Immunohistochemical study on the expression of biologically active substances in the endocrine pancreas of the European bison.

Previous morphological studies of mammalian pancreatic islets have been performed mainly in domestic and laboratory animals. Therefore, the present immunohistochemical investigation was conducted in a wild species, the European bison, using antibodies against glucagon-like peptide-1 (GLP1), glucagon, insulin and somatostatin. Morphological analyses revealed that the mean area of the endocrine pancreas constituted 2.1±0.1% of the whole area of the pancreas, while the mean area of a single pancreatic islet was 13301.5±686.5 µm2. Glucagon-immunoreac- tive cells accounted for 22.4±1.1% and occupied 19.4±0.4% of the average islet area. As many as 14.3±1.4% of pancreatic islet cells were shown to express GLP1, which constituted 12.6±0.8% of the mean area of the islet. Insulin expression was confirmed in 67.6±0.7% of pancreatic islet cells, which represented 62.3±4.9% of the mean total area of the pancreatic islet. As many as 8.5±1.3% of cells stained for somatostatin. The somatostatin-immunoreactive cell area was 4.9±0.3% of the mean pancreatic islet area. In summary, we have determined in detail for the first time the morphometry and islet composition of the European bison pancreas. The distri- bution patterns of immunoreactivities to the substances studied in the European bison show many similarities to those described in other ruminant species.

Changes in growth hormone secretion and leptin receptor mRNA expression under the influence of leptin and adrenocorticotropin in pituitary cells of early weaned ewe lambs.

Early weaning of ewe lambs strongly stimulates the hypothalamic-pituitary-adrenal axis and is associated with suppressed growth rate despite the increased food intake. At the same time, plasma leptin concentration increases only slightly or undetectably. To better understand this atypical interdependence among somatic stress, leptin, and lamb growth rate, we analyzed impact of leptin and/or adrenocorticotropic hormone (ACTH) on growth hormone (GH) secretion as well as the effect of ACTH on mRNA expression of two splice variants of leptin receptor (LEPRa, LEPRb) in pituitary cells isolated from early weaned ewe lambs. The GH secretion under the influence of leptin and/or ACTH depended on the timing of exposure and hormone concentration. After 6 - 30 h, GH secretion increased under 10-11 - 10-8 M leptin (P ≤ 0.05). However, after 24 - 30 h, GH secretion significantly increased only in cells exposed to both leptin and ACTH compared to culture with leptin only. Simultaneously, there was a significant (P ≤ 0.05) decrease in leptin receptor mRNA expression under the influence of ACTH at 10-8 - 10-6 M after 12 - 30 and 24 - 30 h for LEPRa and LEPRb, respectively. ACTH-related downregulation of LEPR mRNA was associated with a significant (P ≤ 0.05) reduction in leptin-stimulated GH secretion, also after 24 - 30 hours. Thus, the timing of ACTH exposure, followed by decreased leptin receptor mRNA, converged with the timing of decreased GH secretion under the influence of leptin with ACTH. The ACTH-induced downregulation of LEPR mRNA therefore may underlie the decrease in GH. These results show a direct role for leptin, ACTH, and leptin receptor expression in modulation of pituitary GH secretion in early weaned ewe lambs. During the early weaning-induced stress response, the ACTH-mediated decrease in sensitivity of pituitary cells to leptin may abolish a stimulatory effect of leptin on GH secretion and explain in part, the reduction in lamb growth rate.

Adropin, nesfatin-1 and angiotensin II receptor expression in the abdominal aorta in ovariectomized rats after nesfatin-1 treatment.

The aim of the research was to assess the effect of nesfatin-1 on the structure, flexibility parameters, and expression of adropin, nesfatin-1, and angiotensin II receptor type 1 (AT1R) in the abdominal aorta in ovariectomized rats. Fragments of aortas were collected after euthanasia of female sham-operated (CONT) and ovariectomized Wistar rats (EXP), which were administered intraperitoneal injection of physiological saline (CONT, n = 7; EXP-O, n = 7) or nesfatin-1 (EXP-N, n = 7) in an amount of 2 µg/kg b.w. once a day for 8 weeks. The samples of aortas were collected for measurement of elasticity as well as histomorphometric, immunohistochemical, FTIR, and Raman spectroscopy analysis. The ovariectomy caused a significant increase in the thickness of the total wall and its particular layers in the aorta, in comparison to the CONT and EXP-N groups. However, the ovariectomy led to a decrease in the amount of elastin, collagen (mature, immature collagen, collagen maturity ratio 1660 - 1690 cm-1), and amides, with a simultaneous increase in lipids, especially in the tunica intima-media of the abdominal aorta compared to the other groups. The use of nesfatin-1 significantly increased the amount of collagen, elastin and amides with a simultaneous decrease in the amount of lipids and the expression of AT1R, adropin and nesfatin-1 in the abdominal aorta of ovariectomized rats. In conclusion, our study showed that the ovariectomy surgery induced changes in the abdominal aorta wall characteristic for aging females. Application of nesfatin-1 may prevent the negative consequences in the vessel wall structure in females in conditions of estrogen deficiency and prevent atherosclerotic changes in the cardiovascular system.

Supplementation with camelina oil prevents negative changes in the artery in orchidectomized rats.

The aim of the research was to examine the influence of orchidectomy on the elasticity and wall structure of the abdominal aorta in male rats and to check whether camelina oil treatment has an effect on aorta wall characteristics in orchidectomized rats. Forty 2-month-old male Wistar rats were used in the experiment: 10 animals underwent a sham testis repositioning operation (SHO) and 30 rats were orchidectomized (ORX). After the convalescence period, the SHO and ORX1 rats were given physiological saline intragastrically for 8 weeks; simultaneously, the other rats received camelina oil at the dose of 5 g/kg/b.w. (ORX2) or 9 g/kg/b.w. (ORX3) once a day. At the end of experiment, the animals were euthanized and fragments of the aorta were sampled for elasticity measurement and for histomorphometric and immunohistochemistry analysis. Orchidectomy caused a significant increase in the thickness of the total wall and its particular layers, mean intensity of elastin fluorescence in the tunica intima-media, and the volume of collagen I in tunica adventitia of the abdominal aorta in comparison to the other groups. The mean intensity of collagen I fluorescence in the tunica adventitia and tunica intima-media was significantly lower in the aorta of the orchidectomized rats. The values of the histomorphometric parameters of animals receiving camelina oil were lower than in the ORX1 group and higher than in the SHO rats. The values of the other parameters analyzed after the camelina oil treatment were similar to those in the SHO rats. In conclusion, our study showed that orchidectomy induced changes in the abdominal aorta wall characteristic for aging. Supplementation with camelina oil prevents negative consequences in the vessel wall structure in males with impaired endocrine function.

Acrylamide-induced prenatal programming of intestine structure in guinea pig.

Potential effects of prenatal administration of acrylamide (ACR) on postnatal development of the small intestine were not examined experimentally yet. The aim of this study was to establish changes of morphological parameters of the small intestine damaged by prenatal action of ACR in guinea pigs. The 3 mg/kg body weight of ACR was given in drinking water every day during the last 35 days of the pregnancy in guinea pigs. The histomorphometry of the duodenum and jejunum was determined. Immunohistochemical staining with anti cadherin antibody was performed. Maternal treatment with ACR led to the decrease of the expression of cadherin in the epithelium. Maternal ACR treatment increased the number of total, divided and inactive crypt, and the number of damaged villi in the duodenum and jejunum of newborn guinea pigs. The thickness of myenteron and submucosa, mucosa fractal dimension and the depth of crypts in the duodenum were increased by ACR. Additionally, in offspring born by mothers administered with ACR the decrease of villi epithelium thickness and active crypt number was observed. Moreover, ACR decreased goblet cells and inact villi number in the duodenum, mucosa thickness and crypts width in the jejunum. Intestine absorptive surface was affected by ACR in the jejunum as well. Results of measurements showed that maternal ACR treatment had negative influence on small intestine histomorphometry. ACR acting prenatally influenced small intestine nervous plexuses that became enlarged by 2.5 times compared with the control group. In conclusion, our results showed the negative impact of maternal ACR treatment on histological structure, integrity and innervation of small intestine wall as well as on absorptive function of small intestine mucosa.

Morphological changes of the cartilage and bone in newborn piglets evoked by experimentally induced glucocorticoid excess during pregnancy.

The study examined articular and growth plate cartilages as well as bone tissues in the offspring of sows treated with glucocorticoid during the last 45 days of pregnancy (dexamethasone at the dose of 0.03 mg/kg body weight intramuscularly, every second day). The offspring were tested at the birth and basal morphology for both articular and growth plate cartilages, and the histomorphometry of trabeculae of the epiphysis and metaphysis of femur and tibia were established. The concentration of selected cytokines and the activity of bone alkaline phosphatase were determined in blood serum. Maternal dexamethasone (DEX) administration reduced the thickness of proliferative, resting and hypertrophic zones of growth plate of femur and tibia of male piglets when compared with the control. DEX significantly reduced the thickness of the resting zone in both bones. It also elongated proliferative and hypertrophic zones of the growth plate in the femur as well as the hypertrophic zone in the tibia of female piglets when compared with the control group. Moreover, DEX decreased the articular cartilage thickness of the tibia in female piglets and enhanced the articular cartilage thickness of the femur in male piglets. Articular cartilage was highly cellular, and chondrocytes were separated by thin septa of matrix. An analysis of the trabecular bone architecture in male piglets showed a loss of the trabecular bone by thinning and DEX-related increase in trabecular porosity. Moreover, the cortical bone looked similar to the trabeculae because of trabecularization of the cortex. There was a DEX that reduced serum osteocalcin and BAP concentrations in both female and male newborn piglets, whereas the serum IL-1 and Il-6 was reduced only in male piglets. The obtained results demonstrated that DEX administration to sows during the last 45 days of pregnancy might cause the growth to slow and eventually to stop, especially in male piglets. It might lead to an alteration within the cartilage during its normal function, and with the time, arthritic changes can follow.

Role of feed-regulating peptides on pancreatic exocrine secretion.

In recent two decades a group of feed intake-regulating peptides (i.e., leptin, apelin, ghrelin, obestatin and orexins) have been discovered. Besides the central nervous system these regulatory peptides are produced and released by the gastrointestinal (GI) endocrine cells and neurons, and functional receptors were found in the GI tract and the pancreas. High expression of feed intake-regulating peptides was found in the stomach; however, they may be expressed in other GI tissues too. The peptides control gastrointestinal functions, modulate orexigenic drive and energy metabolism via different mechanisms. Basal leptin, apelin, ghrelin and obestatin plasma concentrations correlated with BMI, and we observed significant reduction of ghrelin and leptin concentrations following fundectomy in rats. We have shown previously that exogenous leptin and ghrelin (a peptide derived from the same preprohormone as obestatin) inhibit the secretion of rat pancreatic juice through a neurohormonal mechanism. Intravenous obestatin was found to stimulate pancreatic protein output in anaesthetized rat via a CCK-vagal-dependent mechanism, whilst a direct action of obestatin on rat pancreatic acini in vitro resulted in opposite effect. Intravenous boluses of apelin reduced the juice volume, protein and trypsin outputs in a dose-dependent manner. However, apelin administered into the duodenal lumen significantly increased pancreatic protein and trypsin outputs through a vagal mechanism. Orexin A and B were found to stimulate insulin release, though on the rat exocrine pancreas orexin A had no effect, and the effect of orexin B was weak. Concluding, feed intake-regulating peptides participate in controlling the exocrine pancreas.

Obestatin stimulates the secretion of pancreatic juice enzymes through a vagal pathway in anaesthetized rats - preliminary results.

Obestatin is a 23 amino acid peptide derived from the preproghrelin precursor, and originally purified from the rat stomach mucosa. It was shown that obestatin may counteract the effects of its sister peptide, ghrelin, on food intake and gastrointestinal motility but the other roles in controlling the gastrointestinal function remain unknown. The aim of the present study was to determine the influence of exogenous obestatin on the secretion of pancreatic juice. In anesthetized male Wistar rats the external jugular vein was catheterized, and the common biliary-pancreatic duct was cannulated with polyethylene tubing for collection of pancreatic-biliary juice (P-BJ). Obestatin boluses (30, 100 and 300 nmol/kg b. wt.) were injected intravenously or intraduodenally every 30 min. Obestatin was also administered in vagotomized (subdiaphragmatic vagotomy) rats. In the examined rats, obestatin intravenous and intraduodenal boluses did not affect the P-BJ volume. On the other hand, obestatin boluses increased the protein output and trypsin activity. Vagotomy abolished the effects of exogenous obestatin administration. In conclusion, the present study demonstrates for the first time that exogenous obestatin may stimulate the secretion of pancreatic juice enzymes. The effect is dose-dependent and requires intact vagal supply.

The ghrelin pentapeptide inhibits the secretion of pancreatic juice in rats.

Ghrelin, a 28 amino acids polypeptide was recognized as an endogenous ligand for the growth hormone secretagogue receptor. It turned out that the entire sequence of ghrelin is not necessary for performing the above-mentioned functions. It was suggested that 5 residues (Gly-Ser-Ser(n-octanoyl)-Phe, pentaghrelin) constituted functionally active part of the full-length polypeptide. Ghrelin-28 was found to inhibit pancreatic enzyme output in rats, though the effect of pentaghrelin was not studied so far. The study aimed to determine the involvement of pentaghrelin in pancreatic juice secretion in anaesthetized rats. Male Wistar rats (220 +/- 20 g body weight, b. wt.) were anesthetized, the external jugular vein and common biliary-pancreatic duct were cannulated. Pentaghrelin boluses (i.v., 1.2, 12, and 50 nmol kg(-1) b. wt.) were injected every 30 min with or without CCK-8 infusion, duodenal mucosal CCK(1) receptor blockade with tarazepide, vagotomy and capsaicin pretreatment. Pentaghrelin boluses reduced the volume of pancreatic-biliary juice, protein and trypsin outputs both under basal and CCK-8-stimulated conditions in a dose-dependent manner. However, exogenous pentaghrelin failed to affect the pancreatic secretion in rats subjected to vagotomy, capsaicin deactivation of afferents or pretreatment with Tarazepide. In conclusion, pentaghrelin may control exocrine pancreas secretion by affecting duodenal neurohormonal mechanism(s) involving CCK and vagal nerves in rats.

The effect of fundectomy on pancreatic secretion in anaesthetized rats.

The aim of present study was to evaluate the influence of surgical fundectomy and exogenous leptin on the secretion of pancreatic juice in anesthetized rats. In male Wistar rats major part of the gastric fundus was surgically removed, and 60 days afterwards the external jugular vein and the pancreatic-biliary duct were catheterized under general anesthesia. The pancreatic-biliary juice (PBJ) was collected in 15 min intervals without introducing it into the duodenum. Intravenous leptin infusions (0.1, 1.0 and 10 microg/kg body weight) were made every 30 min. The same protocol was used in control non operated rats. The PBJ volume was significantly lower in fundectomized rats as compared to control rats and showed no significant effect to exogenous leptin. The PBJ protein output but not trypsin activity was lower in fundectomized rats as compared to control. Leptin reduced the PBJ protein and trypsin outputs in a dose-related manner in the control and experimental group. The inhibition was, however, more evident in the fundectomized rats. Plasma gastrin was higher in fundectomized rats, while plasma leptin and ghrelin were lower. In conclusion, fundectomy seems to reduce the non stimulated pancreatic secretion and modifies the response to leptin in anesthetized rats.