Figure 1.
Immunolocalization of OX1R in intrapancreatic nerves.
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.
Figure 2.
Co-localization of orexin-1 receptor (OX1R) with either insulin (INS) or glucagon (GLU) in pancreatic islets.
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
Figure 3.
Mean number of pancreatic OX1R-immunoreactive cells containing either insulin or glucagon.
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).
Figure 4.
Comparison of OX1R-immunoreactivity in the endocrine pancreas of Wistar and GK 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.
Figure 5.
Localization of OX1R in the pancreatic islets of Wistar rats with acute, short- and long-term diabetes mellitus.
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.
Figure 6.
Expression of orexin-1 receptor (OX1R) protein in the pancreatic islets of Wistar rats with acute, short- and long-term diabetes mellitus.
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't-test) higher in the pancreas of long-term (8 and 15 months) rats compared to that of rats with acute and short-term diabetes mellitus (n = 6). 1: Control 8 months; 2: Diabetic 8 months; 3: Control 15 months; 4: Diabetic 15 months 5, 6 and 7: Diabetic 12 h; 9 Diabetic 24 h.
Figure 7.
Co-localization of orexin-1 receptor (OX1R) with cleaved caspase 3 in pancreatic islet cells of wild type (C57BL/6) mice, orexin knockout mice, and normal and diabetic 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.
Figure 8.
Distribution of OX1R-, cleaved caspase 3- and OX1R/cleaved caspase 3- immunopositive cells in pancreatic islet of C57BL/6 mice, orexin knockout mice, normal and diabetic Wistar rats.
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.
Figure 9.
Expression of OX1R protein and PARP (poly-ADP-ribosome polymerase) in the pancreas of control or streptozotocin (STZ)-treated OX−/− and C57BL/6 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.
Figure 10.
Glucose tolerance test in control or streptozotocin (STZ)-treated 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 (p<0.01) lower blood glucose levels compared to those of diabetic wild type at 60 and 180 min after glucose load.
Figure 11.
Schematic diagram of putative mechanism of orexin-induced increase in food and water intake, wakefulness and arousal.
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.