How Estrogens Prevent From Lipid-Induced Insulin Resistance

Premenopausal women have generally lower amounts of central adipose tissue and lower incidence of obesity-related metabolic abnormalities, which are frequently summarized as metabolic syndrome. Classical components of the metabolic syndrome are linked to impaired insulin action, also termed insulin resistance. Several mechanisms are currently discussed to underlie insulin resistance and comprise abnormal lipid metabolism with ectopic accumulation of triacylglycerols (TAGs), termed lipid overflow and lipotoxicity (1, 2), abnormal mitochondrial function (3), and oxidative stress as well as inflammation and endoplasmic reticulum stress (4). All these abnormalities can contribute to increased cardiovascular morbidity and mortality. Indeed, it is well known that premenopausal women exhibit a lower risk of cardiovascular mortality than age- and body-mass-index–matched men. This protective effect is lost in the postmenopausal state, when the circulating estrogen levels decline and women become more susceptible to obesity-associated insulin resistance. Decreasing estrogen production in female animal models by ovariectomy leading to a surgical menopause or by gene manipulation leading to down-regulation of estrogen synthesis has similar effects. These observations imply a relevant role of estrogens in the regulation of glucose metabolism and in the pathogenesis of insulin resistance. During the reproductive period, 17ß-ethinyl-estradiol is the predominant estrogen and exerts its physiological actions through the two estrogen receptors (ERs), ERα and ERβ. Although estrogen mainly acts via nuclear estrogen-responsive elements and recruitment of other coregulatory proteins to regulate gene transcription in all metabolically important tissues, there is also evidence for nongenomic effects via membrane-bound ER (reviewed in Ref. 5). Global ERα deletion leads to increased body weight and insulin resistance, suggesting that this receptor is of critical importance for the metabolic estrogen action. In adipose tissue, 17ß-ethinyl-estradiol was found to prevent fat (TAG) accumulation due to suppression of its target gene, lipoprotein lipase (LPL) an enzyme catalyzing lipogenesis. Aside from lower lipogenesis, 17ß-ethinyl-estradiol increases expression of hormone-sensitive lipase (HSL), resulting in increased lipolysis, and stimulates lipid oxidation by increasing phosphorylation of AMP-activated protein kinase (AMPK) and expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), and uncoupling protein 2 (UCP2) (5). In the liver, 17ß-ethinyl-estradiol can reduce TAG accumulation, gluconeogenesis, and inflammatory pathways. Using ERα inactivation showed that estrogens modulate functions of macrophages, of pancreatic ß-cells to maintain insulin secretion via an antiapoptotic activity, and of hypothalamic neurons involved in energy homeostasis, maybe by regulating caloric intake and/or energy expenditure. In skeletal muscle, 17ß-ethinyl-estradiol improves glucose disposal by modulating expression and the insulin-sensitive trafficking of glucose transporters [glucose transporter 4, (GLUT4)]. Finally, 17ß-ethinyl-estradiol is thought to protect endothelial function and induce anti-inflammatory effects in vascular tissue. Despite its multiplicity and complexity of effects, the exact molecular mechanisms linking 17ß-ethinyl-estradiol with improved insulin signaling and whole-body glucose and lipid disposal have not yet been resolved. Recently, several research teams addressed the role of 17ß-ethinyl-estradiol on a systemic level by assessing whole-body and tissue-specific insulin resistance in ovariectomized rodents with and without a high-fat diet (HFD). Of note, estrogen deficit by ovariectomy does not affect body weight and slowly impairs insulin sensitivity after 26 weeks (6). Hypercaloric diets enriched in saturated fats such as HFD not only consistently and rapidly reduce insulin sensitivity in rodents but also mirror key features of the pathogenesis of the metabolic syndrome in humans. Thus, the HFD model is used to accelerate and enhance the metabolic effects of 17ß-ethinyl-estradiol deficiency and repletion (Figure 1). Tissue-specific stimulatory (+, green) and inhibitory (−, red) effects of 17ß-ethinyl-estradiol with relationship to glucose and lipid metabolism. Ovariectomized mice on HFD for 10 weeks showed greater body weight and lower overall insulin sensitivity, as roughly assessed by the insulin tolerance test, which was reversed in mice receiving dietary supplementation of 17ß-ethinyl-estradiol (7). This improvement was accompanied by beneficial modulation of adipocyte biology, such as reduced adipocyte size, lower expression of the inflammatory markers TNFα and monocyte chemoattractant protein 1 and a lower degree of oxidative stress as assessed from phosphorylated histone variant H2AX-stained cell nuclei (Figure 2). Interestingly, serum TAGs were lower, whereas free fatty acids were also increased likely due to augmented lipolysis, in line with the observed increase in activity of HSL and expression of adipose triglyceride lipase (ATGL). Finally, liver histology and serum transaminases indicated that 17ß-ethinyl-estradiol replacement could also improve liver steatosis. Taken together, this paper suggests that the local effects in adipose tissue will result in lowering systemic inflammation and thereby ameliorate whole-body glucose. Why this effect is seen in the presence of higher circulating free fatty acids, which are generally accepted to induce insulin resistance, remained unanswered. Metabolic effects of 17ß-ethinyl-estradiol as derived from studies in hepatocytes, adipocytes, and skeletal myocytes of HFD-fed ovariectomized female mice. Direct (peripheral) effects are indicated by blue diamonds showing the responsible ER, whereas indirect (central nervous) effects are indicated by blue flash symbols. The pathways are described in detail in the text. Abbreviations: ACC, acetyl-coenzyme A carboxylase; ATGL, adipose triglyceride lipase; FAS, fatty acid synthase; G6P, glucose-6-phosphate; GLUT, glucose transporter; IR, insulin receptor; IRS, IR substrate; LPL, lipoprotein lipase; MCP1, monocyte chemoattractant protein 1; PEPCK, phosphoenol-pyruvate-carboxykinase; PI3K, phosphoinositide-3-kinase; PLTP, phospholipid transfer protein; R, receptor; ROS, reactive oxygen species. Studying ovariectomized mice after 5 weeks on HFD, another study found that 17ß-ethinyl-estradiol improved both muscle and hepatic insulin sensitivity using the hyperinsulinemic-euglycemic clamps (8), the gold-standard technique for assessing tissue-specific insulin sensitivity in vivo. Employing [14C]glycerol to trace fatty acid metabolism, they concluded that 17ß-ethinyl-estradiol reduces hepatic steatosis by limiting de novo synthesis of TAGs and diacylglycerols (DAGs) along with blocking insulin signaling to ACC carboxylase and maintaining efficient TGA export as very low-density lipoproteins along with reduced phospholipid transfer protein, which seemed to depend on ERα. These data suggested but did not prove that accumulation of hepatic DAGs, which are known to induced insulin resistance in other rodent models (2), could also be responsible for the insulin-sensitizing effects of 17ß-ethinyl-estradiol in HFD-fed mice. In this issue of Endocrinology, Camporez and colleagues (9) contribute a mechanistic explanation of the effects of 17ß-ethinyl-estradiol on glucose homeostasis in ovariectomized mice after 4 weeks of HFD. Again, these mice showed decreased muscle and hepatic insulin sensitivity, which was normalized by estrogen treatment. Of note, hepatic insulin sensitivity was almost abolished by HFD only so that estrogen depletion did not have a detrimental effect. Interestingly, estrogen treatment resulted in a mild increase in 17ß-ethinyl-estradiol levels above those observed in control mice and completely restored hepatic insulin sensitivity, indicating that 17ß-ethinyl-estradiol is able to antagonize the impact of HFD on hepatic glucose metabolism. But one main finding of this paper is that the improvement of insulin sensitivity by 17ß-ethinyl-estradiol is paralleled by lower tissue DAG content in both membranes and cytosol of liver and muscle. This was accompanied by reduced activities of the novel serine/threonine protein kinase C (nPKC) isoforms, PKCϵ in liver and PKCθ in skeletal muscle, and regeneration of protein kinase B phosphorylation in both tissues (Figure 2). These findings support the concept that the DAG/nPKC pathway is the major mechanism inducing lipid-mediated insulin resistance, as previously demonstrated in several (2) but not all models (10). Of note, ceramides, another class of toxic lipid intermediates that have been implicated in insulin resistance (11), were unchanged in this study. Elevation of tissue DAGs will stimulate nPKC, which in turn decreases tyrosine phosphorylation of the insulin receptor substrate 1 and followed by decreased activation of phosphoinositide-3-kinase and protein kinase B, which will reduce insulin stimulation of muscle glucose uptake and insulin suppression of hepatic gluconeogenesis. But how can 17ß-ethinyl-estradiol regulate tissue DAG content? The accumulation of lipids and lipid intermediates is prevented by a balance between lipid synthesis, ß-oxidation, and tricarboxylic acid cycle activity (12). The present study and other recent studies point to acceleration of lipid oxidation and reduction of lipid synthesis by 17ß-ethinyl-estradiol. Estrogen replacement restored whole-body energy expenditure to control levels, which might result from direct or indirect effects on the transcription of thermogenic genes like uncoupling protein 1 (Ucp1), Cidea, and Prdm16 in white adipose tissue. Interestingly, hepatocytes isolated from 17ß-ethinyl-estradiol–treated mice feature greater oxygen consumption in line with increased mitochondrial activity. Some recent studies revealed that ERs are localized in the mitochondrial membrane and that estrogens stimulate biogenesis of the mitochondrial electron transport system by regulating both its nuclear and mitochondrial DNA-encoded genes (13). Although all these observations are in accordance with decreasing intracellular lipid contents, it is puzzling why such a decrease should be selective for DAG. 17ß-Ethinyl-estradiol may exert different effects in the central and peripheral nervous system (14). Subcutaneous and intracerebroventricular estrogen delivery were equally effective to reduce body weight and increase insulin and glucose tolerance in ovariectomized mice on HFD. However, centrally administered 17ß-ethinyl-estradiol beneficially affected expression of enzymes involved in hepatic gluconeogenesis and energy expenditure and increased HSL in adipose tissue (Figure 2), and peripheral 17ß-ethinyl-estradiol reduced fatty acid synthesis and adipose tissue inflammation (14). Finally, 17ß-ethinyl-estradiol replacement in ovariectomized ApoE−/− mice, which are prone to accelerated arteriosclerosis, did not affect systemic metabolism, as expected in the absence of HFD, but reduced reactive oxygen species generation, monocyte recruitment, and atherosclerotic lesion formation and improved endothelial function, likely due to normalization of vascular expression of preoxisome proliferator-activated receptor gamma (15). One might speculate that heterogeneous effects of peroxisome proliferator-activated receptor gamma activation might also contribute to the divergent estrogen effects on cardiovascular outcomes in premenopausal women and postmenopausal women on hormone replacement therapy. To summarize, there is growing evidence for beneficial metabolic effects of 17ß-ethinyl-estradiol replacement at least in rodent models. These effects need to be counterbalanced against results of clinical trials showing not only increased risk of endometrial and breast cancer but also greater cardiovascular mortality. However, the better understanding of mechanisms of 17ß-ethinyl-estradiol action in metabolically active tissues might lead to developing novel receptor-specific as well as tissue-specific 17ß-ethinyl-estradiol agonists. T. J. is supported by a Leibniz-DAAD (German Academic Exchange Service) Fellowship. The research of M. R. is funded in part by funding of the German Ministry of Health and North-Rhine Westfalia Ministry for Innovation, Science, Research, and Technology to the Leibniz Center for Diabetes Research and a grant from the German Federal Ministry of Education and Research to the German Diabetes Center for Diabetes Research. Disclosure Summary: The authors have no potential conflict of interest to disclose with regard to this paper. diacylglycerol estrogen receptor high-fat diet hormone-sensitive lipase novel serine/threonine protein kinase C triacylglycerol