Conceived and designed the experiments: EA MFC BaR. Performed the experiments: EA MFC RH HEH MEW TA BaR. Analyzed the data: EA MFC TA BaR. Contributed reagents/materials/analysis tools: EA MFC BaR. Wrote the paper: EA MFC BaR.
The authors have declared that no competing interests exist.
Recent studies have shown that orexins play a critical role in the regulation of sleep/wake states, feeding behaviour, and reward processes. The exocrine and endocrine pancreas are involved in the regulation of food metabolism and energy balance. This function is deranged in diabetes mellitus. This study examined the pattern of distribution of orexin-1 receptor (OX1R) in the endocrine cells of the pancreas of normal and diabetic Wistar (a model of type 1 diabetes), Goto-Kakizaki (GK, a model of type 2 diabetes) rats and in orexin-deficient (OX−/−) and wild type mice. Diabetes mellitus (DM) was induced in Wistar rats and mice by streptozotocin (STZ). At different time points (12 h, 24 h, 4 weeks, 8 months and 15 months) after the induction of DM, pancreatic fragments of normal and diabetic rats were processed for immunohistochemistry and Western blotting. OX1R-immunoreactive nerves were observed in the pancreas of normal and diabetic Wistar rats. OX1R was also discernible in the pancreatic islets of normal and diabetic Wistar and GK rats, and wild type mice. OX1R co-localized with insulin (INS) and glucagon (GLU) in the pancreas of Wistar and GK rats. The number of OX1R-positive cells in the islets increased markedly (p<0.0001) after the onset of DM. The increase in the number of OX1R-positive cells is associated with a high degree of co-localization with GLU. The number of GLU- positive cells expressing OX1R was significantly (p<0.0001) higher after the onset of DM. The tissue level of OX1R protein increased with the duration of DM especially in type 1 diabetes where it co-localized with cleaved caspase 3 in islet cells. In comparison to STZ-treated wild type mice, STZ-treated OX−/− animals exhibited reduced hyperglycemia and handled glucose more efficiently in glucose tolerance test. The findings suggest an important role for the OX-OX1R pathway in STZ-induced experimental diabetes.
Recent studies have shown that orexins A and B play a critical role in the regulation of sleep/wake states, feeding behaviour, and reward processes
OX1R and OX2R are present in the lateral hypothalamic area (LHA), which is rich in orexin-positive nerves
The expression of orexin receptor mRNA is not confined only to the central nervous system. Low OX1R mRNA expression has been observed in the adrenal gland of the rat
Since diabetes mellitus is associated with a dysfunction of glucose metabolism, the aim of this study was to examine whether diabetes mellitus will induce changes in the pattern of distribution of OX1R in the endocrine cells of Wistar and GK rats and mice pancreas.
Male Wistar rats (12-week old) obtained from United Arab Emirates University breeding colony, weighing approximately 250 g were used in this study. The study was approved by the Animal Ethics Research Committee at the Faculty of Medicine & Health Sciences. The guidelines set by this committee for animal husbandry and welfare, based on Helsinki Declaration of 2006 was followed. The rats were divided into two groups, streptozotocin (STZ)-induced diabetics and age-matched controls. Diabetes was induced by a single intraperitoneal injection of STZ (Sigma, Poole, UK) at 60 mg kg−1 (200 mg kg−1 for mice) prepared in 5 mM citrate buffer pH 4.50
The colony of GK rats were obtained from Taconic Inc. (Ejby, Denmark) and bred in our laboratory. GK rats are non-obese and exhibits similar metabolic, hormonal and vascular disorders to the human disease
Founder orexin deficient (OX−/−) mice were on a C57BL/6J-129/SvEV background, and their offspring were backcrossed with C57BL/6J mice for 6–8 generations
Pancreatic tissue fragments from six normal and diabetic rats from each group (time point), were trimmed free of adherent fat and connective tissue and cut into small pieces (2 mm3) and fixed overnight in Zamboni's solution
In order to determine whether diabetes mellitus influences the number and pattern of distribution of OX1R-, insulin-, glucagon- and caspase-immunoreactive cells, the total number of cells in the islets of Langerhans of normal and diabetic rats was counted using Axiovision® Image Analysis System (Zeiss, Gottingen, Germany) attached to a fluorescent microscope. Thereafter, OX1R-immunoreactive cells and those co-localized with either insulin (INS) or glucagon (GLU) in a given islet were also counted. The number of OX1R-immunoreactive cells or those that co-localized with INS or GLU was divided by the total number of cells to give the percentage distribution. Isolated OX1R-postive cells or those co-localized with INS- or GLU-positive cells were counted in visible sections at X40 magnification. A total of 10 random islets were taken from a total of 6 slides for each group. The values obtained from sections of the islets of Langerhans of normal rat were compared with those of diabetic rat.
The co-localization of OX1R with either INS or GLU in pancreatic islets of normal and diabetic rats was performed with double-labeled immunofluorescence method as described by Adeghate and Ponery
Some studies
Pancreatic tissue samples from different experimental time points were homogenized in 1 ml of lysis buffer [1% NP-40, 25 mM Tris pH 7.4, 0.1 mM EDTA, 1 mM DTT, protease inhibitor cocktail set III (Calbiochem, San Diego, CA, USA) on ice. After incubating the homogenates on ice for 15 min, samples were centrifuged at 13000 rpm for 15 min at 4°C. Clarified cell lysates were collected and total protein concentrations were measured by the Bradford method (Bio-Rad protein assay, Bio-Rad laboratories, Munich, Germany). The lysates (200 µg protein/lane) were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane (Bio-Rad), and blocked with 5% nonfat milk powder in TBS containing 0.01% Tween 20 at room temperature for 1 h. Membranes were incubated with OX1R antibody (1∶1000, goat-anti-rat orexin R-1, Santa Cruz Biotechnology, USA) overnight at 4°C, washed, and incubated with horse radish peroxidase-conjugated anti-goat IgG secondary antibody (Sigma, UK) for 1.5 h at room temperature. Blots were washed and developed using SuperSignal substrate (Pierce, Rockford, IL, USA).
In a bid to examine a possible link between OX1R and apoptosis, pancreatic tissue samples from control OX−/− and C57BL/6 and STZ-treated OX−/− and C57BL/6 mice were homogenized and processed for Western blotting as described above. Membranes were incubated overnight at 4°C with either anti orexin R-1 or anti β-actin antibodies (1∶1000, Santa Cruz Biotechnology, USA), or anti-PARP antibody (1∶1000, R&D Systems Inc., Minneapolis, USA). The blots were further processed as described above and developed using SuperSignal substrate.
In order to investigate the ability of orexin knockout (OX−/−) and wild type (C57BL/6) mice to handle glucose challenge, control OX−/− and C57BL/6 and STZ-treated OX−/− and C57BL/6 mice were fasted overnight (12 h) prior to the test. Each mouse was given a glucose load of 3 g/kg body weight intra-peritoneally, according to Zhang and Tan
All values were expressed as mean±standard error of the mean (SEM). Statistical significance was assessed using Student's
Peripheral nerves located in the interlobular, perivascular, and periductal regions of the pancreas of both normal and diabetic rats contain OX1R (
Light micrographs showing OX1R-immunoreactive nerve fibers (arrow) in the pancreas of normal (a), and diabetic (b, d) rats. OX1R expression is equally present in the nerve fibers of both normal and diabetic (4 weeks after the onset of diabetes) rats. Ganglion cells (arrow) of normal pancreas (c) expressed OX1R. Magnification: ×400.
Immunofluorescence images showing orexin-1 receptor (OX1R)-immunoreactive cells (red) with either, (a) INS (green) or (d) GLU (green) in the pancreatic islet of normal rats. Many cells (orange-yellow) contain both OX1R and INS in the pancreas of normal Wistar rats (a). Only few cells contain both OX1R and GLU in the endocrine pancreas of normal Wistar rats (d). In diabetic (4 weeks after the onset of diabetes) rats, the number of INS cells decreased (b). However, some surviving INS-positive cells also contained OX1R (orange-yellow) (b). OX1R also co-localized (orange-yellow) with INS in the pancreatic islets of GK rats (c). A large number of GLU-positive cells expressed OX1R after the onset of streptozotocin-induced diabetes (e). There was little to no co-localization of GLU and OX1R in the islet of GK rats (f). INS = insulin, GLU = glucagon; GK = Goto Kakizaki. Magnification: ×200
In contrast to the small degree of co-localization observed between OX1R and INS pancreatic islet cells of diabetic Wistar rats, a much larger subset of GLU-positive cells expressed OX1R in diabetic Wistar rats (
OX1R co-localized with INS in a large number of INS-producing beta cells in the pancreas of normal Wistar rat. The number of OX1R-containing cells that contain INS decreased significantly (p<0.001) after the onset of diabetes. Although some OX1R-positive cells also contained GLU, the percentage distribution of GLU-immunoreactive cells that co-localized with OX1R rose significantly (p<0.0001) after the onset of diabetes when compared to normal (
Histograms of the mean of the distribution of OX1R-immunoreactive cells containing either INS or GLU in normal and diabetic (4 weeks after the onset of diabetes) rat pancreas. *(p<0.01: OX1R/INS in control versus diabetic); **(p<0.0001: OX1R/GLU in control versus diabetic). INS = insulin, GLU = glucagon. (10 islets from 6 animals/group).
The Goto-Kakizaki (GK) rat is a model of type 2 diabetes with similar metabolic disorders compared to human diabetes. GK rats are hyperglycaemic and have an impaired insulin secretion. Immunohistochemistry was performed to examine whether the pattern of distribution of OX1R is similar in the pancreatic islets of GK compared to Wistar rats. The pancreatic islets of GK rats contain numerous OX1R-imunoreactive cells similar to that observed in the islet of diabetic Wistar rats (
Light micrographs showing OX1R-immunoreactive cells in the pancreatic islet of normal Wistar (a) and Goto Kakizaki (b) rats. Note that the islet cells of Goto Kakizaki stains more intensely for OX1R compared to Wistar. Magnification: ×200.
The expression of OX1R in the pancreas of diabetic rats was investigated at different time points after the induction of DM. The expression of OX1R increased with the duration of DM from pale staining 12–24 h after the onset of DM to intense expression 8–15 months after the induction of DM (
Light micrographs showing OX1R-immunoreactive cells in the pancreatic islet of Wistar 12 h (a); 24 h (b); 8 months (c) and 15 months (d) after the onset of diabetes. Note the large number of islet cells containing OX1R in long-term diabetes (c) and (d). Magnification: ×200.
Western blot analysis of pancreas segments from normal and diabetic rats was performed to quantify the tissue content of OX1R. The expression of OX1R protein was highest in the pancreas of rats with long-term DM, namely 8 and 15 months and low in the pancreas of rats with acute to short-term (12–24 h) DM (
Western blot analysis of OX1R protein expression in the pancreas of normal and diabetic rats at different time points of diabetes. Note that the tissue level of OX1R protein was significantly (p < 0.0001, Students'
In order to examine a possible link between OX1R and cleaved caspase 3 in pancreatic islet of OX−/− and C57BL/6, we used immunofluorescence method to simultaneously localize OX1R and cleaved caspase 3. Cleaved caspase 3-positive cells were located in the peripheral region of pancreatic islets in OX−/−, C57BL/6 mice and normal Wistar rats (
Immunofluorescence images showing co-localization of orexin-1 receptor (OX1R) (green) with cleaved caspase 3 (red) in pancreatic islet cells of C57BL/6 mice (a), orexin knockout mice (b), normal Wistar (c) and diabetic Wistar (d) rats. OX1R-positive cells are located in the peripheral region of the islets of C57BL/6 and orexin knockout mice and normal Wistar rats. However, in diabetic Wistar rats (d), there is a high degree of co-localization (orange-yellow) between OX1R and caspase 3 in many pancreatic islet cells. It is worth noting that the number of OX1R (green) and cleaved caspase 3 (red) immunoreactive cells are significantly reduced in orexin knockout mice. Magnification: ×200.
Cleaved caspase 3-positive cells were relatively high in the islets of normal Wistar rats compared to wild type and OX−/− mice. However, the number of OX1R- and cleaved caspase 3-immunoreactive cells increased significantly (p<0.001) after the onset of STZ-induced diabetes. The degree of co-localization of OX1R with cleaved caspase 3 was more than 90%. This values was significantly higher in Wistar diabetic rats than those obtained in orexin deficient or wild type mice (
Histograms of the pattern of distribution of OX1R-positive (green), cleaved caspase 3-immunoreactive cells (red) and cells containing both OX1R and cleaved caspase 3- in the pancreas of in pancreatic islet cells of C57BL/6 mice, orexin knockout mice, normal Wistar and diabetic Wistar rats. Note the direct correlation between the number of OX1R-positive cells and that of cleaved caspase 3. The number of cells containing both OX1R cleaved caspase 3 is significantly elevated in streptozotocin-induced diabetic Wistar rats.
To further elucidate the link between OX1R and apoptosis, we used Western blotting technique to semi-quantify pancreatic tissue levels of OX1R protein and the apoptotic marker PARP polymerase, a DNA repair enzyme which is targeted by active caspase 3 during apoptosis.
OX1R expression was not enhanced in the pancreas of wild type mice but was upregulated in the pancreas of OX−/− mice after the onset of STZ-induced diabetes. In contrast, the expression of PARP, an indication of proteolysis by active caspase 3, was robust in the pancreas of C57BL/6 mice but decreased markedly in OX−/− mice (
Western blot analysis of OX1R protein and PARP (poly-ADP-ribosome polymerase) expression in the pancreas of control or STZ-treated OX−/− and C57BL/6 mice are shown in lanes 1 and 2. Lane 1 shows that the OX1R expression was not enhanced in the pancreas of wild type mice but was upregulated in the pancreas of OX−/− mice after the onset of STZ-induced diabetes. In contrast, the expression of PARP was robust in the pancreas of C57BL/6 mice but decreased markedly in OX−/− mice treated with STZ (Lane 2). Lane 3 shows the expression of the control protein, beta actin.
We observed that OX1R co-localizes with INS in pancreatic beta cell and it is up-regulated in diabetes. Control OX−/− and C57BL/6 and STZ-treated OX−/− and C57BL/6 mice were challenged with intra-peritoneal glucose load to examine whether the orexin/OX1R system has a role to play in glucose metabolism. Blood glucose levels of control OX−/− and C57BL/6 mice were similar at time 0, 30, 60, 180 min. In contrast, blood glucose level of STZ-induced diabetic OX−/− mice was slightly but not significantly lower than that of diabetic C57BL/6 mice at 0 min. Diabetic OX−/− mice displayed a significantly lower blood glucose levels compared to those of diabetic wild type at 60 and 180 min after glucose challenge. The efficacy of glucose handling by STZ-treated OX−/− mice approached that of control OX−/− and C57BL/6 mice (
The graph shows blood glucose levels in control or STZ-treated OX−/− and C57BL/6 mice 0, 30, 60 and 180 min after intra-peritoneal glucose challenge (3 g/kg body weight, given intra-peritoneally). Blood glucose levels of control OX−/− and C57BL/6 mice were similar at time 0, 30, 60, 180 min. Note that blood glucose level of STZ-induced diabetic OX−/− mice was slightly but not significantly lower than that of diabetic C57BL/6 mice at 0 min. Diabetic OX−/− mice displayed a significantly (
The observations of this study showed that OX1R is present in the intrinsic neuronal ganglion and nerve fibers innervating the pancreas of both normal and diabetic Wistar rats. There was no difference between the pattern of distribution of OX1R-postive nerves in normal and diabetic Wistar rat pancreas. The presence of OX1R-immunopositive nerves confirms the result of previous studies from our laboratory
The present study and those reported previously
It was interesting to observe OX1R expression in the islet of GK rats, an animal model of type 2 diabetes. The expression of OX1R in the pancreas of GK rats was higher than those in the pancreas of normal Wistar rat but similar to that seen in the pancreas of diabetic Wistar rats. This observation probably points to a common denominator that induces increased OX1R expression in the pancreatic islet of type 1 diabetic rats.
Several pancreatic beta cells of normal Wistar rats contained OX1R. In diabetic Wistar rat pancreas, the number of INS-positive cells expressing OX1R decreased with a concomitant increase in the number of OX1R-positive cells. It is well known that the number of INS-positive cells is decreased in diabetes
The co-localization of OX1R and INS is further confirmed by the fact that a similar pattern was seen in the islet of GK rats, an animal model of type 2 diabetes, where hyperglycaemia is much milder. All of these support a functional role of OX1R and the orexinergic system in the regulation of carbohydrate metabolism.
This is the first report on the co-localisation of INS and GLU and OX1R. The number of GLU-positive cells expressing OX1R increased significantly after the onset of type 1 diabetes. Although, OX1R is present in the pancreatic islets of GK rats, a model of type 2 diabetes, the degree of co-localization with GLU was minimal compared to that STZ-induced diabetic rat (a model of type 1 diabetes). This may be due to the fact that the hyperglycaemia is much milder in GK compared to STZ-treated Wistar rats. The co-localization of OX1R with INS and GLU suggests a pivotal role for INS and GLU in the biological actions of orexins. Moreover, orexins evoked the release of INS
OX1R was also upregulated in PP-positive islets cells after the onset of diabetes (result not shown). The reason for the increase in the expression of OX1R in pancreatic polypeptide (PP)-immunoreactive cells is unknown. Since OX1R is present in PP-positive cells, orexin may stimulate the release of PP, which may in turn enhance appetite. It has been shown that centrally administered PP elicits food intake via NPY receptors
Diabetes mellitus, with the resulting decrease in intracellular glucose, leads to increased expression of OX1R in pancreatic islet cells such as glucagon (GLU) and pancreatic polypeptide (PP). All of these, in combination with circulating and neural-derived orexins stimulate GLU and PP release. GLU induces gluconeogenesis and glycogenolysis resulting in energy utilization, wakefulness and arousal. Moreover, GLU and PP from the circulation and via paracrine effect may stimulate insulin (INS) release resulting in increased intracellular glucose, and energy utilization. DM = diabetes mellitus; OX1R = orexin-1 receptor; INS = insulin; GLU = glucagon; PP = pancreatic polypeptide. (+) = stimulate; (−) = inhibit.
This study showed a larger increase in OX1R expression in the pancreatic islets of rats with long-term DM compared to those with short-term diabetes. The reason for this observation is not clear, however, it may be due to the irreparable damage done to pancreatic tissue due to persistent hyperglycaemia and other inductors of oxidative stress. Oxidative stress is a major cause of late complications of DM and may be more pronounced in the pancreas of rats with long and established DM
Cleaved caspase 3, indicative of an activated enzyme, is a critical step in the apoptosis pathway. Expression of active caspase 3 was discernible in the cells of the peripheral regions of the islets of OX−/− and C57BL/6 mice and normal Wistar rats. The number of caspase 3-positive islet cells was significantly fewer in OX−/− mice compared to C57BL/6 mice. It is worth noting that OX−/− mice have significantly fewer OX1R compared to wild type. It does appear that the number of OX1R-positive cells is directly proportional to that of caspase 3. The use of OX−/− mice shows that the fewer the number of OX1R-immunoreactive cells, the lower the number of caspase 3-positive cells becomes.
In contrast, the number of OX1R- and caspase 3-postive cells increased significantly after the onset of STZ-induced diabetes in Wistar rats. A significant number of pancreatic islet cells of this animal model contain both OX1R- and caspase 3. This shows a link,
A link between OX1R and apoptosis has been described by Voisin et al,
Western blot technique showed the expression of PARP in the pancreas of both orexin knockout and wild type mice. The tissue level of PARP decreased after the induction of experimental diabetes in orexin-deficient mice. It is not clear why PARP was well expressed in the pancreas of normal wild type and knockout mice at least when they were not subjected to any toxins that could possibly induce oxidative stress and therefore apoptosis.
The pancreas of orexin knockout mice contained a small quantity of OX1R compared to the pancreas of the wild type. However, the tissue level OX1R increased after the onset of diabetes in the mutant, but not in control mice. The reason for the increase in not known, but it may be due to the modulation of the orexinergic system by STZ, especially in the absence of orexin in the mutant mice. The increased expression of OX1R after the onset of diabetes in the mutant mice was accompanied by a decrease in the expression of PARP when compared to control, non-diabetic mutant mice and control as well as diabetic wild type. The reason for this inverse relationship is unclear but it may be due to the absence of orexin, a major trigger of apoptosis.
We showed for the first time that STZ-induced diabetic orexin knockout mice shows a more efficient glucose handling compared to control OX−/− and C57BL/6 and STZ-treated C57BL/6 mice. Since STZ causes oxidative stress and apoptosis
The number of cells expressing OX1R is increased in pancreatic islets after the onset of diabetes. The longer the duration of diabetes the more intense the OX1R expression becomes. OX1R-positive islet cells co-localizes with INS and GLU in Wistar rats and with cleaved caspase 3 after the onset of diabetes in Wistar rats treated with STZ. The expression of OX1R and caspase 3 is significantly lower in the pancreas of orexin knockout mice compared to wild type and Wistar rats.
Orexin knockout mice treated with STZ have the ability to handle glucose load more effectively that control OX−/− and C57BL/6 and STZ-treated C57BL/6 mice. STZ requires the orexinergic system to cause severe apoptosis that can consequently lead to an overt diabetes.
The authors are grateful to Dr. Tom Scammell, Department of Neurology, Harvard University Medical School, Boston, MA 02215, USA for kindly providing orexin knockout mice.