Ghrelin attenuates heat-induced degenerative effects in the rat testis

This study was conducted to examine the efficacy of ghrelin in prevention of deleterious effects of heat stress in rat testicular tissue. Forty five adult male rats were scheduled for this study and were divided equally into three groups: heat-saline, heat-ghrelin and control-saline. The scrota of heated-designed rats were immersed once in water bath at 43 °C for 15 min. Immediately upon heating, 2 nmol of ghrelin were given subcutaneously to heat-ghrelin animals every other day up to day 60 and physiological saline to the other two groups using the same method. The animals were sacrificed at 10, 30 and 60 days after heat treatment and their testes were taken for later photomicrograph and immunohistochemical analysis. Testicular histopathology revealed a significant reduction in the means of seminiferous tubules and Sertoli cell nucleus diameters as well as germinal epithelium height on day 10 in both heated groups. Furthermore, other testicular components including miotic index, spermatogenesis rate, presence of spermatocytes and volume densities were dramatically decreased following heat exposure. Notably, ghrelin caused a partial recovery in all of the above-mentioned parameters and accelerated testicular regeneration process by day 30 compared to the heat-saline group (P<0.05). Because of testicular progressive recovery, these indices were similar among groups on day 60 (P>0.05). However, immunohistochemistry evaluation for in situ detection of Bcl-2 protein did not exhibit any germ cells-positive of this factor among groups at different experimental days. In conclusion, the results of the present study indicate for the first time the novel evidences of ghrelin ability in attenuation of heat-induced testicular damage and also that ghrelin therapy may be useful as a suppressor of degenerative effects following testicular hyperthermia.     

Endotoxemia in newborn rats attenuates acute pancreatitis at adult age.

Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day) during 5 consecutive days. Two months later these rats have been subjected to i.p. cearulein infusion (25 microg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1beta (IL-1 beta), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dismutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and alpha-amylase activities, as well as plasma concentrations of IL-1beta and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E. coli or S. typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to the increased concentration of antioxidative enzyme SO in the pancreatic tissue and to the modulation of cytokines production in these animals.     

Ghrelin stimulates myocyte development.

Ghrelin, a gastric peptide hormone, may regulate mesenchymal cell development. Here we show that ghrelin promotes myogenesis in vitro in the mouse myoblast cell line C2C12 cells. Cells expressing ghrelin demonstrated a significant increase in the differentiation of premyocytes into myocytes. The mean myogenic index in cells stably expressing ghrelin increased significantly relative to control cells (42 +/- 2% vs. 16 +/- 1%, p<0.05). Western blotting showed that expression of MHC protein was elevated in cells expressing ghrelin compared to control cells. MyoD expression increased after treatment of C2C12 cells with exogenous ghrelin (10(-7) M). The stimulatory effect of ghrelin on myogenesis was abolished by either replacement of the third amino acid serine with alanine or deletion of its first nine amino acids. This study demonstrated that ghrelin is a potent stimulator for myogenesis.     

Caerulein-induced acute pancreatitis in the rat. Pancreatic secretory response to cholecystokinin.

The response of pancreatic exocrine secretion to cholecystokinin (CCK), has been studied in experimental acute pancreatitis induced in rats by supramaximal doses of caerulein. Several doses of caerulein were used (4, 20 and 40 micrograms/Kg) and each one was administered by four subcutaneous injections over 3 h at hourly intervals. Pancreatic juice was collected 9 h after the first injection. The caerulein-treated animals showed a statistically significant increase in serum amylase levels. Secretory activity of ductular cells remained unchanged in all the caerulein-treated animals, but total protein and amylase secretion decreased significantly at all the caerulein doses used, both in resting conditions and under stimulation with CCK (1.25 micrograms/Kg/h). Despite this the acinar cells of rats treated with the lowest dose of caerulein retained a certain degree of secretory function since amylase activity in pancreatic juice was greater than in other groups of rats treated with higher doses of caerulein. Moreover, the percentage of increase observed in total protein and amylase in response to CCK respect to basal secretion is similar to that of the untreated animals. At higher doses (20 and 40 micrograms/Kg) the secretory capacity in response to CCK was inhibited. Therefore CCK administration in slight acute pancreatitis could be used as a therapy since it favours the secretion of pancreatic enzymes at percentual levels similar to those of the controls.     

Dysregulation of the calpain-calpastatin system plays a role in the development of cerulein-induced acute pancreatitis in the rat

Calpain, a calcium-dependent cytosolic cysteine protease, is implicated in a multitude of cellular functions but also plays a role in cell death. Recently, we have shown that two ubiquitous isoforms, termed micro-calpain and m-calpain, are expressed in rat pancreatic acinar cells and that calcium ionophore-induced calpain activation leads to acinar cell injury. On the basis of these observations, we have now investigated the role of both calpain forms and the endogenous calpain inhibitor calpastatin in acute pancreatitis. After treatment of rats either without or with calpain inhibitor Z-Val-Phe methyl ester (ZVP; 60 mg/kg i.p.), pancreatitis was induced by cerulein injections (10 microg/kg i.p.; 5 times at hourly intervals). Calpain activation and calpastatin expression in the pancreatic tissue were studied by Western blot analysis. Pancreatic injury was assessed by plasma amylase activity, pancreatic wet/dry weight ratio (edema), histological and electron-microscopic analyses, as well as fluorescence labeling of actin filaments. Cerulein caused an activation of both micro-calpain and m-calpain, accompanied by degradation of calpastatin. Prophylactic administration of ZVP reduced the cerulein-induced calpain activation but had no effect on calpastatin alterations. In correlation to the diminished calpain activity, the severity of pancreatitis decreased as indicated by a decline in amylase activity (P < 0.01), pancreatic edema formation (P < 0.05), histological score for eight parameters (P < 0.01), and actin filament alterations. Our findings support the hypothesis that dysregulation of the calpain-calpastatin system may play a role in the onset of acute pancreatitis.     

Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat.

The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism.     

Ghrelin in growth and development

Exogenous administration of ghrelin increases caloric intake and stimulates growth hormone (GH) secretion, two effects that are mediated through binding of ghrelin to the GH secretagogue receptor (GHS-R). In addition, ghrelin is thought to inhibit adipogenesis by GHS-R-independent mechanisms. In adults, ghrelin is mainly produced by the stomach. In contrast, in the fetal and early postnatal period, ghrelin gene expression is abundant in the pancreas but not in the stomach. While knockout animal studies demonstrate that ghrelin is not required for perinatal development under normal nutritional conditions, the characteristics of ghrelin metabolism during fetal development suggest that ghrelin could contribute to the programming of mechanisms involved in energy balance, such as beta-cell maturation, orexigenic pathways and adipogenesis. In humans, ghrelin concentrations progressively decrease during childhood and adolescence, as well as with advancing puberty. In adolescents, similar to adults, ghrelin concentrations are inversely related to body mass index and to circulating insulin. One notable exception is the presence of elevated ghrelin concentrations in subjects with Prader-Willi syndrome, raising the possibility that ghrelin could be part of the etiology of excess food intake in this condition. These data raise a number of fascinating questions on the potential physiologic role of this hormone during growth and development.     

Correlation between the glucose level and the development of acute pancreatitis

Objective

To investigate the correlation between the level of glucose in serum and the development of acute pancreatitis (AP).

Vitamin E attenuates biochemical and morphological features associated with development of chronic pancreatitis

The objective was to investigate the effects of vitamin E on collagen deposition induced by Cyclosporin A (CsA) administration in rats with caerulein (Cr) pancreatitis. CsA transforms the fully regenerative, self-limited form of Cr pancreatitis into a chroniclike disease in conjunction with increased transforming growth factor (TGF)-beta and myofibroblast proliferation. Vitamin E inhibits TGF-beta release in mesangial cells and reduces CsA cytotoxicity. Wistar rats received CsA daily (20 mg/kg), and CR pancreatitis was induced on days 1 and 8 (Cr + CsA group). In a separate group, vitamin E (600 mg.kg(-1).day(-1)) was administered starting 4 days before CsA. Three other groups received either vehicle, CsA, or Cr alone. Thiobarbituric acid-reactive substance (TBARS), 8-isoprostanes, and hyaluronic acid were measured in plasma obtained on the day the animals were killed (day 15). Pancreases were weighed and processed for light microscopy to assess connective tissue and myofibroblast number. Pancreatic homogenates were also assayed for collagen (hydroxyproline) and TBARS content. TBARS, 8-isoprostane, and TGF-beta were elevated in CsA and Cr + CsA rats. Vitamin E treatment greatly decreased these parameters. Vitamin E also decreased the fall in pancreatic weight observed in Cr + CsA pancreas. Pancreatic hydroxyproline and plasma hyaluronic acid were increased in Cr + CsA rats but were effectively reduced by vitamin E. Morphology showed improvement in fibrosis score and a decreased number of myofibroblasts in vitamin E-treated rats. Vitamin E reduces oxidative stress and collagen deposition during the development of experimental chronic pancreatitis. Adjuvant antioxidants may be of value in the treatment of chronic pancreatitis.     

Patterns of incidence in acute pancreatitis.

A review of acute pancreatitis occurring over a 20-year period in the Bristol clinical area is reported. A total of 590 cases were available for analysis. The yearly incidence was 53-8 per million population at risk, with a mortality of 9-0 per million. This compares favourably with 11-4 deaths per million for England and Wales as a whole during the same period but the difference is not statistically significant. When the deaths occurring in the Bristol clinical area were expressed in terms of case mortality rate the figure was 17%. In contrast the mortality for recurrent acute pancreatitis was only 1-5%, and the benign nature of this second condition is confirmed. Aetiological factors and age and sex distribution were also analysed in relation to each other and to mortality. An increase in acute pancreatitis secondary to chronic alcoholism was confirmed and steroid pancreatitis also emerged as a definite entity in this survey. The pattern of recurrence in patients with idiopathic pancreatitis was studied in detail and is analysed on an actuarial basis.     

Pathobiology of experimental acute pancreatitis.

Pancreatic duct obstruction, even in the absence of biliary obstruction and/or bile reflux into the pancreatic duct, can trigger acute hemorrhagic necrotizing pancreatitis. The earliest changes are seen within acinar cells. Early derangements in acinar cell biology include inhibition of digestive enzyme secretion and the co-localization of lysosomal hydrolases with digestive enzyme zymogens. Under appropriate conditions, this co-localization could lead to digestive enzyme activation within acinar cells.     

Ghrelin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the ERK pathway

Vascular calcification results from osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and is a major risk factor for cardiovascular events. Ghrelin is a newly discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagog receptor (GHSR). Several studies have identified the protective effects of ghrelin on the cardiovascular system, however research on the effects and mechanisms of ghrelin on vascular calcification is still quite rare. In this study, we determined the effect of ghrelin on osteoblastic differentiation of VSMCs and investigated the mechanism involved using the two universally accepted calcifying models of calcifying vascular smooth muscle cells (CVSMCs) and beta-glycerophosphate (beta-GP)-induced VSMCs. Our data demonstrated that ghrelin inhibits osteoblastic differentiation and mineralization of VSMCs due to decreased alkaline phosphatase (ALP) activity, Runx2 expression, bone morphogenetic protein-2 (BMP-2) expression and calcium content. Further study demonstrated that ghrelin exerted this suppression effect via an extracellular signal-related kinase (ERK)-dependent pathway and that the suppression effect of ghrelin was time dependent and dose dependent. Furthermore, inhibition of the growth hormone secretagog receptor (GHSR), the ghrelin receptor, by siRNA significantly reversed the activation of ERK by ghrelin. In conclusion, our study suggests that ghrelin may inhibit osteoblastic differentiation of VSMCs through the GHSR/ERK pathway.     

Effect of secretin on pancreatic juice proteins in caerulein-induced acute pancreatitis in the rat

Nine hours after the start of treatment with caerulein in rats, an increase in the weight of the pancreas and an increase in serum amylase levels were observed. Likewise, a significant increase in endogenous secretin occurred in rats with acute pancreatitis. A dramatic reduction in the secretion of total protein and amylase was also observed. A partial recovery of this latter effect was achieved after an infusion of high doses of secretin. Under our experimental conditions, the volume of secretion did not vary in caerulein-treated rats wtih respect to controls, either in resting conditions or under secretin stimulation, which indicates that the ductular cells were not significantly affected. Isoelectrofocusing (IEF) and crossed-immunoelectrophoresis (CIE) studies revealed important alterations in the proteins of the pancreatic juice of rats with caerulein-induced acute pancreatitis. Trypsinogen appeared to be particularly affected, showing an increase in the T2 acidic form with an IEP of 4.4 and a decrease in the basic form T3 with an IEP of 8.0, which splits in other forms with a clear antigenic community. A hydrolase was also observed with an IEP of 6.2. In this sense, secretin administration may also be said to induce a significant improvement in established acute pancreatitis, since it tended to normalize the structure and proportion of the proteins secreted.