Am J Physiol Endocrinol Metab 306: E1188–E1197, 2014. First published April 1, 2014; doi:10.1152/ajpendo.00579.2013.

Estrogen signaling prevents diet-induced hepatic insulin resistance in male mice with obesity Lin Zhu,2 Melissa N. Martinez,3 Christopher H. Emfinger,1,2 Brian T. Palmisano,3 and John M. Stafford1-3 1

Tennessee Valley Healthcare System, 2Division of Diabetes, Endocrinology, and Metabolism, Department of Internal Medicine, and 3Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee Submitted 17 October 2013; accepted in final form 1 April 2014

Zhu L, Martinez MN, Emfinger CH, Palmisano BT, Stafford JM. Estrogen signaling prevents diet-induced hepatic insulin resistance in male mice with obesity. Am J Physiol Endocrinol Metab 306: E1188–E1197, 2014. First published April 1, 2014; doi:10.1152/ajpendo.00579.2013.—The development of insulin resistance in the liver is a key event that drives dyslipidemia and predicts diabetes and cardiovascular risk with obesity. Clinical data show that estrogen signaling in males helps prevent adiposity and insulin resistance, which may be mediated through estrogen receptor-␣ (ER␣). The tissues and pathways that mediate the benefits of estrogen signaling in males with obesity are not well defined. In female mice, ER␣ signaling in the liver helps to correct pathway-selective insulin resistance with estrogen treatment after ovariectomy. We assessed the importance of liver estrogen signaling in males using liver ER␣-knockout (LKO) mice fed a high-fat diet (HFD). We found that the LKO male mice had decreased insulin sensitivity compared with their wild-type floxed (fl/fl) littermates during hyperinsulinemic euglycemic clamps. Insulin failed to suppress endogenous glucose production in LKO mice, indicating liver insulin resistance. Insulin promoted glucose disappearance in LKO and fl/fl mice similarly. In the liver, insulin failed to induce phosphorylation of Akt-Ser473 and exclude FOXO1 from the nucleus in LKO mice, a pathway important for liver glucose and lipid metabolism. Liver triglycerides and diacylglycerides were also increased in LKO mice, which corresponded with dysregulation of insulin-stimulated ACC phosphorylation and DGAT1/2 protein levels. Our studies demonstrate that estrogen signaling through ER␣ in the liver helps prevent whole body and hepatic insulin resistance associated with HFD feeding in males. Augmenting hepatic estrogen signaling through ER␣ may lessen the impact of obesity on diabetes and cardiovascular risk in males. estrogen; estrogen receptor-␣; sex differences; males; insulin resistance; liver lipid metabolism; and hyperinsulinemic euglycemic clamp

are well studied with regard to metabolism in females in part because females are protected against insulin resistance and cardiovascular disease compared with males of the same age or body mass index (BMI) (14, 15). This relative protection is diminished in postmenopausal women (15, 26, 45). Males also express estrogen receptor in many tissues, and aromatase can metabolize androgens to generate estrogen metabolites locally. An increasing body of evidence suggests that estrogens have important biological functions in males with regard to the control of metabolism and adiposity (12, 13, 21, 23, 29, 34, 37), although the pathways

ESTROGEN AND ESTROGEN SIGNALING

Address for reprint requests and other correspondence: J. M. Stafford, 7445D Medical Research Bldg. IV, Nashville, TN 37232-0475 (e-mail: [email protected]). E1188

that mediate estrogen’s protective effects in males are not well defined. A recent clinical study in humans demonstrated that the aromatization of testosterone to estradiol is responsible for many of the physiological effects that had previously been attributed to testosterone, such as increasing libido and prevention of visceral adiposity (12). Many of the effects of estrogen in males may be attributed to estrogen receptor-␣ (ER␣)mediated signaling. For example, polymorphisms in ER␣ are associated with increased risk of type 2 diabetes and cardiovascular disease in males (13, 21, 29). Furthermore, males with disruptive mutations in either the estrogen receptor-␣ or aromatase genes have impaired glucose tolerance, hyperglycemia, and hyperinsulinemia (23, 34, 37). The metabolic syndrome associated with aromatase deficiency is also improved by estrogen treatment (23, 34). Collectively, these human studies suggest that estrogen signaling through ER␣ may be metabolically protective in males as well as for females in the setting of obesity. The onset of liver insulin resistance, even in the setting of normal blood glucose, is an important contributor to the development of diabetes and obesity-associated cardiovascular risk. Numerous studies have shown that liver insulin resistance, or even an elevated serum alanine aminotransferase, is a significant predictor of type 2 diabetes independent of BMI or muscle insulin sensitivity (4, 17, 43). The failure of insulin to suppress hepatic glucose production contributes to impaired fasting glucose in humans. Metabolomic data suggest the presence of liver insulin resistance up to a decade before the diagnosis of diabetes (32, 44). Liver insulin resistance is the major driver of obesity-associated dyslipidemia, including elevated triglycerides in VLDL and low cholesterol levels in HDL and increased cardiovascular risk (10, 36, 40). Liver, but not muscle insulin resistance, contributes to impairments in the composition of HDL particles, an effect modulated by estrogen-signaling pathways (24). Reciprocally, pharmacological strategies that target estrogen signaling to the liver improve metabolic syndrome in animal models (11, 19). Thus, clinical and basic science evidence supports that understanding the pathways influencing the development of liver insulin resistance will likely be important toward understanding the early changes with obesity that give rise to the risk of diabetes and cardiovascular disease. The use of mouse models has begun to establish tissues and pathways by which estrogen signaling may protect males from complications of obesity. Global loss of estrogen signaling through knockout of ER␣ leads to obesity, increased inflammation, and hyperlipidemia in male mice (16, 28, 33). Estrogen

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LIVER ESTROGEN SIGNALING AND INSULIN SENSITIVITY IN MALES

signaling through ER␣ in adipose promotes adipose triglyceride (TG) storage in males, as demonstrated in an adiposespecific knockout of ER␣ in mice (7). In humans, liver fat accumulation is a major contributor toward both dyslipidemia and impaired glucose metabolism in males (10), but the role of estrogen signaling in the liver in males is not well established. In females, estrogen signaling through ER␣ has prominent signaling functions in the liver. Global ER␣-knockout and ovariectomy in mice result in increased expression of lipogenesis genes in the liver and development of steatosis (33, 49). In female liver ER␣-knockout mice, estrogen limits liver fat accumulation and promotes insulin signaling under high-fat diet (HFD) feeding (49). Given the importance of liver estrogen signaling in minimizing liver fat and improving insulin sensitivity in females, we postulated that estrogen signaling in the liver might mediate part of the protective effects of estrogen signaling in males. We used mice with liver-specific ER␣-knockout (LKO) to investigate the importance of liver ER␣ toward the metabolic adaptation to HFD feeding in males. In our study, we used the hyperinsulinemic euglycemic clamp technique to determine whole body insulin resistance. Our clamp study design was optimized to define insulin action in the liver. We demonstrate that male LKO mice develop hepatic insulin resistance and accumulate hepatic triglycerides and diacylglycerols, which are associated with impaired liver insulin signaling. Muscle glucose uptake in response to hyperinsulinemia is unchanged. These studies demonstrate an important role of hepatic estrogen signaling to prevent the metabolic complications of obesity in males. MATERIALS AND METHODS

Animals and experimental design. Mice with liver-specific deletion of ER␣ (LKO) were generated as reported before (8, 9, 49). LKO mice compared with wild-type (WT) littermates for these studies are ER␣ flox/flox littermates. All mice were 12–14 wk old at the onset of diet (n ⱖ 10/group) and were housed at 22 ⫾ 1°C in a 12:12-h light-dark cycle. The Institutional Animal Care and Use Committee at Vanderbilt University approved the protocols. Four cohorts of LKO mice and their floxed/floxed WT controls were used for these studies. Cohort 1 (before HFD) chow-fed. Mice fasted for 5 h and were then euthanized to define genotype effects in the absence of HFD feeding. There were no differences in body weight or body composition in chow-fed mice. Cohort 2 (HFD/no insulin). Mice were fed HFD for 12 wk (D08060104 60% fat from lard, 20% protein, and 20% carbohydrate from corn starch, 5.24 kcal/g; Research Diets) and then fasted for 5 h and euthanized. This cohort was included to obtain tissues that were not insulin treated for Figs. 2– 4. Cohort 3 (HFD/insulin clamp). Mice were fed HFD for 12 wk. At week 11, catheters were implanted by the Vanderbilt Mouse Metabolic Phenotyping Center in the left common carotid artery and right jugular vein for sampling and infusions, as described previously (3). Mice were maintained on HFD for 1 wk of recovery and then fasted for 5 h before hyperinsulinemic euglycemic clamp study. This cohort was included to define insulin sensitivity and obtain insulin-treated tissues for Figs. 2– 4. Cohort 4 (HFD for 5 wk). Mice were fed HFD for 5 wk. After fasting for 5 h, mice underwent an intraperitoneal (ip) injection of insulin (0.75 units of regular human insulin diluted in 0.5 ml of saline) or saline and were euthanized 15 min later. This study was included to define the importance of liver estrogen signaling at an intermediate

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time point of HFD feeding and to define acute insulin signaling in liver, muscle, and adipose tissues. Hyperinsulinemic euglycemic clamps. Five days after catheter placement, hyperinsulinemic euglycemic clamps were performed in unrestrained 5-h-fasted mice (cohort 3). A primed (5.4 ␮Ci), continuous (0.135 ␮Ci/min) infusion of [3-3H]glucose was initiated at t ⫽ ⫺90 min. This basal period was followed by hyperinsulinemia started at t ⫽ 0 (2.5 mU·kg⫺1·min⫺1, Humulin R; Eli Lilly, Indianapolis, IN). Our clamp study design with an insulin infusion of 2.5 mU·kg⫺1·min⫺1 was optimized to define hepatic insulin action, because this insulin dose does not maximally suppress endogenous glucose production (EndoRa; an index of hepatic glucose production) (2). At t ⫽ 0 min, the infusion rate was increased to 0.27 ␮Ci/min. Euglycemia (⬃150 mg/dl) was maintained by measuring blood glucose every 10 min starting at t ⫽ 0 min and adjusting the infusion of 50% dextrose as necessary. Mice received saline-washed erythrocytes from donors to prevent a fall in hematocrit. At t ⫽ 120 min, mice were euthanized and tissues flash-frozen. To obtain non-insulin-treated samples, a parallel set of experiments was performed where mice were fed a HFD for 12 wk, fasted for 5 h, and then euthanized (cohort 2). Plasma processing and calculations. Insulin levels were determined by ELISA (no. EZRMI-13K; Millipore, St. Charles, MO). [3-3H]glucose-specific activities were determined by liquid scintillation counting after plasma deproteination. Glucose disappearance rate (Rd) and EndoRa were determined, and insulin sensitivity index was calculated as described (1, 3, 38). Serum estradiol and testosterone values were measured by the Vanderbilt Hormone Assay Core Facility. Liver TG/diacylglycerol content analysis. Liver lipid was extracted using Folch methodology as described (50). TG and diacylglycerol (DAG) were separated using TLC according to Zhu et al. (49). Total liver TG and DAG amount was quantified as reported previously (49). Protein immunoblots. Antibodies for Akt and p-Akt Ser473 were from Cell Signaling Technology (Beverly, MA), antibodies for ER␣ (sc-7207), diaclyglycerol acyltransferase (DGAT1; sc-31680), DGAT2 (sc-66859), FoxO1 (sc-11350), and ␤-actin (sc-47778) were from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody for apolipoprotein B (apoB)-100 was from Lifespan Biosciences (LS-c20729). Anti-microsomal triglyceride transfer protein (MTP) antibody was provided by Dr. Larry Swift (41). Primary antibody was incubated at 4°C overnight with the dilution recommended in the manufacturer’s database. Anti-mouse or anti-rabbit antibody was incubated with the dilution of 1:15,000 at room temperature for 1 h. Nuclear extracts were prepared according to the manufacturer’s instructions (NE-PER; Thermo Scientific). Imaging and densitometry were performed using the Odyssey imaging system (Li-COR, Lincoln, NE) and Image J processing program. Liver glycogen quantification. Liver glycogen extraction was performed as established by Chan and Exton (6), with minor modifications. Briefly, 50 –100 mg of liver tissue was homogenized in 0.03 N HCl, using 0.5 mm of zinc oxide beads (no. ZrOB05; Next Advance) in a Bullet-Blender. Homogenized tissues were incubated at 80°C for 10 min, and homogenate was blotted onto chromatography paper strips (no. 0303614; Whatman). Strips were washed three times in 70% ethanol for 40 min, rinsed in acetone, and then dried overnight. Dried strips were digested using 0.1 mg/ml amyloglucosidase (no. A7420; Sigma) in 0.04 M sodium acetate for 3 h in a 37°C shaking water bath. Glucose in glycogen was quantified by enzymatic assay. Twenty microliters/well of a 96-well plate of glucose-enriched digest solution was loaded (no. 12565501; Fisher) with 250 ␮l of enzyme solution. The plate was incubated for 15 min at room temperature. Enzyme solution contained 70 mg of ATP (no. 100008; MP Biomedical), 24 ml of 200 mM Tris·HCl, 500 ␮l of 500 mM MgCl2, 50 mg of ␤-NADP (no. 10128058001; Roche), 50 ␮l of hexokinase (no. 11426362001; Roche), and 125 ␮l of glucose-6-phosphate dehydrogenase (no. 10737232001; Roche). Plate absorbance was read at 340 nm to quantify glucose in digest solution. Oyster glycogen (Sigma no. G8751) and glucose (Sigma

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00579.2013 • www.ajpendo.org

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Fig. 1. Liver estrogen signaling prevents insulin resistance in males with high-fat diet (HFD) feeding. A: Western blots show that estrogen receptor-␣ (ER␣) protein levels are decreased in liver-specific ER␣-knockout (LKO) mouse liver but not in muscle. B: schematic of the hyperinsulinemic euglycemic clamp study design. C: euglycemia was maintained at ⬃150 mg/dl during the hyperinsulinemic clamp. D: glucose infusion rate (GIR) to maintain euglycemia was lower for LKO mice than for fl/fl mice. E: insulin sensitivity index was lower in LKO mice; n ⫽ 6 – 8/group. *P ⬍ 0.05. Differences between groups were determined by t-test.

no. G6918) were used as standard controls for the extraction and quantification procedures, respectively. Statistical analysis. Data are presented as means ⫾ SD. Differences between groups were determined by two-way ANOVA, followed by Bonferroni posttests, or by Student’s t-test as appropriate. Significance was considered as P ⬍ 0.05. The specific statistical tests used are indicated in the figure legends. RESULTS

Male LKO mice exhibit liver and whole body insulin resistance compared with their WT (fl/fl) littermates after HFD feeding. To define the importance of liver estrogen signaling for glucose and lipid metabolism in males, 12-wk-old male

LKO mice and their fl/fl littermates were fed a HFD for 12 wk. The fl/fl littermates for these studies are ER␣ flox/flox littermates. In LKO mice, liver ER␣ expression was reduced ⬎90% from whole liver extracts but unchanged in muscle (Fig. 1A). The HFD feeding caused increased body weight and adiposity in fl/fl and LKO male mice, with the LKO mice gaining more adiposity (Table 1). Fasting glucose and insulin levels were not different between fl/fl and LKO mice at baseline on chow diet (Table 1). HFD feeding caused an increase in fasting glucose and insulin levels for both groups, with no significant differences between groups; however, LKO mice tended to have higher insulin values (Table 1).

Table 1. HFD-induced obesity and insulin resistance for LKO and fl/fl mice fl/fl

Body weight, g (12-wk study) %Adiposity (12-wk study) %Muscle (12-wk study) Fasting glucose, mg/dl (12-wk study) Fasting insulin, ng/ml (12-wk study) Clamp insulin, ng/ml (12-wk study) Clamp C-peptide, ng/ml (12-wk study) Serum estradiol, pg/ml Serum total testosterone, ng/ml Body weight, g (5-wk study) %Adiposity (5-wk study) %Muscle (5-wk study)

LKO

Chow fed

After HFD

Chow fed

After HFD

22.5 ⫾ 2.6 10.8 ⫾ 3.9 76.2 ⫾ 4.5 98.2 ⫾ 11.7 0.43 ⫾ 0.07

35.2 ⫾ 3.7* 23.5 ⫾ 4.1* 64.7 ⫾ 7.6* 144.8 ⫾ 21.3* 3.38 ⫾ 0.71* 5.35 ⫾ 0.6* 2.29 ⫾ 0.11* 67.0 ⫾ 9.5 16.7 ⫾ 1.0 33.5 ⫾ 1.2 25.9 ⫾ 1.1 74.3 ⫾ 2.8

23.1 ⫾ 3.2 9.2 ⫾ 2.3 79.6 ⫾ 5.8 105.9 ⫾ 13.2 0.47 ⫾ 0.11

37.8 ⫾ 4.3* 28.6 ⫾ 3.9*# 61.7 ⫾ 6.4* 132.8 ⫾ 20.5* 4.52 ⫾ 1.02* 7.94 ⫾ 1.59# 7.41 ⫾ 0.29** 79.0 ⫾ 2.4 0.46 ⫾ 0.22** 32.2 ⫾ 1.1 23.7 ⫾ 1.6 75.6 ⫾ 1.7

Values are means ⫾ SE. HFD, high-fat diet; LKO, liver ER␣-knockout. *P ⬍ 0.05, comparison between “before HFD” and “after HFD”; #P ⬍ 0.05, comparison of fl/fl-HFD with LKO-HFD; **,P ⬍ 0.005, comparison of fl/fl-HFD with LKO-HFD. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00579.2013 • www.ajpendo.org

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To determine the importance of ER␣ toward whole body insulin sensitivity, we performed hyperinsulinemic euglycemic clamps in LKO mice and WT littermates (Fig. 1B). In the setting of hyperinsulinemia, a greater glucose infusion rate (GIR) indicates greater insulin sensitivity. Similarly, a lower GIR indicates greater insulin resistance. During the clamp, the GIR was lower for LKO mice compared with the WT littermates (Fig. 1, C and D), indicating that male LKO mice were more insulin resistant than WT littermates. Fasting insulin, clamp phase insulin, and clamp phase C-peptide levels are shown in Table 1. Since insulin infusion does not suppress endogenous insulin production in mice, clamp phase plasma insulin and C-peptide levels were significantly higher in LKO mice, consistent with whole body insulin resistance (Table 1). We calculated an insulin sensitivity index by normalizing the GIR-to-plasma insulin concentrations during the clamp period. The insulin sensitivity index was significantly lower in LKO mice than in fl/fl littermates (P ⬍ 0.01; Fig. 1E). These results demonstrated that the absence of liver estrogen signaling in males worsens whole body insulin resistance induced by HFD feeding. Hepatic insulin signaling is impaired in male LKO mice. We used [3-3H]glucose as a tracer to assess liver glucose metabolism during the clamp studies. The dose of insulin infusion during the clamp was optimized to define liver glucose metabolism (2.5 mU·kg⫺1·min⫺1; reviewed in Ref. 3). EndoRa was suppressed by hyperinsulinemia during the clamp in fl/fl litter-

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mates (Fig. 2A). In contrast, insulin failed to suppress EndoRa in male LKO mice, demonstrating hepatic insulin resistance (Fig. 2A). To obtain non-insulin-treated samples so that we could define insulin-signaling pathways, we repeated a cohort of LKO and fl/fl mice that were fed HFD for 12 wk and euthanized after a 5-h fast. In fl/fl mice, insulin during the clamp induced an increase in p-Akt Ser473 (Fig. 2, B and C). In LKO mice, insulin failed to induce p-Akt Ser473. In the insulinsensitive liver, p-Akt Ser473 phosphorylates the transcription factor forkhead box protein O1 (FoxO1), resulting in nuclear exclusion of FoxO1 (46). Nuclear levels of FoxO1 were increased in LKO mice compared with fl/fl (Fig. 2, D and E). Insulin signaling through Akt and FoxO1 is an important pathway for the regulation of hepatic glucose and TG metabolism (46). LKO male mice have altered liver glycogen metabolism. Diabetes is characterized by impaired glycogen synthesis and metabolism (18, 20). In our study, LKO male mice had significantly reduced fasting liver glycogen compared with fl/fl controls (9.6 ⫾ 2.1 vs. 17.5 ⫾ 3.2 ␮g/mg, P ⬍ 0.05; Fig. 2F). Insulin-stimulated glycogen content was also impaired in LKO mice (17.4 ⫾ 1.0 vs. 30.68 ⫾ 4.9 ␮g/mg, P ⬍ 0.05; Fig. 2F). This impaired hepatic glycogen metabolism likely contributes to the impaired suppression of EndoRa that we observed in the clamp study. Muscle glucose disposal is not impaired in LKO mice. An index of muscle insulin action is the Rd in the clamp period,

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Fig. 2. Hepatic insulin signaling is impaired in LKO mice. A: insulin failed to suppress hepatic glucose production (EndoRa). B and C: Western blots from whole liver extracts show that Akt Ser473 phosphorylation by insulin was decreased in LKO mice. D and E: Western blots show increased forkhead box protein O1 (FoxO1) from nuclear extracts of liver from LKO mice. F: liver glycogen levels were lower in LKO mice during fasting and hyperinsulinemia; n ⫽ 4 – 6/group for bar graphs. *P ⬍ 0.05 by t-test; **P ⬍ 0.005 by t-test; #P ⬍ 0.005 by genotype by 2-way ANOVA. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00579.2013 • www.ajpendo.org

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which was similar for fl/fl and LKO mice during the clamp (Fig. 3A). In muscle, insulin induced phosphorylation of Akt Ser473 to a similar degree in WT and LKO mice (Fig. 3, B and C). Collectively, tracer analysis and tissue-specific insulin signaling suggest that hepatic insulin resistance caused the whole body insulin resistance in LKO mice. Liver ER␣ signaling protects against liver TG and DAG accumulation with HFD feeding. To define the effects of hepatic estrogen signaling on liver fat accumulation with HFD feeding, we measured liver TG and DAG content in both chow-fed and HFD-fed mice. There were no differences in hepatic TG or DAG content in a cohort of mice that was chow-fed and euthanized after a 5-h fast (liver TG: 15.3 ⫾ 6.3 ␮g/mg for fl/fl and 16.1 ⫾ 4.7 ␮g/mg for LKO; liver DAG: 0.31 ⫾ 0.05 ␮g/mg for fl/fl and 0.29 ⫾ 0.1 ␮g/mg for LKO). Hepatic steatosis developed in both WT and LKO mice following 12 wk of HFD, and this steatosis was more pronounced in LKO mice (Fig. 4A). Similarly, liver DAG content was also increased in HFD-fed LKO mice (Fig. 4B). These results demonstrate that liver estrogen signaling protects against liver TG and DAG accumulation in male mice with HFD feeding. Estrogen signaling protects against dyslipidemia in males after HFD feeding. In the liver, insulin signaling promotes dephosphorylation of acyl-coA carboxylase (ACC), increasing fatty acid synthesis. Our previous work showed that liver estrogen signaling limited liver lipid accumulation in part by preventing insulin-mediated dephosphorylation of ACC, which promoted fatty acid oxidation rather than synthesis (49). We compared ACC phosphorylation during fasting and hyperinsulinemia and found that in fl/fl animals with intact liver estrogen signaling, insulin did not dephosphorylate ACC, consistent with our previous results seen in females. In contrast, insulin did dephosphorylate ACC in LKO mice, which would promote fatty acid synthesis and is consistent with increased liver fat content (Fig. 4, A, C, and D). The acyl-CoA-DGAT enzymes catalyze TG synthesis by transferring fatty acid to DAG. DGAT1 and DGAT2 are from different gene families and have distinct, often reciprocal, regulation in the liver (5; also reviewed in Ref. 48). Insulin increases DGAT2 to promote TG synthesis and reduce hepatic free fatty acid and DAG levels, which may help to prevent hepatic insulin resistance (27, 47). In fl/fl mice we saw appro-

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priate insulin stimulation of DGAT2 protein levels, whereas in LKO mice insulin failed to upregulate DGAT2 (Fig. 4, E and F). Reciprocally, DGAT1 is increased in fasting and reduced with hyperinsulinemia (27). Fasting levels of DGAT1 were increased in LKO mice compared with fl/fl, consistent with fatty liver in the LKO mice (Fig. 4, E and G). There were no differences in either mature or processed forms of sterol regulatory element-binding protein-1c (data not shown). Collectively, these data suggest that ER␣ signaling is involved in both fasting and insulin regulation of DGAT1/2, which likely contributes to limiting liver fat accumulation. Increased fasting plasma TG level in LKO mice. Increased liver fat content is a major driver of increased production of VLDL (10). In the liver, the enzyme MTP adds TG onto the nascent apoB-100 particle as it is translocated into the lumen of the endoplasmic reticulum for secretion as a VLDL (31). We observed that during fasting, liver apoB-100 and MTP protein levels were significantly higher in LKO mice than in fl/fl littermates and were correlated with increased levels of plasma TG in LKO mice (Fig. 5, A–D). Insulin reduces hepatic apoB-100 levels. Insulin suppressed expression of apoB-100 in both LKO mice and their fl/fl littermates (Fig. 5, A and B). There was no significant difference in plasma cholesterol levels between LKO and fl/fl mice during fasting, and the fasting plasma cholesterol levels were suppressed by hyperinsulinemia during the clamp in both groups of mice (Fig. 5E). These findings suggest that liver estrogen signaling in males prevents liver fat accumulation, resulting in improvements in both dyslipidemia and glucose metabolism. LKO male mice have impaired insulin signaling to liver and muscle at 5 wk of HFD feeding. We next aimed to define the importance of liver ER␣ signaling at an intermediate duration of HFD feeding for 5 wk compared with 12 wk in the previous study. There were no differences in body weight or adiposity at 5 wk of HFD feeding (n ⫽ 9 –10/group; Fig. 6A). To define acute insulin signaling, we administered intraperitoneal insulin or saline and euthanized mice 15 min later. We used this approach because insulin downregulates its own signaling with chronic administration. We assessed insulin signaling to Akt in liver, muscle, and adipose tissues. In the liver, we found that fl/fl mice responded to insulin and had a robust induction of phosphorylated Akt Ser473 (Fig. 6, B and C). In LKO mice,

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Fig. 3. Muscle insulin action is not impaired in LKO mice. A: insulin-stimulated glucose disappearance rate (Rd) was not different between LKO and fl/fl mice. B and C: insulin (Ins)-stimulated phosphorylation from muscle extracts of Akt Ser473 was similar for LKO and fl/fl mice; n ⫽ 4 – 6/group for bar graphs. **P ⬍ 0.005. Differences between groups were determined by t-test. AJP-Endocrinol Metab • doi:10.1152/ajpendo.00579.2013 • www.ajpendo.org

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Fig. 4. Liver ER␣ signaling protects against triglyceride (TG) and diacylglycerol (DAG) accumulation with HFD feeding in male LKO mice. A and B: liver TG and DAG levels were increased in LKO mice compared with their littermates. C and D: acyl-CoA carboxylase (ACC) phosphorylation (p-ACC) was decreased by insulin in LKO mice, shown by Western blotting from whole liver extracts. E–G: Western blotting of diacylglycerol acyltransferase (DGAT)1/2 impaired insulin regulation of DGAT levels in liver from LKO mice; n ⫽ 4 – 6/group for bar graphs. *P ⬍ 0.05 by t-test; **P ⬍ 0.005 by t-test.

baseline p-Akt Ser473 was reduced, and insulin failed to induce p-Akt Ser473. This finding is similar to the results of our 12-wk study for liver. Total Akt levels were unchanged in the liver between LKO and fl/fl. In muscle, for the fl/fl mouse, insulin also induced robust induction of p-Akt Ser473. In LKO mice, baseline p-Akt Ser473 was increased, and insulin had diminished capacity to induce p-Akt Ser473. In adipose, baseline phosphorylation of Akt Ser473 was decreased in LKO mice, and the insulin induction of Akt was more robust. These results confirm that loss of liver ER␣ signaling impairs insulin signaling to Akt in the liver following 5 wk of HFD feeding. Overall, this study confirms the importance of liver estrogen signaling in protecting the liver from insulin resistance and also protecting against whole body insulin resistance with HFD feeding in male mice. DISCUSSION

Accumulating evidence supports the importance of estrogen and estrogen signaling in males with regard to metabolic

control. A recent clinical study found that many of the benefits attributed to testosterone in males actually require aromatization of testosterone to estrogen (12). The target tissues responsible for estrogen’s beneficial effects in males are not yet clear. Insulin resistance in the liver is a major contributor to both impaired glucose metabolism and dyslipidemia with obesity. Because of the prominent role of hepatic estrogen signaling in limiting the adverse effects of high-fat feeding in females (16, 19, 49), we hypothesized that liver estrogen signaling may also limit the adverse effects of high-fat feeding in males. We discovered that male mice with liver ER␣-knockout (LKO) developed insulin resistance and impaired insulin signaling in liver. This liver insulin resistance was associated with 1) increased levels of proteins involved in fatty acid and TG synthesis in the liver, 2) decreased liver glycogen synthesis, and 3) increased hepatic glucose production during hyperinsulinemia. These results demonstrate that some of the protective metabolic effects of estrogen in males are mediated through hepatic ER␣ signaling.

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Fig. 5. Increased fasting plasma TG level in LKO mice. A and B: Western blotting shows that liver apolipoprotein B (apoB)-100 protein levels were suppressed during insulin clamps for LKO and fl/fl mice. A and C: microsomal TG transfer protein (MTP) levels were increased in LKO mice. D: fasting plasma TG was increased in LKO mice, but insulin reduced serum TG in both LKO and fl/fl mice. E: cholesterol levels were reduced with insulin for LKO mice and their fl/fl controls; n ⫽ 4 – 6/group for bar graphs. *P ⬍ 0.05 by t-test; **P ⬍ 0.005 by t-test; #P ⬍ 0.05 by genotype by 2-way ANOVA.

We demonstrate that the liver is an important target of estrogen signaling in males for preventing whole body insulin resistance, using mice with liver-specific ER␣ knockout and hyperinsulinemic euglycemic clamp techniques. Our clamp study design with insulin infusion of 2.5 mU·kg⫺1·min⫺1 was optimized to define hepatic insulin action (2). In LKO mice, insulin failed to suppress hepatic glucose production (EndoRa) in LKO mice, a measurement of hepatic insulin action. Correspondingly, we saw impaired insulin-stimulated phosphorylation of Akt in the liver of LKO mice. The LKO mice in the cohort that was fed HFD for 12 wk had a slightly higher adiposity than fl/fl mice, which would bias toward finding insulin resistance in the LKO group. To address this, we repeated a cohort of LKO and fl/fl mice on an intermediate duration of HFD feeding, i.e., 5 wk. In these mice, there were no differences in adiposity; in fact, the LKO mice were slightly leaner. The LKO mice still had a significant impairment in insulin signaling to Akt in the liver, confirming the finding that estrogen signaling through liver ER␣ promotes liver insulin sensitivity with HFD feeding. In the clamp study, there was minimal apparent effect of liver ER␣ knockout on muscle, as insulin-mediated glucose disposal (Rd) was not altered. However, we did see that muscle insulin signaling was impaired with an intraperitoneal injection of insulin in our cohort of mice fed HFD for 5 wk. The

difference in these two studies is likely in part because the longer duration of HFD feeding diminished the muscle insulin response in both groups (insulin stimulated p-Akt was 4.2-fold in fl/fl mice at 5 wk but just 2.8-fold at 12 wk) and possibly because the duration of insulin infusion downregulated p-Akt in the clamp study. In the clamp study, we did see elevated C-peptide levels in LKO mice, which is also consistent with muscle insulin resistance. Thus, there is likely a muscle phenotype induced by loss of hepatic ER␣. The low total testosterone levels seen in the LKO mice may contribute to impaired insulin action in muscle, as testosterone is known to promote muscle glucose uptake (Table 1 and Refs. 30 and 35). However, we were unable to measure sex hormone-binding globulin, which may have been reduced in the LKO mice. There were no differences in serum estradiol. One published report did not show a major phenotype in older mice lacking liver ER␣ in either males or females using intraperitoneal glucose tolerance testing (GTT) (25). Our clamp approach provides an advantage over the GTT because blood glucose and insulin levels are held constant, helping to avoid the counterregulatory changes that occur during the GTT. We additionally verified significant liver insulin resistance by loss of liver ER␣ with a second model, i.e., ip insulin vs. ip saline after 5 wk of HFD feeding (Fig. 6). Collectively, by coupling tissue-specific knockout of ER␣ with techniques optimized to study liver

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Fig. 6. Hepatic insulin signaling is impaired in LKO mice with 5 wk of HFD feeding. A: there were no differences in adiposity or lean mass after 5 wk of HFD feeding between LKO and fl/fl male mice. B–E: after a 5-h fast, mice underwent an intraperitoneal (ip) insulin or saline injection. Western blotting of p-Akt Ser473 and total Akt show insulin resistance in liver and muscle but not adipose tissue; n ⫽ 5– 6/group for bar graphs. *P ⬍ 0.05 by t-test; **P ⬍ 0.005 by t-test; #P ⬍ 0.05 by genotype by 2-way ANOVA.

insulin action, we were able to show an important role of hepatic estrogen signaling in preventing HFD-induced insulin resistance in males. Our results suggest that many of the protective effects of liver ER␣ signaling with regard to lipid metabolism in females are also present in males. Estrogen signaling in the liver likely arose to couple reproduction to with nutritional signals. In cycling female mice, the mRNA levels of lipogenic genes in liver change significantly during the 4-day-long estrus cycle (42). Furthermore, amino acids regulate liver ER␣ and contribute to both the metabolic and reproductive effects of estrogen (8). Reciprocally, the absence of liver ER␣ signaling leads to lipid accumulation with overnutrition (49). We show that in males liver estrogen signaling through ER␣ also limits liver TG and DAG accumulation with HFD feeding. In fl/fl male mice, with intact liver estrogen signaling, the reduction in both liver TG and DAG likely improved liver insulin sensitivity. Dysregulation of DGAT2 by insulin in LKO mice may contribute to the DAG accumulation and liver insulin resistance. DGAT2 is involved in the bulk of TG synthesis in the liver (39). Insulin increases fatty acid synthesis and concordantly increases DGAT2 protein levels, which promote the

synthesis of TG from fatty acids and DAG. This coordinate synthesis of fatty acids with induction of DGAT2 minimizes the accumulation of lipotoxic DAG species. LKO mice had increased dephosphorylation of ACC (which promotes fatty acid synthesis) with insulin treatment but impaired insulin induction of DGAT2. Liver estrogen signaling seems to temper the insulin-stimulated accumulation of lipotoxic lipids by decreasing ACC-mediated fatty acid synthesis and by increasing DGAT2-mediated conversion of fatty acids and DAGs to a more innocuous TG molecule. Studies in humans have shown that estrogen signaling protects from obesity-related complications in males (12, 13, 21, 23, 29, 34, 37). Our data suggest that estrogen signaling through ER␣ in the liver likely contributes to this protection. Women are relatively protected against cardiovascular disease compared with age- or BMI-matched men (14, 22). Our laboratory and others’ have shown that liver estrogen signaling protects against complications of high-fat feeding in females. Our studies highlight the opportunity to define pathways that confer cardiovascular protection for females, which can then be targeted to lessen obesity complications in males. Strategies to augment hepatic estrogen signaling pathways in males may

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be a promising approach to reduce liver fat and improve insulin signaling associated with obesity. ACKNOWLEDGMENTS We acknowledge Drs. David Wasserman and Owen McGuinness for their assistance in the design of the clamp studies. We acknowledge excellent assistance by the Vanderbilt Mouse Metabolic Phenotyping Center (DK59637) and the Vanderbilt Hormone Assay and Analytical Services Core (DK-59637 and DK-20593). GRANTS This work was supported by the Department of Veterans Affairs Career Development Award and Merit Award (BX002223), the American Heart Association (10GRNT3650024), the Vanderbilt Diabetes Research and Training Center Pilot and Feasibility Program (P-30-DK-20593), and the Vanderbilt Digestive Diseases Research Center Pilot and Feasibility Program (DK058404). B. T. Palmisano was supported by Public Health Service award T32-GM-07347 from the National Institute of General Medical Studies for the Vanderbilt Medical Scientist Training Program. DISCLOSURES The authors disclose no conflicts of interest, financial or otherwise. AUTHOR CONTRIBUTIONS L.Z., M.N.M., and J.M.S. conception and design of research; L.Z., M.N.M., C.H.E., and B.T.P. performed experiments; L.Z., M.N.M., C.H.E., B.T.P., and J.M.S. analyzed data; L.Z., M.N.M., C.H.E., B.T.P., and J.M.S. interpreted results of experiments; L.Z., M.N.M., and J.M.S. prepared figures; L.Z. drafted manuscript; L.Z., M.N.M., B.T.P., and J.M.S. edited and revised manuscript; L.Z., M.N.M., C.H.E., B.T.P., and J.M.S. approved final version of manuscript. REFERENCES 1. Ayala JE, Bracy DP, Julien BM, Rottman JN, Fueger PT, Wasserman DH. Chronic treatment with sildenafil improves energy balance and insulin action in high fat-fed conscious mice. Diabetes 56: 1025–1033, 2007. 2. Ayala JE, Bracy DP, Mcguinness O, Wasserman DH. Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes 55: 390 –397, 2006. 3. Ayala JE, Samuel VT, Morton GJ, Obici S, Croniger CM, Shulman GI, Wasserman DH, McGuinness OP. Standard operating procedures for describing and performing metabolic tests of glucose homeostasis in mice. Dis Model Mech 3: 525–534, 2010. 4. Bonnet F, Ducluzeau PH, Gastaldelli A, Laville M, Anderwald CH, Konrad T, Mari A, Balkau B; RISC Study Group. Liver enzymes are associated with hepatic insulin resistance, insulin secretion, and glucagon concentration in healthy men and women. Diabetes 60: 1660 –1667, 2011. 5. Cases S, Stone SJ, Zhou P, Yen E, Tow B, Lardizabal KD, Voelker T, Farese RV Jr. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members. J Biol Chem 276: 38870 – 38876, 2001. 6. Chan TM, Exton JH. A Rapid Method for the Determination of Glycogen Content and Radioactivity in Small Quantities of Tissue or Isolated Hepatocytes. Anal Biochem 71: 96 –105, 1976. 7. Davis KE, D Neinast M, Sun K, M Skiles W, D Bills J, A Zehr J, Zeve D, D Hahner L, W Cox D, M Gent L, Xu Y, V Wang Z, A Khan S, Clegg DJ. The sexually dimorphic role of adipose and adipocyte estrogen receptors in modulating adipose tissue expansion, inflammation, and fibrosis. Mol Metab 2: 227–242, 2013. 8. Della Torre S, Rando G, Meda C, Stell A, Chambon P, Krust A, Ibarra C, Magni P, Ciana P, Maggi A. Amino acid-dependent activation of liver estrogen receptor alpha integrates metabolic and reproductive functions via IGF-1. Cell Metab 13: 205–214, 2011. 9. Dupont S, Krust A, Gansmuller A, Dierich A, Chambon P, Mark M. Effect of single and compound knockouts of estrogen receptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. Development 127: 4277–4291, 2000. 10. Fabbrini E, Magkos F, Mohammed BS, Pietka T, Abumrad NA, Patterson BW, Okunade A, Klein S. Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity. Proc Natl Acad Sci USA 106: 15430 –15435, 2009.

11. Finan B, Yang B, Ottaway N, Stemmer K, Müller TD, Yi CX, Habegger K, Schriever SC, García-Cáceres C, Kabra DG, Hembree J, Holland J, Raver C, Seeley RJ, Hans W, Irmler M, Beckers J, de Angelis MH, Tiano JP, Mauvais-Jarvis F, Perez-Tilve D, Pfluger P, Zhang L, Gelfanov V, DiMarchi RD, Tschöp MH. Targeted estrogen delivery reverses the metabolic syndrome. Nat Med 18: 1847–1856, 2012. 12. Finkelstein JS, Lee H, Burnett-Bowie SA, Pallais JC, Yu EW, Borges LF, Jones BF, Barry CV, Wulczyn KE, Thomas BJ, Leder BZ. Gonadal steroids and body composition, strength, and sexual function in men. N Engl J Med 369: 1011–1022, 2013. 13. Fox CS, Yang Q, Cupples LA, Guo CY, Atwood LD, Murabito JM, Levy D, Mendelsohn ME, Housman DE, Shearman AM. Sex-specific association between estrogen receptor-alpha gene variation and measures of adiposity: the Framingham Heart Study. J Clin Endocrinol Metab 90: 6257–6262, 2005. 14. Freedman DS, Otvos JD, Jeyarajah EJ, Shalaurova I, Cupples LA, Parise H, D’Agostino RB, Wilson PW, Schaefer EJ. Sex and age differences in lipoprotein subclasses measured by nuclear magnetic resonance spectroscopy: the Framingham Study. Clin Chem 50: 1189 –1200, 2004. 15. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai S, Ford ES, Fox CS, Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Huffman MD, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Magid D, Marcus GM, Marelli A, Matchar DB, McGuire DK, Mohler ER, Moy CS, Mussolino ME, Nichol G, Paynter NP, Schreiner PJ, Sorlie PD, Stein J, Turan TN, Virani SS, Wong ND, Woo D, Turner MB; American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics—2013 update: a report from the American Heart Association. Circulation 127: e6 –e245, 2013. 16. Handgraaf S, Riant E, Fabre A, Waget A, Burcelin R, Liere P, Krust A, Chambon P, Arnal JF, Gourdy P. Prevention of obesity and insulin resistance by estrogens requires ER␣ activation function-2 (ER␣AF-2), whereas ER␣AF-1 is dispensable. Diabetes 62: 4098 –4108, 2013. 17. Hanley AJ, Williams K, Festa A, Wagenknecht LE, D’Agostino RB Jr, Kempf J, Zinman B, Haffner SM; insulin resistance atherosclerosis study. Elevations in markers of liver injury and risk of type 2 diabetes: the insulin resistance atherosclerosis study. Diabetes 53: 2623–2632, 2004. 18. Hwang JH, Perseghin G, Rothman DL, Cline GW, Magnusson I, Petersen KF, Shulman GI. Impaired net hepatic glycogen synthesis in insulin-dependent diabetic subjects during mixed meal ingestion. A 13C nuclear magnetic resonance spectroscopy study. J Clin Invest 95: 783– 787, 1995. 19. Kim JH, Meyers MS, Khuder SS, Abdallah SL, Muturi HT, Russo L, Tate CR, Hevener AL, Najjar SM, Leloup C, Mauvais-Jarvis F. Tissue-selective estrogen complexes with bazedoxifene prevent metabolic dysfunction in female mice. Mol Metab 3: 177–190, 2014. 20. Krssak M, Brehm A, Bernroider E, Anderwald C, Nowotny P, Man CD, Cobelli C, Cline GW, Shulman GI, Waldhäusl W, Roden M. Alterations in postprandial hepatic glycogen metabolism in type 2 diabetes. Diabetes 53: 3048 –3056, 2004. 21. Linnér C, Svartberg J, Giwercman A, Giwercman YL. Estrogen receptor alpha single nucleotide polymorphism as predictor of diabetes type 2 risk in hypogonadal men. Aging Male 16: 52–57, 2013. 22. Lloyd-Jones D, Adams R, Carnethon M, De Simone G, Ferguson T, Flegal K, Ford E, Furie K, Go A, Greenlund K, Haase N, Hailpern S, Ho M, Howard V, Kissela B, Kittner S, Lackland D, Lisabeth L, Marelli A, Mcdermott M, Meigs J, Mozaffarian D, Nichol G, O’donnell C, Roger V, Rosamond W, Sacco R, Sorlie P, Stafford R, Steinberger J, Thom T, Wasserthiel-Smoller S, Wong N, Wylie-Rosett J, Hong Y. Heart disease and stroke statistics—2009 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation 119: e21–e181, 2009. 23. Maffei L, Murata Y, Rochira V, Tubert G, Aranda C, Vazquez M, Clyne CD, Davis S, Simpson ER, Carani C. Dysmetabolic syndrome in a man with a novel mutation of the aromatase gene: effects of testosterone, alendronate, and estradiol treatment. J Clin Endocrinol Metab 89: 61–70, 2004. 24. Martinez MN, Emfinger CH, Overton MH, Hill S, Ramaswamy TS, Cappel DA, Wu K, Fazio S, McDonald WH, Hachey DL, Tabb DL, Stafford JM. Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones. J Lipid Res 53: 379 –389, 2012.

AJP-Endocrinol Metab • doi:10.1152/ajpendo.00579.2013 • www.ajpendo.org

LIVER ESTROGEN SIGNALING AND INSULIN SENSITIVITY IN MALES 25. Matic M, Bryzgalova G, Gao H, Antonson P, Humire P, Omoto Y, Portwood N, Pramfalk C, Efendic S, Berggren PO, Gustafsson JÅ, Dahlman-Wright K. Estrogen signalling and the metabolic syndrome: targeting the hepatic estrogen receptor alpha action. PLoS One 8: e57458, 2013. 26. Matthews KA, Meilahn E, Kuller LH, Kelsey SF, Caggiula AW, Wing RR. Menopause and risk factors for coronary heart disease. N Engl J Med 321: 641–646, 1989. 27. Meegalla RL, Billheimer JT, Cheng D. Concerted elevation of acylcoenzyme A:diacylglycerol acyltransferase (DGAT) activity through independent stimulation of mRNA expression of DGAT1 and DGAT2 by carbohydrate and insulin. Biochem Biophys Res Commun 298: 317–323, 2002. 28. Ohlsson C, Hellberg N, Parini P, Vidal O, Bohlooly YM, Rudling M, Lindberg MK, Warner M, Angelin B, Gustafsson JA. Obesity and disturbed lipoprotein profile in estrogen receptor-alpha-deficient male mice. Biochem Biophys Res Commun 278: 640 –645, 2000. 29. Peter I, Huggins GS, Shearman AM, Pollak A, Schmid CH, Cupples LA, Demissie S, Patten RD, Karas RH, Housman DE, Mendelsohn ME, Vasan RS, Benjamin EJ. Age-related changes in echocardiographic measurements: association with variation in the estrogen receptor-alpha gene. Hypertension 49: 1000 –1006, 2007. 30. Pitteloud N, Mootha VK, Dwyer AA, Hardin M, Lee H, Eriksson KF, Tripathy DJ, Yialamas M, Groop L, Elahi D, Hayes FJ. Relationship between testosterone levels, insulin sensitivity, and mitochondrial function in men. Diabetes Care 28: 1636 –1642, 2005. 31. Raabe M, Véniant MM, Sullivan MA, Zlot CH, Björkegren J, Nielsen LB, Wong JS, Hamilton RL, Young SG. Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice. J Clin Invest 103: 1287–1298, 1999. 32. Rhee EP, Cheng S, Larson MG, Walford GA, Lewis GD, McCabe E, Yang E, Farrell L, Fox CS, O’Donnell CJ, Carr SA, Vasan RS, Florez JC, Clish CB, Wang TJ, Gerszten RE. Lipid profiling identifies a triacylglycerol signature of insulin resistance and improves diabetes prediction in humans. J Clin Invest 121: 1402–1411, 2011. 33. Ribas V, Nguyen MT, Henstridge DC, Nguyen AK, Beaven SW, Watt MJ, Hevener AL. Impaired oxidative metabolism and inflammation are associated with insulin resistance in ER␣-deficient mice. Am J Physiol Endocrinol Metab 298: E304 –E319, 2010. 34. Rochira V, Madeo B, Zirilli L, Caffagni G, Maffei L, Carani C. Oestradiol replacement treatment and glucose homeostasis in two men with congenital aromatase deficiency: evidence for a role of oestradiol and sex steroids imbalance on insulin sensitivity in men. Diabet Med 24: 1491–1495, 2007. 35. Sato K, Iemitsu M, Aizawa K, Ajisaka R. Testosterone and DHEA activate the glucose metabolism-related signaling pathway in skeletal muscle. Am J Physiol Endocrinol Metab 294: E961–E968, 2008. 36. Schindhelm RK, Dekker JM, Nijpels G, Bouter LM, Stehouwer CD, Heine RJ, Diamant M. Alanine aminotransferase predicts coronary heart disease events: a 10-year follow-up of the Hoorn Study. Atherosclerosis 191: 391–396, 2007.

E1197

37. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331: 1056 –1061, 1994. 38. Steele R, Wall JS, De Bodo RC, Altszuler N. Measurement of size and turnover rate of body glucose pool by the isotope dilution method. Am J Physiol 187: 15–24, 1956. 39. Stone SJ, Myers HM, Watkins SM, Brown BE, Feingold KR, Elias PM, Farese RV Jr. Lipopenia and skin barrier abnormalities in DGAT2deficient mice. J Biol Chem 279: 11767–11776, 2004. 40. Sung KC, Wild SH, Kwag HJ, Byrne CD. Fatty liver, insulin resistance, and features of metabolic syndrome: relationships with coronary artery calcium in 10,153 people. Diabetes Care 35: 2359 –2364, 2012. 41. Swift LL, Kakkad B, Boone C, Jovanovska A, Jerome WG, Mohler PJ, Ong DE. Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism. FEBS Lett 579: 3183– 3189, 2005. 42. Villa A, Della Torre S, Stell A, Cook J, Brown M, Maggi A. Tetradian oscillation of estrogen receptor ␣ is necessary to prevent liver lipid deposition. Proc Natl Acad Sci USA 109: 11806 –11811, 2012. 43. Vozarova B, Stefan N, Lindsay RS, Saremi A, Pratley RE, Bogardus C, Tataranni PA. High alanine aminotransferase is associated with decreased hepatic insulin sensitivity and predicts the development of type 2 diabetes. Diabetes 51: 1889 –1895, 2002. 44. Wang TJ, Larson MG, Vasan RS, Cheng S, Rhee EP, McCabe E, Lewis GD, Fox CS, Jacques PF, Fernandez C, O’Donnell CJ, Carr SA, Mootha VK, Florez JC, Souza A, Melander O, Clish CB, Gerszten RE. Metabolite profiles and the risk of developing diabetes. Nat Med 17: 448 –453, 2011. 45. Woodard GA, Brooks MM, Barinas-Mitchell E, Mackey RH, Matthews KA, Sutton-Tyrrell K. Lipids, menopause, and early atherosclerosis in Study of Women’s Health Across the Nation Heart women. Menopause 18: 376 –384, 2011. 46. Wu K, Cappel D, Martinez M, Stafford JM. Impaired-inactivation of FoxO1 contributes to glucose-mediated increases in serum very lowdensity lipoprotein. Endocrinology 151: 3566 –3576, 2010. 47. Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, Bhanot S, Monia BP, Li YX, Diehl AM. Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis. Hepatology 45: 1366 –1374, 2007. 48. Yen CL, Stone SJ, Koliwad S, Harris C, Farese RV Jr. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J Lipid Res 49: 2283–2301, 2008. 49. Zhu L, Brown WC, Cai Q, Krust A, Chambon P, McGuinness OP, Stafford JM. Estrogen treatment after ovariectomy protects against fatty liver and may improve pathway-selective insulin resistance. Diabetes 62: 424 –434, 2013. 50. Zhu L, Johnson C, Bakovic M. Stimulation of the human CTP:phosphoethanolamine cytidylyltransferase gene by early growth response protein 1. J Lipid Res 49: 2197–2211, 2008.

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