Rat retina shows robust circadian expression of clock and clock output genes in explant culture.

PURPOSE: Circadian rhythms are central to vision and retinal physiology. A circadian clock located within the retina controls various rhythmic processes including melatonin synthesis in photoreceptors. In the present study, we evaluated the rhythmic expression of clock genes and clock output genes in retinal explants maintained for several days in darkness. METHODS: Retinas were dissected from Wistar rats, either wild-type or from the Per1-luciferase transgenic line housed under a daily 12 h:12 h light-dark cycle (LD12/12), and put in culture at zeitgeber time (ZT) 12 on semipermeable membranes. Explants from wild-type rats were collected every 4 h over 3 days, and total RNA was extracted, quantified, and reverse transcribed. Gene expression was assessed with quantitative PCR, and the periodicity of the relative mRNA amounts was assessed with nonlinear least squares fitting to sine wave functions. Bioluminescence in explants from Per1-luciferase rats was monitored for several days under three different culture protocols. RESULTS: Rhythmic expression was found for all studied clock genes and for clock downstream targets such as c-fos and arylalkylamine N-acetyltransferase (Aanat) genes. Clock and output genes cycled with relatively similar periods and acrophases (peaks of expression during subjective night, except c-fos, which peaked around the end of the subjective day). Data for Per1 were confirmed with bioluminescence monitoring, which also permitted culture conditions to be optimized to study the retina clock. CONCLUSIONS: Our work shows the free-running expression profile of multiple clock genes and potential clock targets in mammalian retinal explants. This research further strengthens the notion that the retina contains a self-sustained oscillator that can be functionally characterized in organotypic culture.journal articleresearch support, non-u.s. gov't20142014 06 02importe

SOCS3 deficiency in leptin receptor-expressing cells mitigates the development of pregnancy-induced metabolic changes

Objective: During pregnancy, women normally increase their food intake and body fat mass, and exhibit insulin resistance. However, an increasing number of women are developing metabolic imbalances during pregnancy, including excessive gestational weight gain and gestational diabetes mellitus. Despite the negative health impacts of pregnancy-induced metabolic imbalances, their molecular causes remain unclear. Therefore, the present study investigated the molecular mechanisms responsible for orchestrating the metabolic changes observed during pregnancy. Methods: Initially, we investigated the hypothalamic expression of key genes that could influence the energy balance and glucose homeostasis during pregnancy. Based on these results, we generated a conditional knockout mouse that lacks the suppressor of cytokine signaling-3 (SOCS3) only in leptin receptor-expressing cells and studied these animals during pregnancy. Results: Among several genes involved in leptin resistance, only SOCS3 was increased in the hypothalamus of pregnant mice. Remarkably, SOCS3 deletion from leptin receptor-expressing cells prevented pregnancy-induced hyperphagia, body fat accumulation as well as leptin and insulin resistance without affecting the ability of the females to carry their gestation to term. Additionally, we found that SOCS3 conditional deletion protected females against long-term postpartum fat retention and streptozotocin-induced gestational diabetes. Conclusions: Our study identified the increased hypothalamic expression of SOCS3 as a key mechanism responsible for triggering pregnancy-induced leptin resistance and metabolic adaptations. These findings not only help to explain a common phenomenon of the mammalian physiology, but it may also aid in the development of approaches to prevent and treat gestational metabolic imbalances

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)RAPG[2007/07790-6]RAPGJAS[2007/08733-06]JASACM[2005/60000-8]ACMJCNJCNCROC[04/06767-2]CRO

Suppression of prolactin secretion partially explains the antidiabetic effect of bromocriptine in ob/ob mice

Previous studies have shown that bromocriptine mesylate (Bromo) lowers blood glucose levels in adults with type 2 diabetes mellitus; however, the mechanism of action of the antidiabetic effects of Bromo is unclear. As a dopamine receptor agonist, Bromo can alter brain dopamine activity affecting glucose control, but it also suppresses prolactin (Prl) secretion, and Prl levels modulate glucose homeostasis. Thus, the objective of the current study was to investigate whether Bromo improves insulin sensitivity via inhibition of Prl secretion. Male and female ob/ob animals (a mouse model of obesity and insulin resistance) were treated with Bromo and/or Prl. Bromo-treated ob/ob mice exhibited lower serum Prl concentration, improved glucose and insulin tolerance, and increased insulin sensitivity in the liver and skeletal muscle compared with vehicle-treated mice. Prl replacement in Bromo-treated mice normalized serum Prl concentration without inducing hyperprolactinemia. Importantly, Prl replacement partially reversed the improvements in glucose homeostasis caused by Bromo treatment. The effects of the Prl receptor antagonist G129R-hPrl on glucose homeostasis were also investigated. We found that central G129R-hPrl infusion increased insulin tolerance of male ob/ob mice. In summary, our findings indicate that part of Bromo effects on glucose homeostasis are associated with decrease in serum Prl levels. Because G129R-hPrl treatment also improved the insulin sensitivity of ob/ob mice, pharmacological compounds that inhibit Prl signaling may represent a promising therapeutic approach to control blood glucose levels in individuals with insulin resistance1601193204FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP12/24345-4; 15/10992-6; 12/15517-6; 13/21722-4; 13/25032-2; 14/11752-6; 17/25281-