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To assess the effect of melatonin on bone metabolism in ovariectomized rats, receiving oestradiol therapy or not, melatonin was administered in the drinking water (25 microg/mL water) and oestradiol (10 microg/kg body weight) or vehicle was given subcutaneously 5 days/week for up to 60 days after surgery. Urinary deoxypyridinoline (a marker of bone resorption) and circulating levels of bone alkaline phosphatase activity (a marker of bone formation), as well as serum calcium and phosphorus levels, were measured every 15 days. Bone area (BA), bone mineral content (BMC), bone mineral density (BMD) and total body fat (expressed as 100 g body weight) were measured by dual-energy X-ray absorptiometry at the end of the experiment. Body weight and total body fat were augmented after ovariectomy, and decreased after melatonin or oestradiol treatment. The effect of melatonin on body weight was seen in sham-operated rats only. Ovariectomy augmented, and melatonin or oestradiol lowered, urinary deoxypyridinoline excretion. This effect of melatonin and oestradiol was seen mainly in ovariectomized rats. The efficacy of oestradiol to counteract ovariectomy-induced bone resorption was increased by melatonin. Melatonin or oestradiol lowered serum bone alkaline phosphatase activity. Melatonin inhibition was seen mainly on the increase of bone alkaline phosphatase activity that followed ovariectomy. Serum phosphorus levels decreased after melatonin administration and were augmented after oestradiol injection; overall, melatonin impaired the increase of serum phosphorus caused by oestradiol. Ovariectomy decreased, and oestradiol increased, serum calcium levels while melatonin augmented serum calcium in sham-operated rats only. On day 60 after surgery, BMD and content decreased after ovariectomy and were increased after oestradiol injection. Melatonin augmented BA of spine and BMC of whole of the skeleton and tibia. The highest values observed were those of rats treated concurrently with oestradiol and melatonin. The present results indicate that: (i) melatonin treatment restrained bone remodelling after ovariectomy; (ii) the effect of melatonin required adequate concentrations of oestradiol; (iii) melatonin augmented oestradiol effects on bone in ovariectomized rats; (iv) a counter-regulation by melatonin of the increase in body fat caused by ovariectomy was uncovered. The melatonin doses employed were pharmacological in terms of circulating melatonin levels but not necessarily for some other fluids or tissues.

The present study was undertaken to examine whether the intracerebroventricular (i.c.v.) administration of interferon (IFN)-gamma could modify 24-hour wheel running locomotor activity in the golden hamster. Hamsters implanted with a cannula in the third ventricle received a single i.c.v. injection of 1 microl of murine recombinant IFN-gamma (40 IU/microl) or its vehicle (saline) at ZT 6 or ZT 18 (with ZT 12 defined arbitrarily as the time of lights off) and their activities were monitored during 24 h. The i.c.v. administration of IFN-gamma at ZT 6 produced a significant phase advance in acrophase of rhythm, an effect not seen at ZT 18. Also, IFN-gamma depressed mesor value significantly, the effect was seen at both times. These results clearly showed that the circadian clock could be modified by IFN-gamma microinjections. One explanation could be the presence of IFN-gamma receptor in the rat suprachiasmatic nucleus, supporting a direct effect on the central oscillator. However, another hypothesis could not be ruled out.

The Vacuole Membrane Protein 1 -VMP1- is a pancreatitis-associated transmembrane protein whose expression triggers autophagy in several human diseases. In the current study, we unveil the mechanism through which this protein induces autophagosome formation in mammalian cells. We show that VMP1 autophagy-related function requires its 20-aminoacid C-terminus hydrophilic domain (VMP1-AtgD). This is achieved through its direct binding to the BH3 motif of Beclin 1 leading to the formation of a complex with the Class III phosphatidylinositol-3 kinase (PI3K) hVps34, a key positive regulator of autophagy, at the site where autophagosomes are generated. This interaction also concomitantly promotes the dissociation of Bcl-2, an autophagy inhibitor, from Beclin 1. Moreover, we show that the VMP1-Beclin 1-hVps34 complex favors the association of Atg16L1 and LC3 with the autophagosomal membranes. Collectively, these findings reveal that VMP1 expression recruits and activates the Class III PI3K complex at the site of autophagosome formation during mammalian autophagy.

Autophagy has recently elicited significant attention as a mechanism that either protects or promotes cell death, although different autophagy pathways, and the cellular context in which they occur, remain to be elucidated. We report a thorough cellular and biochemical characterization of a novel selective autophagy that works as a protective cell response. This new selective autophagy is activated in pancreatic acinar cells during pancreatitis-induced vesicular transport alteration to sequester and degrade potentially deleterious activated zymogen granules. We have coined the term “zymophagy” to refer to this process. The autophagy-related protein VMP1, the ubiquitin-protease USP9x, and the ubiquitin-binding protein p62 mediate zymophagy. Moreover, VMP1 interacts with USP9x, indicating that there is a close cooperation between the autophagy pathway and the ubiquitin recognition machinery required for selective autophagosome formation. Zymophagy is activated by experimental pancreatitis in genetically engineered mice and cultured pancreatic acinar cells and by acute pancreatitis in humans. Furthermore, zymophagy has pathophysiological relevance by controlling pancreatitis-induced intracellular zymogen activation and helping to prevent cell death. Together, these data reveal a novel selective form of autophagy mediated by the VMP1-USP9x-p62 pathway, as a cellular protective response. Fil: Grasso, Daniel Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ropolo, Alejandro Javier. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lo Ré, Andrea Emilia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Boggio, Verónica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina Fil: Molejon, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Iovanna, Juan L.. Inserm; Francia Fil: Gonzalez, Claudio D.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina Fil: Urrutia, Raúl. Mayo Clinic; Estados Unidos Fil: Vaccaro, Maria Ines. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Fisiopatología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Young male golden hamsters, made hyperprolactinemic by a pituitary graft under the kidney capsule, were exposed to a light pulse (1,000 lx/30 min) at Zeitgeber time (ZT) 18. Controls included hamsters receiving a sham graft (muscle). Fos immunoreactive cells were counted in both suprachiasmatic nuclei (SCN) of each animal, using an image analyzer system. The Fos immunoreactivity (Fos-ir) of the ventrolateral and dorsomedial SCN regions was greater in the pituitary-grafted hamsters. Indeed, light induced the greatest response in grafted animals in both SCN regions. However, the SCN of pituitary-grafted hamsters in the absence of light showed the lowest Fos-ir in both regions. The results support the occurrence of a dual effect of hyperprolactinemia on Fos-ir in the SCN of hamsters at ZT 18, with inhibition of Fos expression in the absence of light and potentiation of early gene expression when animals were exposed to a light pulse.