Suppression of estrogen receptor beta classical genomic activity enhances systemic and adipose-specific response to chronic beta-3 adrenergic receptor (β3AR) stimulation

Abstract

White adipose tissue (WAT) dysfunction independently predicts cardiometabolic disease, yet there is a lack of effective adipocyte-targeting therapeutics. B3AR agonists enhance adipocyte mitochondrial function and hold potential in this regard. Based on enhanced sensitivity to B3AR-mediated browning in estrogen receptor (ER)alpha-null mice, we hypothesized that ERβ may enhance the WAT response to the B3AR ligand, CL316,243 (CL).Methods: Male and female wild-type (WT) and ERβ DNA binding domain knock-out (ERβDBDKO) mice fed high-fat diet (HFD) to induce obesity were administered CL (1 mg/kg) daily for 2 weeks. Systemic physiological assessments of body composition (EchoMRI), bioenergetics (metabolic chambers), adipocyte mitochondrial respiration (oroboros) and glucose tolerance were performed, alongside perigonadal (PGAT), subcutaneous (SQAT) and brown adipose tissue (BAT) protein expression assessment (Western blot). Mechanisms were tested in vitro using primary adipocytes isolated from WT mice, and from Esr2-floxed mice in which ERβ was knocked down. Statistical analyses were performed using 2 × 2 analysis of variance (ANOVA) for main effects of genotype (G) and treatment (T), as well as GxT interactions; t-tests were used to determine differences between in vitro treatment conditions (SPSS V24).Results: There were no genotype differences in HFD-induced obesity or systemic rescue effects of CL, yet ERβDBDKO females were more sensitive to CL-induced increases in energy expenditure and WAT UCP1 induction (GxT, p < 0.05), which coincided with greater WAT B3AR protein content among the KO (G, p < 0.05). Among males, who were more insulin resistant to begin with (no genotype differences before treatment), tended to be more sensitive to CL-mediated reduction in insulin resistance. With sexes combined, basal WAT mitochondrial respiration trended toward being lower in the ERβDBDKO mice, but this was completely rescued by CL (p < 0.05). Confirming prior work, CL increased adipose tissue ERβ protein (T, p < 0.05, all), an effect that was enhanced in WAT and BAT the female KO (GxT, p < 0.01). In vitro experiments indicated that an inhibitor of ERβ genomic function (PHTPP) synergized with CL to further increase UCP1 mRNA (p = 0.043), whereas full ERβ protein was required for UCP1 expression (p = 0.042).Conclusion: Full ERβ activity appears requisite and stimulatory for UCP1 expression via a mechanism involving non-classical ERβ signaling. This novel discovery about the role of ERβ in adipocyte metabolism may have important clinical applications.

Selective breeding for physical inactivity produces cognitive deficits via altered hippocampal mitochondrial and synaptic function

“…The resulting mitochondrial pellet is resuspended in 150 μL of mitochondrial phosphate buffer (MiPO3). Mitochondria were given 30 min on ice prior to being loaded in the Oroboros for assessment of mitochondrial respiration using high-resolution respirometry (Oroboros Oxygraph-2 k, Oroboros Instruments, Innsbruck, Austria) ( Cunningham et al, 2021 , 2022 ; Queathem et al, 2022 ). The mitochondrial respiration protocol following addition of mitochondria to the Oroboros consisted of malate (2 mM) and glutamate (5 mM) for state 2 respiration (measuring complex I leak with substrate rate limiting), ADP (1,000 μM) for state 3 – Complex I (measuring oxidative phosphorylation through complex I), succinate (10 mM) for state 3 – complex I + II (measuring oxidative phosphorylation capacity through both complex I and II), FCCP (0.25 μM) for uncoupled (maximal oxygen consumption), and Cyto C (5 μM) to verify quality of mitochondrial preparations.…”

Adipocyte Metabolism and Health after the Menopause: The Role of Exercise

“…However, in the study by Porter et al described previously, the protein content of both ERα and ERβ was highly correlated with the mitochondrial protein uncoupling protein 1 (UCP-1) across sexes and ages [ 126 ]. Those same authors, using preclinical models, have demonstrated that both exercise and a selective beta 3 adrenergic receptor ligand, CL316,243, increased the density of ERβ in both subcutaneous and visceral fat, suggesting that stimulation of adipocyte metabolism may cause increased ERβ expression on adipocytes [ 128 , 129 ]. Meanwhile, a series of preclinical studies in rodents have shown that selective activation of ERβ has metabolism-boosting effects [ 108 , 109 , 130 ].…”

“…Bridging this back to whether changes in ER expression may affect exercise responsiveness after the menopause, a preclinical rodent study revealed that ERβ-mutated mice were resistant to the adipose-tissue-specific metabolic adaptations with exercise that were observed in wild-type mice [ 132 ]. Intriguingly, both exercise [ 133 ] and the adipose-tissue-specific “exercise mimetic” CL316,243 [ 53 , 129 , 134 ] increase ERβ density selectively in adipose tissue. Capitalizing on this enhanced ERβ expression, particularly following hormone loss, combining selective ERβ activation with exercise (or drugs that affect adipose tissue metabolism) may be an effective strategy to maximize maintenance of metabolic health following the menopause.…”