Verónica Boggio

Melatonin effects on bone: experimental facts and clinical perspectives

Abstract: Bone formation proceeds through a remodeling process that runs continuously, involving the resorption of old bone by osteoclasts, and the subsequent formation of new bone by osteoblasts. This is controlled by growth factors and cytokines produced in bone marrow microenvironment and by the action of systemic hormones, like parathyroid hormone, estradiol or growth hormone (GH). One candidate for hormonal modulation of osteoblast and osteoclast formation is melatonin. Because circulating melatonin declines with age, its possible involvement in post‐menopausal and senescence osteoporosis is considered. This review article discusses early studies on melatonin–bone relationships and recent data that suggest a direct effect of melatonin on bone. Melatonin could act as an autacoid in bone cells as it is present in high quantities in bone marrow, where precursors of bone cells are located. Melatonin dose‐dependently augmented proteins that are incorporated into the bone matrix, like procollagen type I c‐peptide. Osteoprotegerin, an osteoblastic protein that inhibits the differentiation of osteoclasts is also augmented by melatonin in vitro. Another possible target cell for melatonin is the osteoclast, which degrades bone partly by generating free radicals. Melatonin through its free radical scavenger and antioxidant properties may impair osteoclast activity and bone resorption. At least in one study melatonin was both inhibitory to osteoclastic and osteoblastic cells. Therefore, the documented bone‐protecting effect of melatonin in ovariectomized rats can depend in part on the free radical scavenging properties of melatonin. Additionally, melatonin may impair development of osteopenia associated with senescence by improving non‐rapid eye movement sleep and restoring GH secretion. Whether melatonin can be used as a novel mode of therapy for augmenting bone mass in diseases deserves to be studied.

Zymophagy, a Novel Selective Autophagy Pathway Mediated by VMP1-USP9x-p62, Prevents Pancreatic Cell Death

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.

The VMP1-Beclin 1 interaction regulates autophagy induction

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.

Melatonin increases oestradiol-induced bone formation in ovariectomized rats

Abstract: 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 μg/mL water) and oestradiol (10 μg/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.

Effect of melatonin on bone metabolism in ovariectomized rats

To assess the effect of pharmacological dose of melatonin on bone metabolism in ovariectomized rats, urinary deoxypyridinoline (a marker of bone resorption) and calcium excretion, circulating levels of calcium, phosphorus and bone alkaline phosphatase activity (a marker of bone formation), and bone mineral density (BMD), mineral content (BMC) and bone area (BA) of total body, were measured in adult rats for up to 60 days after surgery. Rats received melatonin in the drinking water (25 microg/ml water) or drinking water alone. Urinary deoxypyridinoline increased significantly after ovariectomy by 51% (30 days after surgery) and by 47% (60 days after surgery). The increase in urinary deoxypyridinoline found 30 days after ovariectomy was not observed in melatonin-treated rats. Urinary calcium concentration was similar in the 4 experimental groups studied, as was the circulating calcium concentration at every time interval examined. Fifteen days after surgery, a significant increase in serum phosphorus and bone alkaline phosphatase levels occurred in ovariectomized rats receiving melatonin as compared to their controls. Sixty days after surgery BMD, BMC and BA decreased significantly in ovariectomized rats, an effect not modified by melatonin. Serum estradiol decreased significantly by 30 days after ovariectomy to attain values close to the limit of detection of the assay by 60 days after ovariectomy. The results support the conclusion that a pharmacological amount of melatonin modifies bone remodeling after ovariectomy and that the effect may need adequate concentrations of estradiol.

Cerebroventricular Administration of Interferon-Gamma Modifies Locomotor Activity in the Golden Hamster

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 µl of murine recombinant IFN-gamma (40 IU/µl) 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.

Effect of unilateral superior cervical ganglionectomy on bone mineral content and density of rat's mandible.

To assess the effect of a local sympathectomy on bone metabolism, the effect of a unilateral superior cervical ganglionectomy (Gx) on growth and bone mineral content and density of the ipsi- and contralateral mandibles was examined in female rats. A significant increase in the hemi-mandibular bone ipsilateral to Gx was found as compared to the contralateral, sham-operated side 30 days, but not 15 days, after surgery. Bone mineral content of the hemi-mandibular bones was significantly lower in the side ipsilateral to Gx in the group of rats killed on the 30th day after surgery. Since no difference in areas between innervated and denervated hemi-mandibles was found, bone mineral density was also significantly lower in the hemi-mandible ipsilateral to Gx. The results further support that a regional sympathectomy causes qualitative alterations in bone modeling and remodeling, leading to bone resorption.

Chemotherapy and autophagy-mediated cell death in pancreatic cancer cells

Autophagy is an evolutionarily preserved degradation process of cytoplasmic cellular constituents and plays important physiological roles in human health and disease. It has been proposed that autophagy plays an important role both in tumor progression and in promotion of cancer cell death, although the molecular mechanisms responsible for this dual action of autophagy in cancer have not been elucidated. Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies with 2-3% five-year survival rate. Its poor prognosis has been attributed to the lack of specific symptoms and early detection tools, and its relatively refractory to traditional cytotoxic agents and radiotherapy. Experimental evidence pointed at autophagy as a pancreatic cancer cell mechanism to survive under adverse environmental conditions, or as a defective programmed cell death mechanism that favors pancreatic cancer cell resistance to treatment. Here, we consider several phenotypical alterations that have been related to increase or decrease the autophagic process in pancreatic tumor cells. We specially review autophagy as a cell death mechanism in response to chemotherapeutic drugs.

Effect of unilateral superior cervical ganglionectomy on mandibular incisor eruption rate in rats.

To assess the effect of sympathectomy on rat tooth eruption, the effect of a unilateral superior cervical ganglionectomy (SCGx) on eruption rate of ipsi- and contralateral lower incisors was examined. Two experiments were performed. In a first experiment, the eruption rate of ipsilaterally denervated incisors was similar to that of contralaterally innervated incisors, when assessed for up to 28 days after surgery. In a second experiment, under conditions of unilateral unimpeded eruption of incisors performed ipsilaterally or contralaterally to a unilateral SCGx, a significantly lower eruption rate of denervated incisors at the impeded eruption side, and a significantly higher eruption rate of denervated incisors at the unimpeded side were observed, when computed every 2 days. Significant differences in individual Students t tests at every time interval occurred mainly during the first and the last week of examination. When average daily eruption rate was computed in weekly intervals, a significant interaction between SCGx and the side of impeded or unimpeded eruption was found in a factorial ANOVA, that is, for each of the 4 weeks of examination, sympathetically denervated incisors showed lower eruption rates at the impeded eruption side, and higher eruption rates at the unimpeded side. These results indicate that incisor eruption is not modified by a local sympathetic denervation unless the contralateral lower rat incisor is cut out of occlusion.

Effect of Chronic Ethanol Feeding on 24-Hour Rhythms of Mitogenic Responses and Lymphocyte Subset Populations in Thymus and Spleen of Peripubertal Male Rats

This work analyzes the effect of chronic ethanol feeding on the 24-hour variation of mitogenic responses and lymphocyte subset populations in thymus and spleen. Animals were maintained under a 12:12-hour light/dark photoperiod and they received a liquid diet for 4 weeks, starting on day 35 of life. The ethanol-fed group received a similar diet to controls except that maltose was isocalorically replaced by ethanol. Ethanol replacement provided 36% of the total caloric content of the diet. Rats were killed at 6 time intervals around the clock, beginning at Zeitgeber time (ZT) 1 (ZT 0 = lights on). Under ethanol intake the splenic and thymic weight decreased. In addition, mean values of the thymic, but not of the splenic T cell number decreased, and mean values of the thymic and splenic CD8+ and CD4+CD8+ number increased. Consequently, the thymic T/B ratio and the thymic and splenic CD4+/CD8+ ratio decreased in ethanol-fed rats. At the same time there was a significant increase in the response of the thymic cells to LPS. The ethanol diet modified the 24-hour rhythmicity of thymic and splenic T, B and CD4+CD8+ cells, thymic CD4+ and splenic CD8+ cells, thymic and splenic T/B and CD4+/CD8+ ratios, as well as of mitogenic responses in both tissues. Chronic ethanol administration presumably affects the endogenous clock that modulates the circadian variation of immune responsiveness in growing rats.