Tissue-selective estrogen complexes with bazedoxifene prevent metabolic dysfunction in female mice

Abstract

Pairing the selective estrogen receptor modulator bazedoxifene (BZA) with estrogen as a tissue-selective estrogen complex (TSEC) is a novel menopausal therapy. We investigated estrogen, BZA and TSEC effects in preventing diabetisity in ovariectomized mice during high-fat feeding. Estrogen, BZA or TSEC prevented fat accumulation in adipose tissue, liver and skeletal muscle, and improved insulin resistance and glucose intolerance without stimulating uterine growth. Estrogen, BZA and TSEC improved energy homeostasis by increasing lipid oxidation and energy expenditure, and promoted insulin action by enhancing insulin-stimulated glucose disposal and suppressing hepatic glucose production. While estrogen improved metabolic homeostasis, at least partially, by increasing hepatic production of FGF21, BZA increased hepatic expression of Sirtuin1, PPARα and AMPK activity. The metabolic benefits of BZA were lost in estrogen receptor-α deficient mice. Thus, BZA alone or in TSEC produces metabolic signals of fasting and caloric restriction and improves energy and glucose homeostasis in female mice.

Keywords: AMPKα, AMP-activated protein kinase α; AUC, area-under the curve; Akt, protein kinase B; BAT, brown adipose tissue; BZA, bazedoxifene; Bazedoxifene; CE, conjugated equine estrogens; E2, 17β-estradiol; ER, estrogen receptor; FAS, fatty acid synthase; FGF21, fibroblast growth factor 21; GIR, glucose infusion rate; H&E, hematoxylin and eosin; HFD, high-fat diet; HGP, hepatic glucose production; ITT, insulin tolerance test; Insulin resistance; LPL, lipoprotein lipase; Lcn2, lipocalin 2; Menopause; Metabolic syndrome; NAFLD, non-alcoholic fatty liver disease; OGTT, oral glucose tolerance test; OVX, ovariectomy; PTT, pyruvate tolerance test; RBP4, retinol binding protein 4; RER, respiratory exchange ratio; Rd, rate of whole-body glucose disappearance; SERM, selective estrogen receptor modulator; TBARS, thiobarbituric acid reactive substances; TG, triacylglycerol; TSEC, tissue-selective estrogen complex; Tissue-selective estrogen complexes; Type 2 diabetes; UCPs, uncoupling proteins; VO2, oxygen consumption; WAT, white adipose tissue..

Figure 1

TSECs and BZA prevent visceral adiposity. Mice were subjected to sham or OVX surgeries and received the indicated drug treatments for 8 weeks. (A) Body weights, (B) weekly weight gains and (C) weights of visceral adipose depots (n=10). (D) Representative H&E stained section of parametrial adipose tissue and distribution histogram of adipocyte size. (E) Average size of adipocytes and (F) relative adipocyte number (n=4). (G) Uterine weights. Means are adjusted for final body mass as covariate using the ANCOVA analysis (n=10). (H) LPL mRNA expressions from WAT (n=6). Values represent means±S.E. ^*Significantly different from all other treatment groups (A) or the OVX vehicle group (B-H) (^⁎P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001). ^†Significantly different from the sham vehicle (P<0.01).

Figure 2

TSECs and BZA improve hepatic and muscle lipid homeostasis. Mice were subjected to sham or OVX surgeries and received the indicated drug treatments for 8 weeks. (A) Representative H&E stained sections of liver. (B) Liver TG contents (n=10). (C) Liver FAS enzyme activity (n=9–12). (D) Liver FAS mRNA expressions (n=6). (E) Liver TBARS contents (n=6). (F) Muscle TG contents (n=10). Relative phospho-AMPKα (Thr 172) was quantified in (G) liver and (H) skeletal muscle (n=6). Values represent means±S.E. ^*Significantly different from the OVX vehicle group (^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001).

Figure 3

TSEC and BZA promote lipid oxidation and increase energy expenditure via different pathways. OVX mice received the indicated drug treatments for 4 weeks prior to the measurements of (A) O₂ consumption, (B) energy expenditure and (C) respiratory exchange ratio (VCO₂/VO₂) (n=6). The O₂ consumption and energy expenditure were normalized to fat-free mass. In the separate study, mice were subjected to sham or OVX surgeries and received the indicated drug treatments for 8 weeks. The following measurements were performed in the fed state (D) FGF21 mRNA, (E) serum FGF21 (n=10), (F) SIRT1 mRNA, (G) PPARα mRNA and (H) mRNAs of FGF15, SHP and PEPCK1 (pck1) (n=6). mRNA were quantified by Q-PCR for liver samples. Values represent means±S.E. ^*Significantly different from the OVX vehicle group (^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001).

Figure 4

TSECs and BZA improve glucose and insulin homeostasis. Mice were subjected to sham or OVX surgeries and received the indicated drug treatments for 4 weeks prior to the measurements of (A) fed blood glucose, (B) fed plasma insulin, (C) 8-h fasting blood glucose, (D) 8-h fasting plasma insulin, (E) glucose concentrations during OGTT, (F) area under the curve (AUC) for glucose for (E), (G) insulin concentrations during OGTT and (H) AUC for insulin for (G). (I) Glucose concentrations during the ITT (Week 7) and (J) AUC for glucose during ITT. Values represent means±S.E. (n=10). ^*Significantly different from the OVX vehicle group (^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001).

Figure 5

BZA improves glucose and lipid homeostasis via ERα. Mice were subjected to OVX surgery and received the indicated drug treatments for 8 weeks. (A) Weight gain, (B) visceral fat depot weights, (C) hepatic TG content, (D) serum leptin/adiponectin ratio. (E) Glucose concentrations during OGTT and (F) area under the curve (AUC) for glucose at Week 4. (G) Glucose concentrations during the ITT and (H) AUC for glucose at Week 7. Values represent means±S.E. (n=6). ^*Significantly different from the corresponding vehicle groups (^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001).

Figure 6

CE and TSEC improve systemic insulin action. The OVX mice received the indicated drug treatments for 4 weeks and subjected to a euglycemic-hyperinsulinemic clamp for measurement of (A) GIR, (B) Rd and (C) insulin suppression of HGP (n=6–9). In the separate study, (D) glucose concentrations were measured during a PTT performed at Week 4; (E) AUC for glucose during PTT (n=6). (F) Insulin-stimulated Akt activity was measured in lysates from liver and muscle collected following the clamp study using western blotting of Akt phosphorylation and expression. (G) Quantification of immunoblot is shown. p-Akt, phosphorylated-Akt; Akt, total Akt (muscle, n=3; liver, n=4). In the separate study, sham or OVX mice received the indicated drug treatments for 8 weeks prior to the measurements of (H) hepatic CC1 mRNA expressions (n=6). (I) CEACAM1 expression (CC1) and phosphorylation (pCC1) were measured by Western blotting using lysates from liver collected following the clamp study (n=4). Values represent means±S.E. ^*Significantly different from the OVX vehicle group (^*P<0.05, ^**P<0.01, ^***P<0.001).

Figure 7

Proposed mechanism of estrogen, BZA and TSEC effects of hepatic lipid metabolism. (A) Estrogen (CE and E2), TSEC and BZA all induce CEACAM1 expression and phosphorylation thus inhibiting FAS activity and preventing hepatic lipogenesis. (B) CE and E2 promote hepatic lipid oxidation through FGF21 production, thereby elevating basal metabolic rate. In contrast, BZA promotes hepatic lipid oxidation by increasing SIRT1and PPARα expression as well as increasing AMPK activity. The combination of estrogen and BZA in a TSEC promotes a state of increased FGF21 production and sensitivity. Altogether, these effects promote lipid combustion in liver that prevents fat storage in skeletal muscle and adipose tissue.