Functional and morphological study of cultured pancreatic islets treated with cyclosporine

Cyclosporine A (CsA), a potent immunosuppressive drug, has been found to induce glucose intolerance through its toxic effect on the endocrine pancreas. It is not exactly known whether CsA has a direct effect on the endocrine pancreas or induces its effect indirectly. The present study was therefore undertaken to examine the function and morphology of isolated pancreatic islets when they are directly exposed in vitro to CsA. Pancreatic islets were isolated from adult male Lewis rats using collagenase ductal perfusion technique. The islets were separated with the discontinuous Ficoll gradient technique and further purified by hand picking of the non-islet tissue. The islets were cultured in RPMI-1640, pH 7.4 and maintained at 37 degrees C in a humid atmosphere of 5% (v/v) carbon dioxide in air. Cyclosporine was added to the culture medium to give a final concentration of 1 microg/ml (therapeutic dose), 5 microg/ml (toxic dose), or vehicle (control). Islets were harvested at 1, 4 and 10 days of culture and processed for functional or histological study. The functional study of the islets cultured with 1 microg/ml CsA showed insulin and C-peptide contents similar to those of the control islets. The islets cultured with 5 microg/ml CsA showed a marked decrease in insulin and C-peptide contents. Glucose-dependent insulin release was variable. C-peptide release was lower than that of the control following both the therapeutic and toxic doses of CsA. Phase contrast microscopy showed that the islets cultured with 1 microg/ml CsA were mostly normal looking with a well-defined regular periphery; a few islets had ill-defined or irregular peripheries. The islets cultured with 5 microg/ml CsA had ill-defined irregular peripheries at 1 day, and were dense and forming clumps at 4 and 10 days following culture. There was a decrease in the islet number following the therapeutic dose; the decrease was more following the toxic dose of CsA. The islet diameters increased after the therapeutic dose, but slightly decreased following the toxic dose of CsA. Islets showed a weakly positive immunoperoxidase reaction for insulin that was weaker following the toxic dose of CsA. It is concluded that CsA has a direct effect on B-cells that was proved by the functional and morphological changes seen in the pancreatic islets cultured in vitro.     

d-Alanine in the islets of Langerhans of rat pancreas

Relatively high levels of d-alanine (d-Ala), an endogenous d-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed d-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180 ± 60 fmol d-Ala per islet (300 ± 100 nmol/g islet), and 10 ± 2.5 nmol/g of wet tissue in pituitary. Additionally, 12 ± 5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of d-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in d-Ala release of 0.6 ± 0.5 fmol per islet. As d-Ala is stored in islets and released in response to changes in extracellular glucose, d-Ala may have a hormonal role.     

Synaptotagmin 11 interacts with components of the RNA-induced silencing complex RISC in clonal pancreatic β-cells

Synaptotagmins are two C₂ domain-containing transmembrane proteins. The function of calcium-sensitive members in the regulation of post-Golgi traffic has been well established whereas little is known about the calcium-insensitive isoforms constituting half of the protein family. Novel binding partners of synaptotagmin 11 were identified in β-cells. A number of them had been assigned previously to ER/Golgi derived-vesicles or linked to RNA synthesis, translation and processing. Whereas the C2A domain interacted with the Q-SNARE Vti1a, the C2B domain of syt11 interacted with the SND1, Ago2 and FMRP, components of the RNA-induced silencing complex (RISC). Binding to SND was direct via its N-terminal tandem repeats. Our data indicate that syt11 may provide a link between gene regulation by microRNAs and membrane traffic.     

GSK-3β Protein Phosphorylates and Stabilizes HLXB9 Protein in Insulinoma Cells to Form a Targetable Mechanism of Controlling Insulinoma Cell Proliferation

Insulinomas (pancreatic islet β cell tumors) are the most common type of functioning pancreatic neuroendocrine tumors that occur sporadically or as a part of the MEN1 syndrome that is caused by germ line mutations in MEN1. Tissue-specific tumor predisposition from germ line mutations in ubiquitously expressed genes such as MEN1 could occur because of functional consequences on tissue-specific factors. We previously reported the proapoptotic β cell differentiation factor HLXB9 as a downstream target of menin (encoded by MEN1). Here we show that GSK-3β inactivates the proapoptotic activity of HLXB9 by phosphorylating HLXB9 at Ser-78/Ser-80 (pHLXB9). Although HLXB9 is found in the nucleus and cytoplasm, pHLXB9 is predominantly nuclear. Both pHLXB9 and active GSK-3β are elevated in β cells with menin knockdown, in MEN1-associated β cell tumors (insulinomas), and also in human sporadic insulinomas. Pharmacologic inhibition of GSK-3β blocked cell proliferation in three different rodent insulinoma cell lines by arresting the cells in G₂/M phase and caused apoptosis. Taken together, these data suggest that the combination of GSK-3β and pHLXB9 forms a therapeutically targetable mechanism of insulinoma pathogenesis. Our results reveal that GSK-3β and pHLXB9 can serve as novel targets for insulinoma treatment and have implications for understanding the pathways associated with β cell proliferation.     

YES,a Src Family Kinase,Is a Proximal Glucose-specific Activator of Cell Division Cycle Control Protein 42 (Cdc42) in Pancreatic Islet β Cells

Second-phase insulin secretion sustains insulin release in the face of hyperglycemia associated with insulin resistance, requiring the continued mobilization of insulin secretory granules to the plasma membrane. Cdc42, the small Rho family GTPase recognized as the proximal glucose-specific trigger to elicit second-phase insulin secretion, signals downstream to activate the p21-activated kinase (PAK1), which then signals to Raf-1/MEK/ERK to induce filamentous actin (F-actin) remodeling, to ultimately mobilize insulin granules to the plasma membrane. However, the steps required to initiate Cdc42 activation in a glucose-specific manner in β cells have remained elusive. Toward this, we identified the involvement of the Src family kinases (SFKs), based upon the ability of SFK inhibitors to block glucose-stimulated Cdc42 and PAK1 activation events as well as the amplifying pathway of glucose-stimulated insulin release, in MIN6 β cells. Indeed, subsequent studies performed in human islets revealed that SFK phosphorylation was induced only by glucose and within 1 min of stimulation before the activation of Cdc42 at 3 min. Furthermore, pervanadate treatment validated the phosphorylation event to be tyrosine-specific. Although RT-PCR showed β cells to express five different SFK proteins, only two of these, YES and Fyn kinases, were found localized to the plasma membrane, and of these two, only YES kinase underwent glucose-stimulated tyrosine phosphorylation. Immunodetection and RNAi analyses further established YES kinase as a proximal glucose-specific signal in the Cdc42-signaling cascade. Identification of YES kinase provides new insight into the mechanisms underlying the sustainment of insulin secretion via granule mobilization/replenishment and F-actin remodeling.     

Effect of chronic psychological stress on insulin release from rat isolated pancreatic islets

Despite documented studies, the exact role of stress on diabetes is still unclear. The present study investigates the effect of chronic psychological stress on insulin release from isolated rat pancreatic islets. Male Wistar rats were divided into two groups of control and stressed (n=8/group). The animals of the stressed group were exposed to restraint stressors (1 h twice daily) for 15 or 30 consecutive days. At the beginning and end of the experimental periods, the animals were weighed and blood samples taken to determine the fasting plasma levels of glucose, insulin and corticosterone. On the following day the pancreatic islets of 5/group of the above animals were isolated and the static release of insulin in the presence of different glucose concentrations (2.8, 5.6, 8.3, 16.7 mM) was assessed. The results showed that in the stressed group, fasting plasma glucose levels were increased significantly on the 15th day as compared to the control group. However there was no significant increase on the 30th day. Fasting plasma insulin was significantly decreased on the 15th and 30th days of the experiment in the stressed group. Stressed rats showed significantly higher fasting plasma corticosterone levels, only on the 15th day, as compared to the control rats. In response to increasing concentrations of glucose, insulin release from islets of the stressed group was increased significantly on the 30th day of the experiment as compared to the control group. We conclude that chronic psychological stress could increase responsiveness of pancreatic beta cells to glucose, in vitro, and thus, low insulin levels of the stressed animals, in vivo, may be due to reason(s) other than the reduction of insulin releasing capacity of pancreatic beta cells.     

Protection of insulin-producing cells against toxicity of dexamethasone by catalase overexpression

Pancreatic β cells are very sensitive to reactive oxygen species (ROS) and this might play an important role in β cell death in diabetes. Dexamethasone is a synthetic diabetogenic glucocorticoid, which impairs pancreatic β cell function. Therefore we investigated the toxicity of dexamethasone in RINm5F insulin-producing cells and its dependence on the expression level of the antioxidant enzyme catalase, which inactivates hydrogen peroxide. This was correlated with oxidative stress and cell death. An increased generation of ROS was observed in dexamethasone-treated cells together with an increase in caspase-3 activity and apoptosis rate. Interestingly, exposure to dexamethasone increased the cytosolic superoxide dismutase Cu/ZnSOD protein expression and activity, whereas the mitochondrial MnSOD isoform was not affected by the glucocorticoid. Catalase overexpression in insulin-producing cells prevented all the cytotoxic effects of dexamethasone. In conclusion, dexamethasone-induced cell death in insulin-producing cells is ROS mediated. Increased levels of expression and activity of the Cu/ZnSOD might favor the generation of hydrogen peroxide in dexamethasone-treated cells. Increased ROS scavenging capacity in insulin-producing cells, through overexpression of catalase, prevents a deleterious increase in hydrogen peroxide generation and thus prevents dexamethasone-induced apoptosis.     

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

The Surface structure of isolated pancreatic islet cells

Summary The surfaces of isolated pancreatic islet cells were studied with the scanning and transmission electron microscopes. Islets were isolated from the pancreas of Wistar rats by collagenase treatment and were incubated either in glucose-free medium or in 300 mg% glucose for one hour. Immunoreactive insulin (IRI) in the media of both control and experimental preparations was assayed. Islets were then transferred to 4% glutaraldehyde, buffered with cacodylate, pH 7.4, and prepared for scanning and transmission electron microscopy. Cell masses average 200 in diameter. Alpha cells appear pyramidal in shape, are about 8 in diameter and appear in groups. Beta cells are round or oval in shape and have an average diameter of 10 . Glucose stimulation raised the IRI value tenfold and increased the number of blebs and other surface irregularities per unit area of beta cell surface. Comparison with transmission electron micrographs suggests that the blebs are related to the process of emiocytosis.Supported by U.S.P.H.S. Grant AM-10151.