Role of ERβ in adipocyte metabolic response to wheel running following ovariectomy

Abstract

Estrogen receptor β (ERb), one of the two major estrogen receptors, acts via genomic and non-genomic signaling pathways to affect many metabolic functions, including mitochondrial biogenesis and respiration. This study assessed the effect of ERb classical genomic activity on adipocyte-specific and -systemic metabolic responses to wheel running exercise in a rodent model of menopause. Female mice lacking the ERb DNA-binding domain (ERbDBDKO, n = 20) and WT (n = 21) littermate controls were fed a high-fat diet (HFD), ovariectomized (OVX), and randomized to control (no running wheel) and exercise (running wheel access) groups and were followed for 8 weeks. Wheel running did not confer protection against metabolic dysfunction associated with HFD+OVX in either ERbDBDKO or WT mice, despite increased energy expenditure. Unexpectedly, in the ERbDBDKO group, wheel running increased fasting insulin and surrogate measures of insulin resistance, and modestly increased adipose tissue inflammatory gene expression (P ≤ 0.05). These changes were not accompanied by significant changes in adipocyte mitochondrial respiration. It was demonstrated for the first time that female WT OVX mice do experience exercise-induced browning of white adipose tissue, indicated by a robust increase in uncoupling protein 1 (UCP1) (P ≤ 0.05). However, KO mice were completely resistant to this effect, indicating that full ERb genomic activity is required for exercise-induced browning. The inability to upregulate UCP1 with exercise following OVX may have resulted in the increased insulin resistance observed in KO mice, a hypothesis requiring further investigation.

Keywords: DNA-binding domain; adipose tissue; estrogen receptor beta; ovariectomy.

Figure 1

Study design. Female ERB_DBDKO (KO) (n = 20) and littermate WT control mice (n = 21) were individually housed under thermoneutral conditions (26–28°C) and a 12 h light:12 h darkness cycle. Beginning at ~11 (range 10.5–11.5) weeks of age, mice were provided a high-fat diet (46.4% fat: 36.0% carbohydrate: 17.6% protein) ad libitum prior to being ovariectomized at 16 weeks of age. Following a 2-week recovery period, mice in the exercise (‘Ex’) group were provided running wheels whereas control (‘Con’) mice were not provided wheels. One week prior to sacrifice, total and resting energy expenditure and spontaneous physical activity were assessed in metabolic chambers, and glucose tolerance was determined via glucose tolerance testing. Body composition was measured via EchoMRI, and after sacrifice, white adipose tissue (WAT) was harvested and analyzed as described. HFD, high-fat diet.

Figure 2

Genotype validation and white adipose tissue estrogen receptor gene and protein expression. (A) Representative PCR indicating genotype validation. (B) Relative mRNA expression of estrogen receptor beta and estrogen receptor alpha (ERa) in perigonadal white adipose tissue (PGAT). Gene expression was normalized to the housekeeping gene, beta actin and expressed relative to WT control (Con) group. (C) Protein expression of ERb and ERa. (C) Representative western blot bands of ERb, ERa, and housekeeping protein beta actin. Error bars indicate S.E.M. P ≤ 0.05 was considered significant; non-significant P values not indicated. N_WTCon = 11, N_WTEx = 10, N_KOCon = 10, N_KOEx = 10. G, genotype effect; T, treatment effect; G×T, genotype × treatment interaction effect.

Figure 3

Measures of energy balance. (A) Body weight throughout study period. (B) Weekly wheel running distance. (C) Total energy expenditure (EE) in darkness cycle (DK) covaried for lean mass. (D) Darkness cycle spontaneous physical activity (SPA). (E) Light cycle resting EE (measured in LT cycle and covaried for lean mass). (F) Light cycle SPA. N_WTCon = 8–11, N_WTEx = 8–10, N_KOCon = 8–10, N_KOEx = 9–10. For all graphs, error bars indicate S.E.M. P ≤ 0.05 was considered significant; non-significant P values are not indicated. G, genotype effect; T, treatment effect; G×T, = genotype × treatment interaction effect.

Figure 4

Insulin resistance (IR) and adipose tissue inflammatory gene expression. (A) HOMA-IR, calculated as the product of fasting blood glucose and plasma insulin levels. (B) Adipo-IR, calculated as the product of non-esterified fatty acid levels and plasma insulin levels. (C) Perigonadal white adipose tissue (PGAT) inflammatory gene expression. Adipo, adiponectin; TNFa, tumor necrosis factor alpha. Gene expression was normalized to the housekeeping gene beta-actin and then expressed relative to WT Con group. Error bars indicate S.E.M. P ≤ 0.05 was considered significant; non-significant P values were not shown. *KO Ex group was significantly different from the other groups. N_WTCon = 11, N_WTEx = 10, N_KOCon = 10, N_KOEx = 10. G, genotype effect; T, treatment effect; G×T, genotype × treatment interaction effect.

Figure 5

Adipose tissue inflammatory protein expression. (A) Protein expression of inflammatory proteins via Western blot. (B) Representative bands. Error bars indicate S.E.M. P ≤ 0.05 was considered significant; non-significant P values were not indicated. NWTCon = 11, NWTEx = 10, NKOCon = 10, NKOEx = 10. G, genotype effect; T, treatment effect.

Figure 6

White adipose tissue browning. (A) Representative images of UCP1 histological staining in perigonadal white adipose tissue (PGAT). (B) Relative PGAT UCP1 staining intensity normalized to WTCon group (n = 4–5/group). (C) PGAT UCP1 protein expression and representative Western blot images. (D) PGAT UCP1 gene expression. (E) Mitochondrial OX PHOS protein expression and representative Western blot images. Gene and protein expression data were normalized to the housekeeping gene/protein, beta-actin and expressed relative to WTCon group. Error bars indicate S.E.M. P ≤ 0.05 was considered significant; non-significant P values were not shown. *WT Ex group was significantly different from all other groups via post hoc Tukey’s test. G, genotype effect; T, treatment effect; G×T, genotype × treatment interaction effect. Arrows indicate bands of interest.

Figure 7

Measures of white adipocyte browning and mitochondrial activity. (A) WAT mitochondrial OX PHOS protein expression. (B) Representative Western blot images. (C) PGAT COX4 (mitochondrial content indicator) gene expression. (D) PGAT O2 consumption normalized to WT Con group. MitoBasal: 100 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, adjusted to pH 7.1 with KOH at 37°C and 1 g/L fatty acid-free BSA, assessment of basal respiration. GMCILeak: glutamate (5 mM) and malate (2 mM) added, assessment of respiration in absence of ADP. GMADPOxPhos: titration of ADP (50–200 uM), assessment of complex I respiration. SuccinateCICII: addition of succinate (7.5 mM), assessment of state 3, complex I and complex II respiration. FCCPCICII: addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (0.25–0.5 uM), assessment of maximal uncoupled respiration (n = 3–4/group). Gene and protein expression data were normalized to the housekeeping gene/protein, beta-actin and expressed relative to WT Con group. Error bars indicate S.E.M. P ≤ 0.05 was considered significant; non-significant P values were not shown. G, genotype effect; T, treatment effect; G×T, genotype × treatment interaction effect. Arrows indicate bands of interest.

Figure 8

The hypothesized mechanism of the exercise-induced increase in insulin resistance (IR) in ERbDBDKO mice. Exercise increases mitochondrial activity, which associates with increased reactive oxygen species (ROS) and inflammation. UCP1 is an insulin-sensitizing protein that is often upregulated by exercise in adipose tissue for reasons that are not clear and via mechanisms that are not fully known. We propose that UCP1 upregulation is protective against adipocyte stress (e.g. exercise) and buffers exercise-induced stress. Further, when exercise is not coupled with an appropriate increase in UCP1, as seen in these mutant mice lacking the ERb DBD, exercise-induced adipose tissue cell stress leads to increased adipocyte and systemic IR. A full color version of this figure is available at https://doi.org/10.1530/JOE-21–0009.