17α‐estradiol acts through hypothalamic pro‐opiomelanocortin expressing neurons to reduce feeding behavior

Summary Weight loss is an effective intervention for diminishing disease burden in obese older adults. Pharmacological interventions that reduce food intake and thereby promote weight loss may offer effective strategies to reduce age‐related disease. We previously reported that 17α‐estradiol (17α‐E2) administration elicits beneficial effects on metabolism and inflammation in old male mice. These observations were associated with reduced calorie intake. Here, we demonstrate that 17α‐E2 acts through pro‐opiomelanocortin (Pomc) expression in the arcuate nucleus (ARC) to reduce food intake and body mass in mouse models of obesity. These results confirm that 17α‐E2 modulates appetite through selective interactions within hypothalamic anorexigenic pathways. Interestingly, some peripheral markers of metabolic homeostasis were also improved in animals with near complete loss of ARC Pomc transcription. This suggests that 17α‐E2 might have central and peripheral actions that can beneficially affect metabolism cooperatively or independently.


K E Y W O R D S
17a-estradiol, aging, food intake, hypothalamus, obesity, pro-opiomelanocortin Weight loss through reduced energy intake curtails disease burden and metabolic perturbations associated with obesity in older age (Waters, Ward, & Villareal, 2013). However, reduced food intake and sustained weight loss are difficult to maintain in humans due to adverse effects with thermoregulation, libido, satiety, and musculoskeletal mass (Dirks & Leeuwenburgh, 2006). These compliance issues have promoted research interest into pharmacological interventions that promote reductions in food intake without having to voluntarily restrict dietary intake. We recently reported that 17aestradiol (17a-E2), a naturally occurring enantiomer of 17b-estradiol, produces beneficial effects on metabolism and inflammation in old male mice (Stout et al., 2017). These effects may contribute to the reported extension of lifespan by 17a-E2 (Harrison et al., 2014;Strong et al., 2016), which may result from central and peripheral effects on food intake and nutrient-sensing pathways (Stout et al., 2017). Here, we demonstrate that 17a-E2 promotes weight loss in male mouse models of obesity, showing that the beneficial effects of 17a-E2 on food intake and body weight require a functional threshold level of Pomc expression in the hypothalamic arcuate nucleus (ARC).
We first assessed the effect of dietary 17a-E2 treatment on body mass and composition, food intake, spontaneous activity, and energy expenditure in male mice maintained on an obesogenic diet.
17a-E2 quickly initiated weight loss (Figure 1a), resulting in a significant decrease in body mass at the end of the study (Figure 1b). The reduction in body mass was observed despite continued high-fat feeding and was attributed to significant declines in fat mass , sparing lean mass as we previously reported (Stout et al., 2017). We also observed significantly enhanced glucose tolerance, evidenced by increased glucose disposal and decreased insulin secretion during an intraperitoneal glucose challenge ( Figure 1e) and reductions in fasting glucose and insulin levels ( Figure 1f). We performed a phenotypic assessment during week 20 of the intervention to determine the cause of weight reduction. 17a-E2 reduced food intake during the week of assessment, with the majority of these effects occurring during the dark cycle (Figure 1g-j). 17a-E2 did not reverse HFD-mediated reductions in locomotor activity (Figure 1k-m), nor did it alter metabolic rate (Figure 1n-o), suggesting that 17a-E2mediated effects on body mass and composition are driven by changes in food intake. Isolation and placement of mice into metabolic cages could potentially alter energy balance; therefore, changes in energy expenditure with 17a-E2 cannot be completely excluded.
To show that changes in food intake did not result from poor diet palatability, we evaluated body mass, body composition, and food intake in mice treated with subcutaneous slow-release 17a-E2 pellets. As with dietary treatment, subcutaneous 17a-E2 treatment initi- We and others have previously reported that 17a-E2 reduces food intake by acting through hypothalamic pathways (Butera, Beikirch, & Willard, 1990;Stout et al., 2017). Pomc-expressing neurons located within the ARC constitute the dominant anorexigenic node of appetite regulating neurons and are viewed as key regulators of energy homeostasis. Activation of these neurons via peripheral appetite regulators such as leptin (Cowley et al., 2001)  week 20 of the study, including (g) energy intake over a representative 24-hour sampling period, (h) cumulative weekly food, and (i) energy intake, (j) average daily energy intake during light and dark periods, (k) daily activity over a representative 24-hour sampling period, (l) cumulative weekly activity, (m) averaged daily activity during light and dark periods, (n) oxygen consumption (VO 2 ) normalized to body mass over a representative 24hour sampling period, and (o) averaged VO 2 normalized to body mass over the 7-day assessment period during light and dark periods. Change in (p) body mass, (q) body composition, and (r) averaged daily energy intake in mice implanted with subcutaneous cholesterol matrix pellets releasing either 0.0 (placebo), 0.1, or 0.3 lg/day 17a-E2. All data are expressed as mean AE SEM (A-0: N = 6/group; P-R: N = 5/group). For A-O, p < .05 considered statistically different from chow (*) or HFD ( # ) treated mice. For P-Q, p < .05 from baseline (*). For R, p < .05 from baseline (*, days À5 to 0), or placebo ( # ) during respective treatment periods Souza et al., 2005). Deletion of nPE2, nPE1, or insertion of a transcription-blocking neo selection cassette into the vicinity of the two hypothalamic neuronal Pomc enhancers reduces hypothalamic Pomc expression to~80%,~30%, or~2% of wild-type controls, respectively (Lam et al., 2015). A reduction in hypothalamic Pomc expression at or below~30% of wild-type controls in these mice results in a functional loss of Pomc-mediated regulation of body mass (Bumaschny et al., 2012;Lam et al., 2015;Zhan et al., 2013). Therefore, we hypothesized that if 17a-E2 were to act selectively by increasing hypothalamic Pomc expression, the treatment effects on body mass and food intake would be disrupted in mutant mice lacking nPE1 (Pomc D1 ) or those containing the Pomc transcription-block- To determine whether 17a-E2-mediated effects on food intake, body mass, and adiposity also modulate metabolic homeostasis, we assessed fasting glucose and insulin at baseline and week 3 of treatment. In alignment with Study 1, 17a-E2 treatment decreased fasting glucose in Pomc wt , Pomc D2 , and Pomc D1 mice (Figure 2i). There was no change in fasting glucose levels in Pomc neo mice, but as previously demonstrated, these mice are resistant to developing hyperglycemia because of a lower renal threshold for glycosuria (Chhabra et al., 2016). Interestingly, 17a-E2 lowered fasting insulin in Pomc neo mice (Figure 2j), an effect mirrored in the HOMA-IR data (Figure 2k). The physiological relevance of this modest reduction remains unclear.
Collectively, these data suggest that metabolic improvements by 17a-E2 may not be solely driven by declines in food intake and body mass. Future studies are needed to definitively determine whether 17a-E2 acts independently of ARC Pomc transcripts to improve systemic metabolic parameters.
We conclude that 17a-E2 acts via hypothalamic Pomc transcripts to reduce food intake, thereby promoting reductions in body mass and adiposity in male mouse models of obesity. By isolating the central effects of 17a-E2 to ARC Pomc, we gained insight into the mechanisms of 17a-E2 actions and established the basis for future experiments to explore beneficial effects of 17a-E2 that may occur independent of central appetite regulation. . The catabolic action of insulin in the brain F I G U R E 2 17a-E2-mediated effects on food intake, body mass, and adiposity are dependent upon hypothalamic Pomc gene transcription. (a) Change in body mass, normalized to baseline, following administration of 17a-E2. (b) Actual (left) and percent change (right) in body mass relative to baseline at necropsy. (c) Hypothalamic Pomc expression in Pomc wt , Pomc D2 , Pomc D1 , and Pomc neo mice at necropsy. (d) Daily energy intake before and following administration of 17a-E2. (e) Percent change in energy intake, normalized to body mass, before and following 17a-E2 treatment. Weekly (f) food intake, (g) body mass, and (h) fat and fat-free mass, normalized to baseline, following 17a-E2 treatment. (i) Fasting glucose, (j) fasting insulin, and (k) homeostatic model assessment of insulin resistance (HOMA-IR) at baseline and week 3 of the study following administration of 17a-E2. All data are expressed as mean AE SEM (Pomc WT N = 12; Pomc D2 N = 7; Pomc D1 N = 9; Pomc neo N = 7) with p < .05 considered statistically different from baseline (*; panels a-b,d-k) or Pomc wt (*; panels b-c)