Hedonic and incentive signals for body weight control.

Here we review the emerging neurobiological understanding of the role of the brain's reward system in the regulation of body weight in health and in disease. Common obesity is characterized by the over-consumption of palatable/rewarding foods, reflecting an imbalance in the relative importance of hedonic versus homeostatic signals. The popular 'incentive salience theory' of food reward recognises not only a hedonic/pleasure component ('liking') but also an incentive motivation component ('wanting' or 'reward-seeking'). Central to the neurobiology of the reward mechanism is the mesoaccumbal dopamine system that confers incentive motivation not only for natural rewards such as food but also by artificial rewards (eg. addictive drugs). Indeed, this mesoaccumbal dopamine system receives and integrates information about the incentive (rewarding) value of foods with information about metabolic status. Problematic over-eating likely reflects a changing balance in the control exerted by hypothalamic versus reward circuits and/or it could reflect an allostatic shift in the hedonic set point for food reward. Certainly, for obesity to prevail, metabolic satiety signals such as leptin and insulin fail to regain control of appetitive brain networks, including those involved in food reward. On the other hand, metabolic control could reflect increased signalling by the stomach-derived orexigenic hormone, ghrelin. We have shown that ghrelin activates the mesoaccumbal dopamine system and that central ghrelin signalling is required for reward from both chemical drugs (eg alcohol) and also from palatable food. Future therapies for problematic over-eating and obesity may include drugs that interfere with incentive motivation, such as ghrelin antagonists.

Transcriptional effects of progesterone receptor antagonist in rat granulosa cells.

Progesterone, acting via the nuclear progesterone receptor (PGR), reduces apoptosis in periovulatory granulosa cells, and is a likely mediator of the anti-atretic actions of LH. The underlying mechanisms, however, have not been clearly defined. In this study, we sought to identify progesterone-mediated transcriptional changes involved in apoptosis regulation. Granulosa cells from immature, gonadotropin-primed female rats were treated in vitro with 100 nM of the PGR antagonist Org 31710. Transcriptional effects were analyzed after 5 and 22 h of incubation using microarrays, and the expression of 85 genes was subsequently measured by quantitative PCR. Follow-up experiments focused on genes related to the functional group "apoptosis". We have identified novel, early gene targets of PGR that may be involved in the control of apoptosis and other biologically significant functions in periovulatory granulosa cells. This study expands our knowledge of events that occur during the processes of ovulation and luteinization.

Dominant role of nuclear progesterone receptor in the control of rat periovulatory granulosa cell apoptosis.

In this study, it was hypothesized that progesterone (P4) acts as a survival factor primarily by actions of the classic nuclear progesterone receptor (PGR) signaling pathway in rat periovulatory granulosa cells. Granulosa cells were isolated from immature female rats primed with equine chorionic gonadotropin/human chorionic gonadotropin and treated in vitro with PGR antagonists. As little as 10 nM of two different PGR antagonists (Org 31710 and RU 486) increased apoptosis measured as caspase 3/7 activity, which was reversed by cotreatment with the progestin R5020. Concurrently, P4 synthesis was decreased. Inhibition of P4 synthesis by cyanoketone similarly induced apoptosis but required greater inhibition of P4 synthesis than that seen after treatment with PGR antagonists. Therefore, the induction of apoptosis by PGR antagonists cannot be explained by decreased P4 synthesis alone. Low concentrations of R5020 also completely reversed the effects of cyanoketone. Inhibition of P4 synthesis was more effective in inducing apoptosis than treatment with PGR antagonists. However, cotreatment with PGR antagonists protected cells from the additional effects of cyanoketone, indicating partial agonist effects of the antagonists and a dominating role for PGR in P4-mediated regulation of apoptosis. Progesterone receptor membrane component 1 (PGRMC1) was expressed in granulosa cells; however, an anti-PGRMC1 antibody did not induce apoptosis in periovulatory granulosa cells. Neither anti-PGRMC1 nor P4 or cyanoketone affected apoptosis of immature granulosa cells. In conclusion, we show that P4 regulates apoptosis in periovulatory granulosa cells by acting via the classic nuclear receptor.

Apoptotic effects of a progesterone receptor antagonist on rat granulosa cells are not mediated via reduced protein isoprenylation.

Progesterone is a survival factor in rat periovulatory granulosa cells. The mechanisms involved are unclear but progesterone receptor (PGR) antagonists have been shown to inhibit cholesterol synthesis and induce apoptosis. Furthermore, reports suggest that statins induce apoptosis by inhibition of protein isoprenylation. Statins inhibit the rate-limiting step of the cholesterol synthesis, thereby reducing availability of intermediates used for the post-translational isoprenylation process. It has been suggested that PGR antagonists in a similar manner induce apoptosis by decreasing cholesterol synthesis and thereby protein isoprenylation. In this study we hypothesized that the mechanism by which the nuclear PGR antagonist Org 31,710 induces apoptosis in rat periovulatory granulosa cells, is by decreasing cholesterol synthesis and thereby general cell protein isoprenylation. Incubation of isolated granulosa cells with Org 31,710 or simvastatin for 22 hr resulted in increased apoptosis and reduced cholesterol synthesis. However, simvastatin caused a substantial inhibition of cholesterol synthesis after 6 hr in culture without inducing apoptosis. In contrast, Org 31,710 had only a modest effect on cholesterol synthesis after 6 hr while it significantly induced apoptosis. Addition of isoprenylation substrates partially reversed apoptosis induced by simvastatin and to a lesser extent apoptosis induced by Org 31,710. In addition, and in contrast to Org 31,710, simvastatin caused a decrease in isoprenylation of a selected isoprenylation marker protein, the Ras-related protein RAB11. In conclusion, we demonstrate that the PGR antagonist inhibits cholesterol synthesis in granulosa cells but reduced protein isoprenylation is not the mediating mechanism of increased apoptosis as previously hypothesized.

Depletion of substrates for protein prenylation increases apoptosis in human periovulatory granulosa cells.

Progesterone receptor (PR) stimulation promotes survival in human and rat periovulatory granulosa cells. PR antagonists, Org 31710 and RU 486, both increase apoptosis and decrease cholesterol synthesis in these cells. The decrease in cholesterol synthesis also causes decreased synthesis of other products branching from the cholesterol synthesis pathway, including substrates for protein prenylation. In this study we focus on the link between apoptosis and prenylation in human periovulatory granulosa cells. A decreased cholesterol synthesis and increased apoptosis was verified in experiments with human periovulatory granulosa cells treated with the PR antagonists Org 31710 or RU 486 by measuring caspase-3/7 activity and incorporation of 14C-acetate into cholesterol and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory human granulosa cells using HMG-CoA reductase inhibitors (lovastatin or simvastatin) increased apoptosis, measured as caspase-3/7 activity. The increase in apoptosis caused by simvastatin or Org 31710 was partially reversed by addition of the protein prenylation precursors farnesol or geranylgeraniol. In addition, the prenylation inhibitors FTI R115777 and GGTI 2147 increased apoptosis in these cells. In conclusion our data suggest that PR antagonists increase apoptosis and reduce cholesterol synthesis in periovulatory granulosa cells and that the resulting depletion of substrates for protein prenylation may contribute to the increased apoptosis sensitivity.

Progesterone-receptor antagonists and statins decrease de novo cholesterol synthesis and increase apoptosis in rat and human periovulatory granulosa cells in vitro.

Progesterone-receptor (PR) stimulation promotes survival in rat and human periovulatory granulosa cells. To investigate the mechanisms involved, periovulatory rat granulosa cells were incubated in vitro with or without the PR-antagonist Org 31710. Org 31710 caused the expected increase in apoptosis, and expression profiling using cDNA microarray analysis revealed regulation of several groups of genes with functional and/or metabolic connections. This regulation included decreased expression of genes involved in follicular rupture, increased stress responses, decreased angiogenesis, and decreased cholesterol synthesis. A decreased cholesterol synthesis was verified in experiments with both rat and human periovulatory granulosa cells treated with the PR-antagonists Org 31710 or RU 486 by measuring incorporation of [14C]acetate into cholesterol, cholesterol ester, and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory rat granulosa cells using 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (lovastatin, mevastatin, or simvastatin) increased apoptosis, measured as DNA fragmentation and caspase-3/7 activity. The increase in apoptosis caused by simvastatin was reversed by addition of the cholesterol synthesis-intermediary mevalonic acid. These results show that PR antagonists reduce cholesterol synthesis in periovulatory granulosa cells and that cholesterol synthesis is important for granulosa cell survival.

Expression of progesterone receptor (PR) A and B isoforms in mouse granulosa cells: stage-dependent PR-mediated regulation of apoptosis and cell proliferation.

The intracellular progesterone receptor (PR) in the mammalian ovary is a part of the physiological pathway that facilitates ovulation. Two PR isoforms (A and B) exist, with different molecular and biological functions. Previous studies have revealed that the cellular ratio of the PR isoforms is important for progesterone-responsive tissues and is under developmental control in different species. However, the relative expression of PR isoforms in the ovary is unknown. In this study we have demonstrated first that the expression of both PR isoforms in mouse granulosa cells was rapidly up-regulated by hCG treatment and dramatically down-regulated when the granulosa cells were undergoing luteinization. The relative level of protein expression of the A and B forms was 2:1 and the highest total PR protein expression was found after hCG stimulation. Second, we demonstrated that the expression of PR protein was specific to granulosa cells of periovulatory follicles and was absent in undifferentiated granulosa cells of growing follicles. It was not detected in other cell types (i.e., corpora lutea or any stage of follicles with features of apoptosis). Third, we demonstrated that treatment with the PR antagonist RU 486 in vivo resulted in down-regulation of both isoforms in parallel with increased activation of caspase-3, a decreased level of proliferating cell nuclear antigen, and a reduced rate of ovulation. Fourth, we demonstrated, in vitro, that the PR antagonists RU 486 and Org 31710 increased internucleosomal DNA fragmentation parallel with a decrease in DNA synthesis in granulosa cells, which express PR. These results indicate that PR and its isoforms participate in regulation of ovulation, along with suppression of granulosa cell apoptosis and promotion of cell survival in the mouse ovary.