Avaliação dos efeitos dos hormônios ovarianos e da alimentação restrita à fase de luz sobre o relógio hepático e parâmetros metabólicos de ratas

Abstract

It has been demonstrated that the time of food intake contributes to control body weight gain and glucose metabolism. One of the direct effects of food offering throughout inactive phase of rodents, that is, the light phase, is alteration in the temporal pattern of the liver’s clock genes. On the other hand, the hepatic clock directly regulates glucose homeostasis, which contributes to the maintenance of glucose levels during fasting. Moreover, estradiol regulates food intake and insulin resistance. Thus, the present study investigated the role of ovarian hormones in the response of the hepatic clock to time restricted (TR) feeding during the light phase, as well as in the tolerance to glucose. To that end, Wistar rats went through SHAM or ovariectomy surgery (OVX). Both groups were submitted to standard chow ad libtum (AD), or TR of 12 hours in the light phase for twenty-one days. Food intake and body weight were measured, and at the end of the protocol, animals were euthanized at Zeitgeber Time 1 (ZT1, one hour after the lights went on in the vivarium), at ZT6, ZT12 and ZT18 for collecting liver and blood. The expression of the clock genes Per1, Bmal1, Reverbα, Cry1 and Cry2 was performed at the four ZTs by qRT-PCR. OVX rats gained more weight than did the SHAM rats and TR feeding reduced the body weight gain of both groups. Food intake in OVX rats was higher and the lack of ovarian hormones did not change the circadian pattern of food intake. Thus, both SHAM and OVX rats under TR feeding showed reduced food intake during the dark phase. TR feeding also reduced the food intake of both groups over the 24 hours. These effects of TR feeding on body weight gain and food intake were accompanied by similar temporal changes in hepatic clock genes expression in SHAM and OVX rats. Therefore, ovarian hormones did not interfere in the response of the hepatic clock to TR feeding. On the other hand, OVX rats presented higher levels of Per1, Bmal1, Cry1 e Cry2 mRNA expression compared to SHAM at ZT1. Hence, we evaluated whether estradiol contributes to the maintenance in the clock genes expression. For this purpose, another group of OVX rats was subcutaneously treated with corn oil (0,2 mL) or cypionate of estradiol (10 μg / 0,2 mL) for three consecutive days and was then euthanized at ZT1 in the next day. The treatment with estradiol reversed the OVX-induced increasing in Per1, Bmal1, Cry1 e Cry2 but it did not alter the mRNA levels of Reverb-α gene. We also evaluated whether ovariectomy or TR feeding alter the temporal pattern of leptin secretion once they affected food intake. Serum leptin was determined by Elisa at ZT1 and ZT12. Leptin levels were lower at ZT12 in SHAM-AD rats however it did not change between ZT1 and ZT12 of SHAM-TR, OVX-AD and OVX-TR rats. Thus, both OVX and TR feeding abolished the temporal variation in leptin levels, what may explain their effects in the food intake. The expression of PPar-α e TRB-3 genes, involved in lipid and glucose metabolism, respectively, was analyzed at ZT1 and ZT12. Neither OVX or TR feeding were capable of affecting the temporal pattern of these clock gene expression in the liver. Intraperitoneal glucose tolerance tests (IPGTT) were performed at ZT1 and ZT13 in NAÏVE (before OVX) and OVX rats. NAÏVE rats presented a better response at ZT13 compared to ZT1 as demonstrated by lower levels of glucose in the area under the curve. Different response over the time was not observed in OVX rats. Beyond abolishing temporal difference in the response to the IPTTG, compared to NAÏVE, OVX rats presented a worse response at ZT13. TR feeding reduced the glucose tolerance of OVX rats. So, we concluded that ovarian hormones contribute to the maintenance of clock genes mRNA levels in liver in a time specific way, without changing, however, the response of the clock liver to TR feeding. Estradiol seems to be the responsible for this maintenance. Ovarian hormones are also important to the glucose homeostasis as ovariectomy abolished the temporal response to ipTTG and it caused a worse response in IPTTG compared to NAÏVE at ZT13. Besides, the lack of ovarian hormones together with TR feeding resulted in a lower glucose tolerance. These results may contribute to the understanding of metabolic alterations caused by the decrease or absence of ovarian hormones like the increase in body weight gain and the impairment in the glucose metabolism commonly observed during menopause.