Orexin B is a 28-amino acid neuropeptide with a molecular weight of 2.94 kd. The structure of orexin B is identical to that of hypocretin 2. It has no cysteine residues and consequently no intrachain disulfide bonds.1 Human orexin B shares 46% amino acid sequence homology with orexin A. Human orexin B consists of 2 α-helices2 from Leu7 to Gly19 and from Ala23 to Met28.2 Sakurai et al3 showed that orexins induce intracellular calcium transients in both receptor-transfected and native-receptor expressing cells. Orexin B increases corticosterone plasma level4 and food intake and appetite5 after subcutaneous and intramuscular injections of orexin B, respectively. In addition, orexin B can modulate the function of macrophages.6
Orexins were initially identified in the dorsal and lateral hypothalamic nuclei of rat brain.7 The lateral hypothalamic area is involved in feeding behavior. Orexins are also present in the prefornical and median eminence of the hypothalamus of the rat.8 These orexin-positive nerves branch to a variety of brain regions, which contains orexin receptors.9
In addition to the localization of orexins to several parts of the central nervous system, orexins have also been demonstrated in several peripheral organs such as the rat testis10 and rat and human kidneys.11 In the kidney, orexins are expressed mainly by tubular cells and released into urine. Takahashi et al11 showed that the kidney concentration of orexin A is 600 times lower than that of the brain. Other studies have shown that fish,12,13 rat,14 and human15 pituitary glands contain orexins. Orexins have also been identified in the pancreas of man16 and the rat lacrimal gland.17
Nakabayashi et al18 observed messenger RNA expression for prepro-orexin in several peripheral tissues including the kidney, the adrenal gland, the placenta, the stomach, the ileum, the colon, and colorectal epithelial cells of rat. The localization of orexin in the gastrointestinal system by molecular techniques was supported by other techniques including immunohistochemistry.19
Orexins have been shown to have effects on many body systems, including cardiovascular, hypothalamic-pituitary-adrenal, reproductive, gastrointestinal.20 Orexins increases the mean arterial pressure and heart rate.21 Stimulation of the hypothalamic-pituitary-adrenal system leads to proliferation of adrenal cells.22 Orexins, especially orexin A, can increase the plasma luteinizing hormone and prolactin levels in rats.23 Studies have shown that orexins stimulate both the peripheral and central nervous systems via the vagus24 to influence gastric secretion, gastrointestinal motility, and pancreatic function. In fact it has been shown that the cardiovascular effects caused by central administration of orexins act via cholinergic transmission.25
Most of the information we know about orexins is based mainly on studies performed with orexin A. The aim of this study was to examine the pattern of distribution of orexin B in the pancreas of normal and diabetic rats to determine whether the onset of diabetes has any effect on the pattern of distribution of orexin B. Moreover, the study examined the mechanism by which orexin B stimulates insulin and glucagon from the rat pancreas in normal and diabetic conditions.
MATERIALS AND METHODS
Animals and Induction of Diabetes Mellitus
Twelve-week-old male Wistar rats weighing approximately 250 g were used in this study. Rats were obtained from the Faculty of Medicine and Health Sciences (United Arab Emirates University, Al Ain, United Arab Emirates) breeding colony. The study was approved by the animal ethics research committee at the Faculty of Medicine and Health Sciences. The guidelines set by this committee for animal husbandry and welfare, based on the Helsinki Declaration of 2006, was followed.
The rats were divided into 2 groups: streptozotocin-induced diabetics and age-matched controls. Diabetes was induced by a single intraperitoneal injection of streptozotocin (Sigma, Poole, United Kingdom) at 60 mg/kg prepared in 5-mmol/L citrate buffer with pH of 4.50.26 The animals were kept in plastic cages and maintained on standard laboratory animal diet with food and water ad libitum. The One-Touch II glucometer (LifeScan; Johnson and Johnson, Milpitas, Calif) was used to measure the blood glucose level for each individual animal. The animals were considered diabetic if the random blood glucose levels were equal to or more than 300 mg/dL. After 4 weeks from the date of induction of diabetes, all of the animals from both groups were euthanized under chloral hydrate general anesthesia (7% chloral hydrate, 6-mL/kg of body weight; injected intraperitoneally). A midline abdominal incision was made, and the pancreas was rapidly removed and placed in ice-cold Krebs solution. The composition of the Krebs solution in millimoles per liter was as follows: KCl, 4.7; NaCl, 118; CaCl2, 2.5; NaHCO3, 23.3; KH2PO4, 1.2; MgSO4, 1.2; and glucose, 10. Representative fragments were taken from the tail end of the pancreas.
Immunohistochemistry and Immunofluorescence
Six rats from each group (control and diabetic) were used for this experiment. The isolated pancreas was trimmed free of adherent fat and connective tissue and cut into small pieces (2 μL) and fixed overnight in freshly prepared Zamboni fixative.27 The tissue samples were embedded in paraffin and sectioned and processed for immunohistochemistry according to a previously described method.28 Briefly, sections of 6-μm thickness were deparaffinized with xylene and incubated for 30 minutes in 0.3% hydrogen peroxide solution in methanol to block endogenous peroxidase activity. The sections were later incubated for 30 minutes in protein blocking reagent and overnight in goat anti-orexin B. The slides were then washed and incubated for 60 minutes with prediluted biotinylated anti-goat immunoglobulin G. After washing in Tris-buffered saline (TBS), the sections were incubated in streptavidin peroxidase conjugate for 90 minutes. After a final wash in TBS, sites of immunoreactivity were revealed by incubating the specimens for 3 minutes in 3,3-diaminobenzidine tetrahydrochloride containing 0.03% hydrogen peroxide in TBS. The slides were later washed, and counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted in Cytoseal 60 (Stephens Scientific, Riverdale, NJ). Immunopositive areas of the tissue sections were photographed using a Zeiss Axiophot (Zeiss, Göttingen, Germany) microscope. An immunofluorescence study was performed according to a previously described method.29
The antiserum to orexin B was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif) and was used at a dilution of 1:500. No specific immunostaining was observed in pancreatic tissue when primary antiserum was omitted.
Quantification of Insulin and Glucagon Release From the Pancreas of Normal and Diabetic Rats
The body and tail end of freshly removed pancreas was trimmed free of adherent fat and connective tissue and minced into small fragments (0.5-1 μL). The pancreatic tissue fragments were placed in 2-mL glass vials containing 1 mL of Krebs buffer (KB) and preincubated for 30 minutes in a water bath at 37°C, to wash away any hormone due to cutting of the tissues. Immediately, after the preincubation step, the KB was carefully drained, and the specimens were subsequently incubated for 1 hour with different concentrations (10−12 to 10−6 mol/L) of orexin B or with orexin B (10−9 mol/L) and atropine (10−6 mol/L), orexin B (10−9 mol/L) and yohimbine (10−6 mol/L), orexin B (10−9 mol/L), orexin B (10−9 mol/L) and diltiazem (10−6 mol/L), and orexin B (10−9 mol/L) and propranolol (10−6 mol/L). In the control experiments, the pancreatic tissue fragments were incubated in KB alone for the same period. During the incubation period, each of the vials was gassed with a mixture of 95% oxygen and 5% carbon dioxide every 3 minutes. At the end of the incubation period, the tissue fragments were removed, blotted, and weighed and the effluent stored at −20°C for insulin and glucagon radioimmunoassay (RIA).
Insulin was determined using a rat insulin RIA kit (Linco Research, St Charles, Mo). All test samples and controls were assayed in duplicates. Briefly, a volume of 100 μL of calibrators, controls, or test samples was pipetted to 100 μL of insulin antibody in previously labeled tubes and incubated overnight at 4°C. This was followed by the addition of 1 mL of I-insulin tracer into all tubes. Each tube was vortexed and incubated for 24 hours at 4°C. After the incubation period, the tubes were decanted for 3 minutes, and radioactivity was counted for 1 minute using a gamma counter (Beckman, Fullerton, Calif). Results were analyzed using a Beckman Immunofit EIA/RIA analysis software, version 2.00. Values were expressed as nanograms per milliliter per 100-mg tissue. The intra-assay coefficient of variability (CV) was less than 5.1%, and the interassay CV was less than 9.0%. The lowest level of rat insulin that can be detected was 0.01 ng/mL. Normal range of fasting insulin level is 0.5 to 2.0 ng/mL.
Effluent glucagon content was determined according to the instruction provided in glucagon RIA kit (DPC, Los Angeles, Calif). All test samples and controls were assayed in duplicates. Briefly, a volume of 200 μL of calibrator, control, or test sample was pipetted into previously labeled tubes. After this, 100 μL of glucagon antiserum was added to all tubes except nonspecific binding and total count tubes and vortexed. After vortexing, the tubes were covered with parafilm and incubated for 24 hours at 4°C. After the first incubation, 100 μL of I-glucagon was added to every tube and vortexed. The samples were incubated for 24 hours at 4°C, and then, 1 mL of cold precipitating solution was added to all tubes (except total count tubes) and centrifuged for 15 minutes at 100g. Radioactivity was counted as described for insulin, and results were expressed in picograms per milliliter per 100-mg tissue. The intra-assay CV was less than 6.5%, and interassay CV was less than 11.9%. The lowest level of rat glucagon that can be detected was 3.7 pg/mL.
Chemicals and Statistical Analysis
Rat orexin B (catalog no. SC1399) was obtained from Neosystem (Strasbourg, France). All values were expressed as mean ± SD. Statistical significance was assessed using the Student t test. One-way analysis of variance (ANOVA) was used when appropriate. Values with P < 0.05 were accepted as significant.
Immunolocalization of Orexin B in the Pancreas of Normal and Diabetic Rats
Orexin B-immunoreactive nerve fibers were observed in the intrapancreatic blood vessels of both normal (Fig. 1A) and diabetic (Fig. 1B) rats. These orexin B-containing perivascular nerves were varicose in nature and more common around medium-sized blood vessels. The pattern of distribution of these vascular orexin B-immunoreactive nerve profiles was similar in both normal and diabetic rats. Many orexin B-immunopositive cells were observed in the central region of the islet of Langerhans (Fig. 1C). The number of orexin B-positive cells decreased after the onset of diabetes. Orexin B colocalized with insulin in many pancreatic β cells (Fig. 1D). As expected, the number of insulin-positive cells was significantly reduced in diabetic rat pancreas. This decrease was associated with a decrease in the number of orexin B-positive cells (Fig. 1E). Figure 1F shows the pattern of distribution of orexin B-positive cells in the islet of Langerhans compared with that of glucagon. Orexin B was observed mainly in islet cells located within the central region, whereas glucagon-containing cells were discerned in the peripheral cells of islets. Orexin was not observed in glucagon-containing cells (Fig. 1G). In the islet of diabetic rats, there was a minimal trace of orexin B-containing cells compared with glucagon-positive cells, which have increased in number.
Effect of Orexin B on Insulin Secretion
The effect of different concentrations (10−12 to 10−6 mol/L) of orexin B on insulin secretion from the pancreatic tissue fragments of normal and diabetic rats is shown in Figure 2. Orexin B evoked significant (P < 0.02) increases in insulin secretion from the pancreas of normal rats compared with basal. In a similar trend, orexin B also stimulated insulin release from pancreatic tissue fragments of diabetic rats at all concentrations used. The effect of cholinergic (atropine), α (yohimbine)-, and β (propranolol)-adrenergic receptor antagonists and calcium channel blocker (diltiazem) on orexin B-evoked insulin release from normal and diabetic rat pancreas is shown graphically in Figure 3. Propranolol significantly (P < 0.04) inhibited orexin B-induced insulin release in the pancreas of normal rat. Cholinergic, α-adrenergic, and calcium channel antagonists were unable to significantly inhibit orexin B-evoked insulin secretion from the pancreas of normal rat. Moreover, propranolol (P < 0.006) and atropine (P < 0.04) also significantly inhibited orexin B-induced insulin release in the pancreas of diabetic rat. However, yohimbine and diltiazem failed to inhibit orexin B-induced increase in insulin release from the pancreas of either normal or diabetic rats.
Effect of Orexin B on Glucagon Secretion
The effect of orexin B on glucagon release from pancreatic segments of normal and diabetic rats is shown in Figure 4. Orexin B significantly (P < 0.04) stimulated glucagon release from the pancreas of normal rats. This orexin B-induced increase in glucagon release was not inhibited by cholinergic, adrenergic, or calcium channel antagonists (Fig. 5). By contrast, orexin B failed to stimulate glucagon release from pancreatic tissue fragments of diabetic rat. The effect of orexin B on glucagon secretion was not influenced by cholinergic, adrenergic, or calcium channel antagonists used in this study.
This study showed that orexin B is present in the pancreas of normal and to a lesser extent in diabetic rats. The presence of orexin B in the pancreas suggests a functional role for this peptide in the pancreas. In fact, orexin B stimulates amylase release from the AR42J cells.30 This functional role of orexin B in the pancreas is not surprising because the result of this study shows that orexin B-immunopositive cells are located in the wall of blood vessels of both normal and diabetic rat pancreas. The presence of orexin B around blood vessels indicates a vascular regulatory role for orexin B. Orexin B may also regulate blood flow to different structures of the endocrine and exocrine pancreas. The presence of orexin B-immunoreactive nerves in the wall of blood vessels may explain why orexin B increases mean arterial blood pressure and heart rate.31,32
The present study also shows that orexin B is present in the pancreatic islet cells of normal rats. The number of orexin B-immunoreactive cells in pancreatic islets decreased significantly after the onset of diabetes. In fact, the pancreatic islets of diabetic rats contained very few orexin B-positive cells. Orexin B colocalized with insulin in many pancreatic β cells of normal rats. However, this pattern was not observed in the islet of diabetic rats. This may be due to the disappearance of orexin B in the islet of diabetic rats. The reason for the absence of orexin B in pancreatic islet cells after the onset of diabetes is unknown. Because it is colocalized with insulin in pancreatic β cell, the disappearance of insulin may lead to a concomitant absence of orexin B. Previous reports have shown that orexin A is present in pancreatic islet cells of normal rats where they colocalized with glucagon.33 In a more recent study, it was shown that orexin A colocalized with insulin in the islets of normal cattle, sheep, and pigs.34 Our study, however, showed that orexin B colocalized with insulin in pancreatic β cells.
Effect of Orexin B on Insulin Secretion
The result of this study showed that orexin B evoked large and significant increases in insulin release from pancreatic tissue fragments of normal and diabetic rats. This observation is similar to that of Nowak et al35,36 and Switonska et al,37 who reported an increased insulin secretion after subcutaneous injection of 1 to 2 nmol of orexins A and B to normal rats. They also described a similar observation after 1 to 2 nmol of orexin B was added to the perfusion solution of pancreas preparation.35 In contrast to these observations, Ouedraogo et al33 reported that orexin A inhibits insulin release from the islets of rats.
It is evident from the literature and the results of this study that orexin B can regulate pancreatic hormone release. However, the mechanism by which orexin B induces insulin secretion has so far remained unknown. In this study, we showed for the first time that orexin B-evoked insulin release from both normal and diabetic rat pancreas is mediated via β-adrenergic pathway. It was surprising, however, to observe that atropine, a cholinergic receptor antagonist, also inhibited orexin B-induced increase in insulin release from the pancreas of a diabetic rat. A possible reason for this observation may be because Ca2+ mobilization (necessary for signal transduction) in the pancreas of diabetic rats is impaired after the onset of diabetes.38 The observed paradoxical effect of cholinergic receptor antagonist in the regulation of insulin release from the pancreas of diabetic rats may therefore be due to diabetes-induced impairment of Ca2+, a key element in the signal transduction pathway of orexin B. Orexin B stimulates dose-dependent increases in intracellular calcium mobilization38 and can modulate macrophage functions through the activation of Ca2+-dependent K+ channels.5
Diltiazem, a calcium channel antagonist, did not inhibit orexin B-evoked insulin release from the pancreas of either normal or diabetic rats. Moreover, yohimbine and α-adrenergic receptor antagonist also failed to inhibit orexin B-induced increase in insulin release from the pancreatic tissue fragments of normal and diabetic rats. It is noteworthy that atropine (10−6 mol/L), yohimbine (10−6 mol/L), propranolol (10−6 mol/L), or diltiazem (10−6 mol/L), when applied alone, did not cause any significant change in insulin secretion.39
Effect of Orexin B on Glucagon Secretion
The results of the present study provide the first evidence that orexin B can stimulate glucagon release from pancreatic segments of normal rat. However, orexin B failed to stimulate glucagon release from the pancreatic tissue fragments of diabetic rats. A possible reason for the failure of orexin B to stimulate glucagon secretion may be due to diabetes-induced impairment of Ca2+ regulatory proteins.40 The orexin B-induced increase in glucagon release was not inhibited by cholinergic, adrenergic receptor antagonists, or calcium channel blocker. This is logical because different signal transduction mechanisms will govern insulin and glucagon releases. Our observation on the effect of orexin B is similar to that of Ouedraogo et al33 on the effect of orexin A in rat pancreas. The mechanism by which orexin B increases glucagon release from the pancreas of normal rats remains to be elucidated. In a recent study, it was reported that orexin A reduced glucagon release from the isolated rat pancreatic islets and clonal pancreatic A-cells (InR1-G9) through the Foxo-1-dependent pathway.41 The reason for the difference between this observation and ours is that (1) isolated pancreatic islets were used. It is well known that the membranes of an islet cell are damaged during collagenase isolation of islet cells, instead of a whole pancreatic tissue fragments, where the exocrine-endocrine interactions are still intact. Moreover, clonal pancreatic A-cells (InR1-G9) were also used. It is suffice to say that cells lines could behave differently from that of intact normal tissue. (2). We examined the effect of orexin B whole pancreatic tissue fragments used in our study. It appears therefore that orexin A may have a different effect on glucagon secretion when compared with orexin B.
It is likely that orexin regulates energy homeostasis via the exocrine and endocrine pancreas because orexins have been shown to stimulate exocrine secretion through the vagus in rats.24 The function of the endocrine pancreas may also be affected by vagal stimulation because the endocrine pancreas is also innervated by the vagus. The presence of orexin receptors in insulin- and glucagon-producing cells of the islet of Langerhans42 supports the role of islet cells in orexin-mediated energy balance.
In conclusion, orexin B-immunoreactive cells are present in the wall of blood vessels of the pancreas of normal and diabetic rats. Orexin B-immunopositive cells are abundant in the islets of normal rats but are reduced significantly after the onset of diabetes. Orexin B colocalizes with insulin but not glucagon in pancreatic islets of rats. Orexin B evoked large and significant increases in insulin release from the pancreas of normal and diabetic rats. In addition, it stimulated glucagon release from the pancreas of normal rats. Orexin B may have an important role in the regulation of pancreatic endocrine function and influence feeding via the pancreas.
The authors thank Mr Abdulsamad Ponery for technical assistance.
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