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Eva Baquedano, Julie A Chowen, Jesús Argente and Laura M Frago

GH and GH secretagogues (GHSs) are involved in many cellular activities such as stimulation of mitosis, proliferation and differentiation. As astrocytes are involved in developmental and protective functions, our aim was to analyse the effects of GH and GH-releasing hexapeptide on astrocyte proliferation and differentiation in the hypothalamus and hippocampus. Treatment of adult male Wistar rats with GH (i.v., 100 μg/day) for 1 week increased the levels of glial fibrillary acidic protein (GFAP) and decreased the levels of vimentin in the hypothalamus and hippocampus. These changes were not accompanied by increased proliferation. By contrast, GH-releasing hexapeptide (i.v., 150 μg/day) did not affect GFAP levels but increased proliferation in the areas studied. To further study the intracellular mechanisms involved in these effects, we treated C6 astrocytoma cells with GH or GH-releasing hexapeptide and the phosphatidylinositol 3′-kinase (PI3K) inhibitor, LY294002, and observed that the presence of this inhibitor reverted the increase in GFAP levels induced by GH and the proliferation induced by GH-releasing hexapeptide. We conclude that although GH-releasing hexapeptide is a GHS, it may exert GH-independent effects centrally on astrocytes when administered i.v., although the effects of both substances appear to be mediated by the PI3K/Akt pathway.

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Ana I Arroba, Alfonso M Lechuga-Sancho, Laura M Frago, Jesús Argente and Julie A Chowen

Poorly controlled diabetes mellitus can result in decreased prolactin production and thus problems with lactation, reproduction, and other physiological processes. This may be due to a loss of lactotrophs, as we have previously shown that long-term (8 weeks) poorly controlled streptozotocin-induced diabetes results in increased death of lactotrophs and that this most likely occurs through the activation of caspase-8 and the extrinsic cell death cascade. However, cell proliferation is also increased in the anterior pituitary at this time, although the cell type undergoing this proliferation and whether it is a response to the increased cell death remains unknown. In order to determine the time-course of increased cell death and proliferation in the anterior pituitary and if this is related to changes in tumor necrosis factor (TNF)-α, a cytokine involved in the activation of the extrinsic cell death pathway, rats were killed at 1, 4, 6, and 8 weeks after the induction of diabetes. Cell death was significantly increased after 4 weeks, as was caspase-8 activation, although circulating levels of TNF-α were increased as early as 1 week. Pituitary levels of TNF-α did not change significantly until 8 weeks after diabetes onset. Similarly, Western-blot analysis of proliferating cell nuclear antigen showed that anterior pituitary cell proliferation increased significantly 8 weeks after diabetes onset, with the majority of proliferating cells, as detected by BrdU incorporation, corresponding to lactotrophs. These results suggest that the increased death of lactotrophs in poorly controlled diabetic rats is followed by increased proliferation of this cell type, even when no treatment is given.

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Ana I Arroba, Alfonso M Lechuga-Sancho, Laura M Frago, Jesús Argente and Julie A Chowen

Cell death is increased in the anterior pituitary of poorly controlled diabetic rats, but anti-apoptotic mechanisms are also activated. We hypothesized that specific cell types are selectively protected against diabetes-induced cell death. To determine when anti-apoptotic mechanisms are activated, streptozotocin-induced diabetic rats were killed after 1, 4, 6 and 8 weeks of evolution. Anterior pituitaries were processed for western blot analysis to determine changes in the intrinsic cell death pathway and upstream kinases involved in cell protection mechanisms. An increase in cell death was detected by ELISA at 4 weeks of diabetes. TUNEL labelling demonstrated that this corresponded to death of primarily lactotrophs, a few somatotrophs, and no thyrotrophs, corticotrophs or gonadotrophs. Levels of phosphorylated (p) Akt were increased at 1 week of diabetes, while pERK1/2 levels increased at 4 weeks and pJNK at 6 weeks. Activation of caspase 3 decreased and anti-apoptotic members of the Bcl-2 protein family increased as early as 1 week after diabetes onset. These changes were coincident with increased IGF-I receptor levels. Levels of X-linked inhibitor of apoptosis protein (XIAP) increased significantly after 6 weeks of diabetes, as did activation of nuclear factor (NF)κB. Double immunohistochemistry indicated that XIAP was expressed in less than 1% of lactotrophs and gonadotrophs, approximately 50% of somatotrophs and more than 90% of corticotrophs and thyrotrophs. These results suggest that some cell survival mechanisms are rapidly activated in the anterior pituitary, even before increased cell death can be detected, while others are more delayed. Furthermore, both pituitary cell death and expression of protective mechanisms such as XIAP are cell-type specific.

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Adrián Plaza, Beatriz Merino, Victoria Cano, Gema Domínguez, Javier Pérez-Castells, M Soledad Fernández-Alfonso, Coralie Sengenès, Julie A Chowen and Mariano Ruiz-Gayo

The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) represses Angptl-4 expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducing Lpl expression. In vivo CCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulates Angptl-4 expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.