through a direct binding of ligand activated ER dimers to estrogen-responsive elements (EREs) in the regulatory regions of estrogen target genes followed by activation of the transcriptional machinery at the transcription start site. Estrogen also modulates gene expression by a second mechanism in which ERs interact with other transcription factors, such as activating protein-1 (AP-1) and stimulating protein-1 (Sp-1), through a process referred to as transcription factor cross-talk. Estrogen may also elicit effects through non-genomic mechanisms, which involve the activation of downstream signaling cascades like protein kinase A (PKA), protein kinase C (PKC) and mitogen-activated protein (MAP) kinase via membrane-localized ERs.
Recently, an orphan G protein-coupled receptor (GPR) 30 in the cell membrane was reported to mediate non-genomic and rapid estrogen signaling (Revankar, et al. 2005; Thomas, et al. 2005). GPR30 is structurally unrelated to ERï¡ and ERï¢ and the rapid effects from stimulation of this receptor include release of intracellular Ca2+ and subsequent activation of calcium-calmodulin-dependent kinases or activation of MAP kinase and phosphoinositide 3-kinase pathways. Human GPR30 is located in chromosome 7p22.3, and is composed of three exons. Exon3 coincides with the amino acid coding region of GPR30. Based on linkage analysis, the region of the chromosome containing GPR30 is thought to be related to familial hypertensive disease in humans. The mRNA for GPR30 appears to be expressed extensively in most tissues as judged from the overall reports.
Fig. 2. Estrogen signaling mechanisms. I. Classical pathway involving activation of the ERs followed by ERE binding. II. Non-classical pathway involving interactions with transcription factors and subsequent indirect ERE binding. III. Non-genomic pathway involving GPR30.
1.4 Estrogen signaling in glucose homeostasis
Estrogen and estrogen signaling have long been known to be important regulators of glucose homeostasis and are implicated in maintaining normal insulin sensitivity. Fluctuations in estrogen levels below the physiological range, as a consequence of menopause or ovariectomy, may promote IR and T2D. In humans, the most consistent effects of oral contraceptives or HRT are decreased levels of fasting plasma glucose and impaired glucose tolerance. Absence of estrogen signaling in men, due to deficiency of the aromatase enzyme or ERα, results in impaired glucose metabolism. It has also been shown that polymorphisms in the ERα gene are associated with development of the metabolic syndrome and T2D.
Estrogens further display inhibitory effects on maltase, sucrose and lactase activities in the intestine. Estrogen supplements have been shown to manifest a range of disaccharidase and lipase inhibitory actions that help to delay the absorption of dietary carbohydrates in the intestine, which will lead to suppression of the increased glucose levels observed after meals.
Several rodent studies link estrogen to glucose regulatory effects. Female mice are protected against hyperglycemia and aromatase knockout (ArKO) mice display severe IR (Jones, et al. 2000). ERα and ERβ have both been suggested to be involved in blood glucose homeostasis. ERα knockout mice (ERαKO) are insulin resistant and ERα 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).
Fig. 3. Estrogen regulation of glucose homeostasis. Bla bla…
1.5 ERs and the role of the central nervous system for glucose homeostasis
The first finding supporting that the central nervous system (CNS) was involved in the regulation of glucose homeostasis was that ruptures in the fourth ventricle resulted in glucosuria (11). This initial study was followed by numerous of others and it is now firmly established that the CNS regulate glucose homeostasis by the hormones insulin, leptin and glucagon-like peptide (GLP)-1, as well as by glucose and fatty acids (FA). A series of complex systems regulate energy homeostasis in order to keep energy levels and body weight stable (Miller DS. Proc. Nutr. Soc. 41(2), 193-202 (1982)). Glucose is the vital energy source for the brain. There are several glucose sensing neurons in the hypothalamus, which have been established to be essential components in the regulation of feeding behavior and hypoglycemic counter regulatory responses. Central brain circuits receive signals from the periphery to indicate satiety, energy levels and energy stores (Morton, Cummings, Baskin, Nature, 2006). The hypothalamus then processes the afferent signals from the gut and brainstem and efferent signals that modulate food intake and energy expenditure. The hypothalamus is subdivided into interconnecting nuclei, including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial nucleus (VMN), dorsomedial nucleus (DMN) and lateral hypothalamic area (LHA) (Simpson, Martin, Bloom, Expert Rev Endocrinol Metab. 2008;3(5):577-592).
The actions of insulin have also been shown to play a role in the CNS since neuron-specific insulin receptor deficient (NIRKO) mice develop mild IR and elevated circulating insulin levels are highly associated with obesity. Injections of insulin directly into the third cerebral ventricle are shown to suppress hepatic glucose production but not to affect body weight or circulating levels of insulin. Further, by inhibiting insulin or its downstream signaling pathway in the CNS (the insulin receptor and phosphatidylinositol-3 kinase (PI3K)), the suppression of glucose production by increased levels of insulin was impaired. Targeted deletion of insulin receptor expression selectively in the hypothalamus induced IR in rats, which is in accordance to the results from the NIRKO mice studies. These studies show that the CNS regulate glucose homeostasis through insulin actions and requires intact insulin signaling pathways involving the binding of insulin to its receptor and subsequent activation of down-stream mediators.
Estrogen is known to be highly involved in the regulation of satiety, energy expenditure and body weight. Ovariectomy and menopause induce an increase in appetite and food intake, which can be reversed with estrogen replacement therapy. The anorectic effects of estrogen are partially mediated through actions in the hypothalamus as proved by direct E2 injections into the PVN area or the ARC/VMN of the hypothalamus effectively reduced food intake. The same study also showed that the hypothalamic neurons, which regulate energy homeostasis were affected by E2 administration. Energy homeostasis and feeding behavior in the hypothalamus also follows the menstrual cycle and food intake in women varies across the cycle with the lowest daily food intake during the peri-ovulatory period when estrogen levels are peaking (Asarian L, Geary N, Philos Trans R Soc Lond B Biol Sci. 2006).
ERα and ERβ are both expressed in the different areas of the hypothalamus. ERα is the major mediator of the estrogenic effects on central regulation of body weight by estrogens but whether this is regulated by food intake or actions on energy expenditure is controversial. Total ERα knockout mice are obese with increased fat accumulation in the absence of increased food intake. Targeted disruption of ERα in the VMN areas in the hypothalamus of female mice leads to weight gain, increased visceral adiposity, hyperphagia, hyperglycemia and impaired energy expenditure.
ERβ knockout mice display similar food consumption patterns as wild-type mice when fed a high fat diet. In contrast, ovariectomized wild-type mice lead to a 10-25% weight gain, which is associated with increased food intake.
1.6 ERs and the role of pancreatic ï¢ cells in glucose homeostasis
The endocrine pancreas is an adapting tissue with capability to quickly respond to the variations in metabolic state of the organism. The β cells in the islets of Langerhans readily adapt to peripheral IR by increasing their secretory response, as well as their cell mass. If β cells fail to adapt, blood glucose concentration will rise to pathological levels. As a consequence metabolic disturbances, and ultimately type II diabetes, will develop.
E2 has been shown to acutely enhance glucose stimulated insulin secretion at physiological concentrations, both in vitro and in vivo (Nadal et al. 1998; Alonso-Magdalena et al. 2006). It has further been demonstrated that E2 triggers the synthesis of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G (PKG). ATP-dependent potassium channels (KATP) then close in a PKG-dependent manner, causing the plasma membrane to depolarize and enhancing glucose-induced [Ca2+]i signals (Ropero et al. 1999). Very likely this process is responsible for the E2-induced insulin secretion mentioned above (Nadal et al. 1998). Additional to its effect on insulin release, the increase of [Ca2+]i by E2 is involved in the rapid activation of cAMP-response element binding protein (CREB) (Quesada et al. 2002), a key transcription factor involved in β cell division and survival (Jhala et al. 2003; Hussain et al. 2006; Jansson et al. 2008). The insensitivity of these rapid responses to the anti estrogen ICI182,780 as well as the different pharmacological profile of a membrane binding site identified in β cells suggest that a non-classical membrane estrogen receptor (ncmER) was responsible for these actions (Nadal et al. 2000; Ropero et al. 2002). Indeed, two membrane molecules have been described as behaving like ncmERs in β cells and therefore they may be the ncmER previously reported (Nadal et al. 2000): the sulphonylurea receptor (SUR1) expressed in β-cells and GPR30. It is of note, however, that both molecules trigger their actions at pharmacological rather than physiological E2 concentrations. Binding to SUR1 and regulation of apoptosis was demonstrated for E2 concentrations as high as 100 μm (Ackermann et al. 2009). Therefore, a physiological role for SUR1 as ncmER is still undemonstrated. Recently, GPR30 was proposed as a novel estrogen receptor (Revankar et al. 2005; Thomas et al. 2005). It is present in β cells and it mediates rapid E2-induced insulin release, although only at supraphysiological concentrations of E2 (5 μm) (Martensson et al. 2009). A new role for GPR30 in the protection of β cells from apoptosis has been recently described at 10 nm E2 (Liu et al. 2009). In any case, whether GPR30 works in β cells as a proper estrogen receptor or it is recruited by membrane ERs (Levin, 2009) is still a matter of debate.
In addition to the acute effects, it has been known for a long time that E2 exerts long-term regulation of insulin biosynthesis (Sutter-Dub, 2002). It has been demonstrated that direct activation of ERα regulates pancreatic insulin levels at physiological concentrations in vivo and in vitro (Alonso-Magdalena et al. 2008). Together with the increase of insulin biosynthesis, islets treated with E2 displayed enhanced glucose stimulated insulin secretion (Adachi et al. 2005; Alonso-Magdalena et al. 2006, 2008).
Estrogenic effects on various physiological aspects of the islet of Langerhans have been known for a long time and estrogens are known regulators of pancreatic ï¢ï€ cell functions. In humans, E2 reverses the effect of menopause on glucose and insulin metabolism, resulting in increased pancreatic insulin secretion, as well as improved IR. Plasma insulin levels are increased in pregnant rats in response to increased levels of estrogen. A recently published study in mice suggested that long-term exposure to E2 increased insulin content, insulin gene expression, and insulin release without changing cell mass.
ERα has been identified as the functional predominant receptor isoform in the murine pancreas. E2-dependent insulin release in cultured pancreatic islets was reduced in ERα-deficient mice, when compared to islets derived from either ERβ-deficient or wt mice. Also, E2 acting mainly through ERα protects pancreatic β cells from apoptosis induced by oxidative stress in mice studies. However, ERβ-deficient mice also display a mild islet hyperplasia and delayed first phase IR.
The membrane bound estrogen-responsive GPR30 is also expressed in the pancreatic islet cells in mice. Studies using adult female GPR30-deficient mice reveal that these mice do not exhibit E2-induced release of insulin, which is consistent with experiments using isolated pancreatic islet cells in vitro. There are no differences in the expressions of glucose-related genes, such as GLUT2 and glucokinase in GPR30 knockout mice as compared with wild type mice. Thus, GPR30 may act as a regulator in the process of insulin release after E2 stimulation. GPR30 mRNA is also expressed in secretory gland cells, which may indicate that GPR30 may be involved in insulin secretion pathways. It has been reported, however, that the regulation of blood glucose levels is unaffected in young GPR30-deficient mice.
1.7 ER and the role of the liver in glucose homeostasis
The liver is the largest organ in the body and possesses purifying and metabolizing functions. One of the most important tasks is to store glucose in the form of glycogen. The liver is capable of containing up to 10% of its volume in glycogen. The liver releases glycogen when nutrients are scarce, as well as regulates the amount of circulating glucose. Liver glycogen is converted into circulating glucose in response to pancreatic signals; in hypoglycemic conditions glucagon is released to stimulate a release of hepatic glycogen. In a hyperglycemic state, the pancreas releases insulin to stimulate the liver to release less glucose. The maintenance of glucose homeostasis is depending on whole body glucose uptake and glucose production by glycogenolysis and gluconeogenesis in the liver.
Estrogens regulate liver glucose homeostasis and hepatic cholesterol output mainly by acting via ERα as shown by studies using ERα-deficient mice. By using euglycaemic-hyperinsulinaemic clamp analysis, the endogenous glucose production revealed that ERα deficiency was associated with a pronounced hepatic IR. Global gene analysis of hepatic tissue isolated from ERα-deficient and control mice revealed ERα-dependent increase of key genes involved in hepatic lipid biosynthesis, and successive decrease 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 hepatic lipogenic genes.
Fig. 4. Schematic overview of liver metabolism.
1.8 ER and the role of skeletal muscle in glucose homeostasis
Skeletal muscle accounts for 40-60% of human body mass and is the major site of glucose disposal, thereby regulating whole body glucose homeostasis. IR in skeletal muscle is thought to be a primary defect in T2D. Insulin-stimulated circulating glucose disposal is mediated through glucose transport across the muscle cell surface and this step is one of the rate-limiting steps for glucose clearance. Glucose crosses the plasma membranes and enters the skeletal muscle through the glucose transporters (GLUT) by facilitated transport. 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 (see fig. 5 for schematic overview).
Fig. 5. Glucose transport in muscles.
ERα and ERβ receptors seem to have opposing effects on the expression of GLUT-4 transporters. ERα was shown to induce and ERβ seems to inhibit GLUT-4 expression in skeletal muscle. Recent studies indicate that tamoxifen-treated ERα knockout mice displayed increased GLUT-4 expression in skeletal muscle, which indicates a pro-diabetogenic effect of ERβ. It appears that both ER isoforms determine the metabolic estrogen actions in skeletal muscle where, in accordance with other tissues, ERα mediates protective actions and ERβ deleterious. Targeted knockout of ERβ in male mice has also been shown to protect against diet induced IR by increasing PPARγ signaling in adipose tissue (Barros, Gustafsson, Am J Physiol Endocrinol Metab. 2009 Jul;297(1):E124-33. Epub 2009 Apr 14)(Foryst-Ludwig, Clemenz, Barros, Gustafsson, Plos Genetics 2008).
1.9 ER and the role of adipose tissue in glucose homeostasis
Adipose tissue is formed by mature adipocytes, stromal pre-adipocytes, immune cells, extracellular matrix and vascular endothelium. The infiltration of macrophages promotes adipose tissue IR, excessive release of free fatty acids (FFA) and fat accumulation. During nutritional excess, hypertrophic adipocytes develop a gene expression pattern that closely resembles that of fat-loaded activated macrophages found in arterial plaques.
Under normal physiologic conditions, insulin concentrations control within a narrow range the balance between fatty acid storage as triglycerides and their release into the circulation during the fasting state. Adipose tissue is very sensitive to insulin concentrations and inhibits lipolysis at insulin concentrations that are much lower than those needed to inhibit hepatic glucose production or stimulate muscle glucose uptake.
Fig. 6. Adipocytes and estrogen.
In obesity and T2D, there is a marked adipocyte resistance to the anti-lipolytic effects of insulin and the circulating FFA concentrations are typically elevated. Chronic over feeding induces a metabolic stress and the adipocytes become hypertrophic and fail to proliferate and differentiate in an adequate way. 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.
When circulating levels of estrogen are high, adipose tissue metabolism is altered resulting in reduced lipogenic rates and fat depot size. ERα-deficient mice exhibit an increased adipose tissue mass without displaying differences in energy intake, suggesting that ERα plays an important role in adipose tissue biology (Heine, Taylor, Iwamoto, PNAS, 2000). This is further supported by studies in 3T3-L1 pre-/adipocytes with stably transfected ERα which show decreased triglyceride accumulation and reduced expression of lipoprotein lipase (LPL), the enzyme that catalyzes the conversion of triglycerides into FFA and glycerol (Homma, Kurachi, Nishio, Takeda, JBC, 2000). Epidemiological observations also demonstrate that serum triglyceride levels increase in postmenopausal women and that the level of LPL activity is reduced by estrogen treatment (Iverius, P. H., and Brunzell., J. D. (1988) J. Clin. Invest. 82, 1106-1112).
E2 is known to suppresse lipogenesis and triglyceride accumulation in adipose tissue and liver in high-fat diet (HFD) fed and leptin deficient female mice (Bryzgalova, Lundholm, Portwood, Am J Physiol End Met 2008). Global genome wide analysis of livers from ERα-deficient and wild-type mice revealed a ERα-dependent increase in the expression of lipogenic genes and a decreased expression of genes regulating lipid transport (Bryzgalova, Gao, Diabetologia 2006). Conversely, E2 suppressed the expression of lipogenic genes in livers of leptin deficient Ob/Ob mice (Gao, Bryzgalova, Hedman, MolEndo, 2006). Studies using ERï¢-deficient female mice indicate that these mice increase more in body weight under HFD feeding than their wild-type littermates (Foryst-Ludwig, Clemenz, Hohmann, Hartge, Plos Genetics, 2008). The higher body weight in ERï¢ knockout mice is a result from increased adipogenesis and subsequent increased in adipose tissue mass and is associated with improved insulin sensitivity. Further, the key adipogenic and lipogenic factor PPAR was negatively regulated by ERï¢ suggesting that PPAR could be a mediator of the metabolic effects observed in ERï¡ knockout mice (Kintscher, Foryst-Ludwig).
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, actions that are mediated through both ER subtypes.