Human and Rodent Cell Lines as Models of Functional Melatonin-Responsive Pancreatic Islet Cells.

Cell culture of different pancreatic islet cell lines, like the murine α-cell line αTC1.9, the rat ß-cell lines INS-1 and INS-1 832/13, and the human δ-cell line QGP-1, can serve as valuable cell models for the analysis of melatonin-dependent modulation of hormone secretion. The paper summarizes in detail the requirements of culture for each cell line and includes batch protocols to stimulate hormone secretion and to treat cells with several melatonin concentrations as previously published. We here describe the processing of collected cell pellets or cell culture supernatants as well as different methods to analyze cell experiments after melatonin treatment on the basis of our own experience. Finally, we outlined for each cell line under which conditions the melatonin treatment should be performed to gain reproducible results.

Influence of Cannabinoid Receptor Deficiency on Parameters Involved in Blood Glucose Regulation in Mice.

Cannabinoids are known to influence hormone secretion of pancreatic islets via G protein­coupled cannabinoid receptor type 1 and 2 (CB1 and CB2). The present study was designed to further investigate the impact of cannabinoid receptors on the parameters involved in insulin secretion and blood glucose recognition. To this end, CB1 and CB2 receptor knockout mice (10-12 week old, both sexes) were characterised at basal state and compared to wild-type mice. The elimination of cannabinoid receptor signalling resulted in alterations of blood glucose concentrations, body weights and insulin levels. Changes were dependent on the deleted receptor type and on the sex. Analyses at mRNA and protein levels provided evidence for the impact of cannabinoid receptor deficiency on the glucose sensing apparatus in the pancreas. Both receptor knockout mouse lines showed decreased mRNA and protein amounts of glucose transporters Glut1 and Glut2, combined with alterations in immunostaining. In addition, pancreatic glucokinase expression was elevated and immunohistochemical labelling was modified in the pancreatic islets. Taken together, CB1 and CB2 signalling pathways seem to influence glucose sensing in ß-cells by affecting glucose transporters and glucokinase. These alterations were more pronounced in CB2 knockout mice, resulting in higher blood glucose and lower plasma insulin levels.

Distribution and density of melatonin receptors in human main pancreatic islet cell types.

Recent investigations of our group established that melatonin modulates hormone secretion of pancreatic islets via melatonin receptor types MT1 and MT2. Expression of MT1 and MT2 has been shown in mouse, rat, and human pancreatic islets as well as in the ß-, α-, and δ-cell lines INS-1, αTC1.9, and QGP-1. In view of these earlier investigations, this study was performed to analyze in detail the distribution and density of melatonin receptors on the main islet cell types in human pancreatic tissue obtained from nondiabetic and type 2 diabetic patients. Immunohistochemical analysis established the presence of MT1 and MT2 in ß-, α-, and δ-cells, but notably, with differences in receptor density. In general, the lowest MT1 and MT2 receptor density was measured in α-cells compared to the 2 other cell types. In type 2 diabetic islets, MT1 and MT2 receptor density was increased in δ-cells compared to normoglycemic controls. In human islets in batch culture of a nondiabetic donor, an increase of somatostatin secretion was observed under melatonin treatment while in islets of a type 2 diabetic donor, an inhibitory influence could be observed, especially in the presence of 5.5 mmol/L glucose. These data suggest the following: i) cell-type-specific density of MT1 and MT2 receptors in human pancreatic islets, which should be considered in context of the hormone secretion of islets, ii) the influence of diabetes on density of MT1 and MT2 as well as iii) the differential impact of melatonin on somatostatin secretion of nondiabetic and type 2 diabetic islets.

Melatonin influences somatostatin secretion from human pancreatic δ-cells via MT1 and MT2 receptors.

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor-transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α-cells. Virtually, nothing is known concerning the melatonin-mediated effects on islet δ-cells. Analysis of a human pancreatic δ-cell model, the cell line QGP-1, and the use of a somatostatin-specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ-cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose-dependently stimulating somatostatin secretion, in experiments employing the membrane-permeable 8-Br-cAMP. 8-Br-cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1 nm melatonin was reversed after incubation of QGP-1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2-selective antagonist 4-P-PDOT (4-phenyl-2-propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ-cells at low glucose concentrations was significantly inhibited during co-incubation with 1 nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ-cells and on somatostatin release.