Estradiol impairs hypothalamic molecular responses to hypoglycemia

Abstract

In rats and humans estradiol attenuates neuroendocrine responses to hypoglycemia. Since neuroendocrine responses to hypoglycemia are mediated by hypothalamic neurons, we assessed if estradiol attenuates hypoglycemia-induced gene expression in the hypothalamus in female ovariectomized mice. As expected, estradiol-implanted ovariectomized mice exhibited increased plasma estradiol, increased uterine weight, decreased body weight, decreased visceral adiposity, and enhanced glucose tolerance with decreased plasma insulin. Estradiol-implanted mice exhibited attenuated hypoglycemia-induced gene expression of both glucose transporter 1 (Glut1) and inhibitor of kappa beta signaling (IkappaB) in the hypothalamus but not in the liver. Estradiol also attenuated hypoglycemia-induced plasma glucagon, pituitary proopiomelanocortin (POMC), and adrenal c-fos, consistent with impaired counterregulatory responses to hypoglycemia. In addition, estradiol inhibited hypothalamic expression of carnitine palmitoyltransferase (CPT1a and CPT1c) and pyruvate dehydrogenase kinase 4 (PDK4), effects that would be expected to enhance the accumulation of long-chain fatty acids and glycolysis. Taken together, these findings suggest hypothalamic mechanisms mediating attenuation of hypoglycemia-induced neuroendocrine responses.

Fig.1. Estradiol treatment decreases body weight gain but not daily food intake

Mice were ovariectomized and implanted with either placebo (OVX-P) or estradiol (OVX-E). Body weight (A) and food intake (B) were monitored weekly. Data are presented as mean±SEM (n=12-14/group) and statistical difference was determined by Student’s t-test (* p<0.05).

Fig.2. Estradiol treatment has no effect on heat production or physical activity but increases respiratory quotient

Parameters of metabolic rate and physical activity were determined by indirect calorimetry over at least one light/dark cycle. Estradiol had no significant effect on (A) heat production or (B) horizontal activity (number of beam breaks due to horizontal movement per 10-min period), but (C) increased respiratory quotient (RQ). Statistical difference was determined by two-way ANOVA followed by Tukey HSD test (n=8; p< 0.05)

Fig.3. Estradiol treatment enhances glucose tolerance

Mice were challenged with glucose (at 0min) after overnight fasting. Blood samples were collected to measure blood glucose (A) and plasma insulin (B) concentrations. Data are presented as mean±SEM (n=12-14/group) and statistical difference was determined by Student’s t-test (*p<0.05)

Fig.4. Estradiol treatment attenuates the induction of hypothalamic gene expression by insulin-induced hypoglycemia

Hypoglycemia was induced by i.p. insulin injection alone, as described in Materials and Methods. Mice were sacrificed 3 hours after insulin injection and tissue samples were collected for gene expression analysis. Total RNA was extracted to measure relative mRNA level by quantitative real-time PCR (qRT-PCR). Data are calculated as fold regulation and presented as mean±SEM (n=5-8). Groups with different letters are statistically different (p<0.05) by two-way ANOVA followed by Tukey HSD test.

Fig.5. Estradiol treatment produces pro-glycolytic hypothalamic gene expression

Hepatic and hypothalamic CPT1a (A), CPT1c (B) and PDK4 (C) mRNA levels were measured by qRT-PCR. Data are calculated as fold regulation and presented as mean±SEM (n=5-8). Statistical differences were determined by two-way ANOVA. There was not a significant interaction between estradiol and hypoglycemia, nor a main effect of hypoglycemia on the expression of these genes except hepatic GK (not shown) and hypothalamic CPT1c (p=0.021), whereas estradiol significantly inhibited the expression of CPT1a (p=0.005), CPT1c (p=0.026) and PDK4 (p=0.0495) in hypothalamus but not in liver.

Fig.6. Estradiol treatment induces hypothalamic expression of AgRP and NPY

Hypothalamic AgRP, NPY, and POMC mRNA levels were measured by qRT-PCR. Data are presented as mean±SEM (n=5-8). Statistical difference was determined by two-way ANOVA. There was no main effect of hypoglycemia, nor an interaction between estradiol and hypoglycemia, whereas estradiol significantly induced expression of both AgRP (p=0.009) and NPY (p=0.004), and inhibited expression of hypothalamic POMC (though this effect narrowly failed to achieve statistical significance, p=0.07).