Midlife estradiol treatment reduces the firing rate of liver-related PVN neurons in ovariectomized high-fat diet-fed mice

Abstract

Estrogen plays a critical role in the regulation of physiological functions, including metabolism, and its involvement in the regulation of insulin sensitivity and glucose homeostasis has major clinical relevance. Despite the importance of the brain-liver pathway in the regulation of glucose metabolism and that postmenopausal women have an increased risk of developing metabolic disorders, the effect of hormone therapy on hypothalamic neurons involved in the regulation of liver metabolism is not known. Here, we tested the hypothesis that in middle-aged, high-fat diet (HFD)-fed female mice, the excitability of liver-related neurons in the paraventricular nucleus (PVN) of the hypothalamus is increased, whereas estradiol treatment attenuates this increase. Mice fed with phytoestrogen-free control (low-fat diet) or HFD were ovariectomized, received a silastic capsule implant containing either estradiol or vehicle, and stayed on their respective diets. Estradiol treatment resulted in less fat mass and lower body weight. Liver-related neurons were identified with a retrograde, transsynaptic viral tracer, and patch-clamp recordings were conducted from identified neurons in the PVN. Our data show that the excitability of liver-related PVN neurons was increased in ovariectomized HFD mice compared with LFD-fed mice. In estradiol-treated HFD mice, the firing of liver-related PVN neurons was significantly reduced compared with vehicle-treated HFD mice, whereas in LFD mice, estradiol treatment did not alter the activity of liver-related PVN neurons. Our findings suggest that midlife estradiol treatment has beneficial effects on liver-related PVN neurons and thus may contribute to the improved metabolic status observed in estradiol-treated HFD mice.NEW & NOTEWORTHY Menopause increases the risk of metabolic disorders, and despite the importance of the brain-liver pathway in the regulation of glucose homeostasis, the effect of estradiol treatment on liver-related neurons is not known. Our data show that in middle-aged, high-fat diet-fed, ovariectomized female mice, the excitability of liver-related neurons in the paraventricular nucleus is increased, whereas estradiol treatment attenuates this increase. These data suggest that midlife estradiol treatment is beneficial for the brain-liver pathway.

Keywords: electrophysiology; estradiol; high-fat diet; liver-related PVN neurons; ovariectomy.

Figure 1:. Experimental design and uterine weight.

A: Eleven weeks old female C57Bl/6J mice were fed a low-fat diet (LFD) for ~7 months. Mice were exposed to a high-fat diet (HFD) or kept on the LFD for ~12 weeks, then bilateral ovariectomy (OVX) was performed with subcutaneous implantation of capsules delivering either 17-β estradiol (E2) or cholesterol (vehicle, VEH). B: The weight of dissected uteri was determined after sacrifice by DXA to confirm successful OVX and E2 treatment. Circles represent individual mice. Data, represented as mean±SEM, were analyzed by two-way ANOVA followed by uncorrected Fisher’s LSD multiple comparisons. Significant effect of treatment was observed ***p<0.001, ****p<0.0001. LFD-VEH: solid orange bars, LFD-E2: striped, orange bars, HFD-VEH: solid blue bars, HFD-E2: striped, blue bars.

Figure 2:. Body composition.

Body composition was measured by DXA in anesthetized mice. A: Body weight of mice following treatment with 17-β estradiol or vehicle. B-C: Estradiol treatment reduced fat mass, whereas lean mass did not change. Data are represented as mean±SEM. Circles represent individual mice. Effect of diet and treatment were analyzed by two-way ANOVA followed by uncorrected Fisher’s LSD multiple comparisons. Significance: *p<0.05, **p<0.01. LFD-VEH: solid orange bars, LFD-E2: striped, orange bars, HFD-VEH: solid blue bars, HFD-E2: striped, blue bars.

Figure 3:. Identification and localization of recorded liver-related PVN neurons.

A: A lower magnification confocal image shows the PVN, whereas the higher magnification, boxed area illustrates a recorded (red) liver-related (green) PVN neuron. PVN: paraventricular nucleus of the hypothalamus; 3V: third ventricle. B: Location of recorded liver-related PVN neurons. Red dots represent the approximate location of the recorded neurons. Schematic of the PVN map is based on Franklin, Keith BJ. “The mouse brain in stereotaxic coordinates.” (third edition 2007).

Figure 4:. Overactivity of liver-related PVN neurons is attenuated in estradiol treated middle-aged, ovariectomized HFD mice.

A: Representative traces illustrate the firing rate of liver-related PVN neurons at baseline. B: Bar graphs illustrate that the frequency of action potentials was significantly higher in HFD-VEH fed mice compared to LFD-VEH mice and estradiol treatment led to a decrease of firing rate in HFD fed mice. C: Representative traces illustrate the firing rate of liver-related PVN neurons. Neurons were hyperpolarized to ~−90mV, then depolarizing current steps were applied to determine the firing of liver-related neurons. D: Current-action potential frequency curves and computed simple linear regression demonstrated that liver-related PVN neurons fire more in HFD mice. E: Representative recordings of sEPSCs in liver-related PVN neurons. F-G: Bar graphs illustrate that the frequency of sEPSCs (F) was similar among the groups, whereas the amplitude of sEPSCs (G) was larger in HFD-E2 mice compared to LFD-E2 and HFD-VEH mice. Circles represent individual neurons. Data were analyzed by two-way ANOVA followed by uncorrected Fisher’s LSD multiple comparisons. Significance *p<0.05, **p<0.01, ****p<0.0001. LFD-VEH: solid orange bars, LFD-E2: striped, orange bars, HFD-VEH: solid blue bars, HFD-E2: striped, blue bars.