Orexin-1 Receptor Co-Localizes with Pancreatic Hormones in Islet Cells and Modulates the Outcome of Streptozotocin-Induced Diabetes Mellitus

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

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.

<p>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.</p

Mean number of pancreatic OX₁R-immunoreactive cells containing either insulin or glucagon.

<p>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).</p

Co-localization of orexin-1 receptor (OX₁R) with either insulin (INS) or glucagon (GLU) in pancreatic islets.

<p>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</p

Schematic diagram of putative mechanism of orexin-induced increase in food and water intake, wakefulness and arousal.

<p>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.</p

Comparison of OX₁R-immunoreactivity in the endocrine pancreas of Wistar and GK rats.

<p>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.</p

Glucose tolerance test in control or streptozotocin (STZ)-treated OX^−/− and C57BL/6 mice.

<p>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 (<i>p</i><0.01) lower blood glucose levels compared to those of diabetic wild type at 60 and 180 min after glucose load.</p

Immunolocalization of OX₁R in intrapancreatic nerves.

<p>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.</p

Expression of OX₁R protein and PARP (poly-ADP-ribosome polymerase) in the pancreas of control or streptozotocin (STZ)-treated OX^−/− and C57BL/6 mice.

<p>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.</p

Localization of OX₁R in the pancreatic islets of Wistar rats with acute, short- and long-term diabetes mellitus.

<p>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.</p