Silencing of estrogen receptor alpha in the ventromedial nucleus of hypothalamus leads to metabolic syndrome

Abstract

Estrogen receptor alpha (ERalpha) plays a pivotal role in the regulation of food intake and energy expenditure by estrogens. Although it is well documented that a disruption of ERalpha signaling in ERalpha knockout (ERKO) mice leads to an obese phenotype, the sites of estrogen action and mechanisms underlying this phenomenon are still largely unknown. In the present study, we exploited RNA interference mediated by adeno-associated viral vectors to achieve focused silencing of ERalpha in the ventromedial nucleus of the hypothalamus, a key center of energy homeostasis. After suppression of ERalpha expression in this nucleus, female mice and rats developed a phenotype characteristic for metabolic syndrome and marked by obesity, hyperphagia, impaired tolerance to glucose, and reduced energy expenditure. This phenotype persisted despite normal ERalpha levels elsewhere in the brain. Although an increase in food intake preceded weight gain, our data suggest that a leading factor of obesity in this model is likely a decline in energy expenditure with all three major constituents being affected, including voluntary activity, basal metabolic rate, and diet-induced thermogenesis. Together, these findings indicate that ERalpha in the ventromedial nucleus of the hypothalamus neurons plays an essential role in the control of energy balance and the maintenance of normal body weight.

Fig. 1.

Increased body weight and adiposity in mice after ERα knockdown in the VMN. (A) Representative images of ERα immunostaining in the hypothalamic regions (Upper) of OVXed mice (Lower) 14 weeks after injections with either AAV.H1.Luc (Left) or AAV.H1.ER1 (Right). Note reduced ERα expression in the VMN but not in the ARC in AAV.H1.ER1-treated mice. (B) Body weight of OVXed mice injected with the indicated vectors and housed in running wheel activity cages. At week 3 all of the animals were treated with 21-day time-release EB pellets (indicated by an arrow). The difference between the groups became significant (P < 0.01) as early as at week 3. (C) A similar increase in body weight was also observed in gonad-intact females (P < 0.001). (D and E) Accumulation of adipose tissue in several fat depot areas in OVXed (D) and gonad-intact (E) animals treated with AAV.H1.ER1 (∗, P < 0.001). Data are means ± SEM.

Fig. 2.

Hyperphagia and impaired glucose tolerance. (A) Food intake of OVXed animals described in Fig. 1B. Notice that whereas estrogen administration (marked by an arrow) reduced food intake in AAV.H1.ER1-treated animals (P < 0.01), they remained hyperphagic compared with control mice throughout the experiment (P < 0.01). (B) An increase in daily food intake was also observed in gonad-intact females treated with AAV.H1.ER1 (∗, P < 0.05). (C) 2-DG-induced hyperphagia. Nonfasted OVXed animals were injected with saline or 2-DG (250 mg/kg, i.p.), and food intake was measured for 4 h during the light phase. AAV.H1.ER1-treated mice demonstrated a stronger hyperphagic response (∗, P < 0.01) compared with AAV.H1.ER1-treated animals. (D) Hyperglycemia in OVXed mice injected with AAV.H1.ER1 under normal feeding condition (∗, P < 0.05). (E) Glucose tolerance test in OVXed mice. Fasting glucose concentrations were similar between the groups. However, after a glucose challenge blood glucose reached higher levels in AAV.H1.ERa-treated mice (∗, P < 0.05). (F) Glucose tolerance test in gonad-intact females produced similar results (∗, P < 0.05). Data represent means ± SEM.

Fig. 3.

Decreased physical activity and diet-induced thermogenesis. (A) Voluntary locomotor activity. OVXed mice from the experiments described in Figs. 1B and 2A were continuously monitored in running-wheel activity cages for 12 weeks. AAV.H1.ER1-injected mice had reduced levels of activity and were resistant to EB treatment. (B and C) Open-field test. Gonad-intact female mice injected with AAV.H1.ER1 demonstrated lower horizontal (B) and vertical (C) activity levels compared with control animals (∗, P < 0.05) during the last 20 min of a 30-min test. (D) Diet-induced thermogenesis. Consumption of food for 1 h after a 24-h fast raised body temperature in control mice (∗, P < 0.05) but not in AAV.H1.ER1-injected mice. (E) An impaired thermogenic response to feeding was also seen in gonad-intact females. Data are means ± SEM.

Fig. 4.

Obese phenotype in rats. (A) A representative photomicrograph of ERα immunoreactivity in the rat hypothalamus 3 weeks after unilateral injections with the indicated vectors. (B–H) Effects of bilateral vector infusions into the VMN of gonad-intact female rats on body weight (B), total adiposity (C), visceral to s.c. fat ratio (D), lean tissue mass (E), total energy expenditure (F), basal metabolic rate (G), and respiratory quotient (H). Data are means ± SEM (∗, P < 0.05).