Cell-specific localization of G protein alpha-subunits in the islets of Langerhans.

G protein alpha-subunits are involved in the transduction of receptor-mediated regulation of insulin and glucagon secretions. To get further insight into the status of G proteins in alpha- and beta-cells of the Langerhans islets, we have used immunohistochemistry to study the distribution of alpha-subunits in pancreas sections from the rat. Our results show that only insulin-immunoreactive beta-cells display immunoreactivity for selective antibodies directed against the different members of the Galphas and Galpha12-families (alphas, alphaolf, and alpha12, alpha13 respectively). Immunoreactivities for antibodies directed against members of the Galphaq- and Galphai-families showed a more diverse localization: alpha11 and alphao2 were only detected in glucagon-immunoreactive alpha-cells, whereas alphai1 was detected in all beta-cells but only in a few alpha-cells. Even though beta-cells showed immunoreactivities for alphao-non-isoform-selective antibodies, we could not identify the isoform(s) present using selective alphao1 and alphao2 antibodies. Other members of the Galphai- and Galphaq-families (alphai3, alphat2, alphaz and alphaq) were detected in both alpha- and beta-cells. In conclusion, our findings demonstrate a clear difference in the localization of G protein alpha-subunits between alpha- and beta-cells, suggesting the involvement of specific receptor transduction pathways for the neuronal/hormonal regulation of alpha- and beta-cell functions.     

Cellular and molecular events in the localization of diabetogenic T cells to islets of Langerhans

Understanding the entry of autoreactive T cells to their target organ is important in autoimmunity because this entry initiates the inflammatory process. Here, the events that lead to specific localization of diabetogenic CD4 T cells into islets of Langerhans resulting in diabetes were examined. This was evaluated in two models, one in which T cells specific for a hen-egg white lysozyme (HEL) peptide were injected into mice expressing HEL on β cells and the other using T cells in the nonobese diabetic mouse strain, which develops spontaneous diabetes. Only T cells specific for β-cell antigens localized in islets within the first hours after their injection and were found adherent to intraislet dendritic cells (DCs). DCs surrounded blood vessels with dendrites reaching into the vessels. Localization of antigen-specific T cells did not require chemokine receptor signaling but involved class II histocompatibility and intercellular adhesion molecule 1 molecules. We found no evidence for nonspecific localization of CD4 T cells into normal noninflamed islets. Thus, the anatomy of the islet of Langerhans permits the specific localization of diabetogenic T cells at a time when there is no inflammation in the islets.     

Transplantation of islets of Langerhans

The present study attempts to evaluate the possibility of using cryopreserved pancreatic islets in rats. Insulin-releasing activity was studied for the purpose of clarifying the function of cryopreserved islets. Cryopreserved pancreatic islets were also transplanted into the portal veins of recipient rats. Rats recovered from the diabetic state, and the normalized condition was maintained for up to 20 weeks, although 4 weeks were needed before blood and urine glucose reached normal levels after transplantation. Intact B cells were found in the transplanted islet cell masses in the liver of the recipients, but B cells of the recipient's pancreases (streptozotocin-treated rats) showed a marked decrease, as well as degenerative changes.     

Metallothionein induction in islets of Langerhans and insulinoma cells

Isolated pancreatic islets from rat and mouse and the insulinoma cell lines, betaHC9 and RINm5F, were investigated to determine the regulation of metallothionein (MT). Dexamethasone (DEX) increased rat and mouse islet and insulinoma cell MT levels in a time- and concentration-dependent manner. Rat islet MT expression was increased with interleukin-1beta (IL-1beta), but not tumor necrosis factor-alpha (TNF). However, MT induction by IL-1beta and TNF was synergistic with DEX in rat islets and insulinoma cells. Mouse islet MT failed to respond to IL-1beta alone, although IL-1beta and TNF were synergistic. IL-1beta and TNF did not synergize with DEX for mouse islet MT induction. Zinc sulfate induced MT in rat islets but not mouse islets. MT messenger RNA levels were significantly increased in rat islets in response to DEX and IL-1beta plus DEX. The inducible nitric oxide synthase inhibitors N(G)-monomethyl-L-arginine and aminoguanidine failed to inhibit IL-1beta induced MT levels in insulinoma cells, and the nitric oxide generating agent sodium nitroprusside failed to significantly affect MT levels. Phorbol dibutyrate increased MT levels in rat islets and betaHC9 cells, but phorbol dibutyrate and IL-1beta effects were not additive. Transgenic MT-null and wild-type mouse islets had similar insulin contents, but basal and glucose-stimulated insulin release from MT-null islets were significantly lower than in wild-type islets. Blood glucose levels in MT-null mice were, however, slightly lower than those in wild-type mice. Thus, MT induction in pancreatic islets and beta-cells is regulated by cytokines and DEX, and protein kinase C activation may play a role. However, regulation of MT induction in mouse and rat islets differs. MT also appears to modulate insulin release from pancreatic islets.     

Plasma and cellular retinoid-binding proteins and transthyretin (prealbumin) are all localized in the islets of Langerhans in the rat.

The immunohistochemical localization of plasma retinol-binding protein (RBP), cellular retinol-binding protein (CRBP), and transthyretin (TTR) was studied in rat pancreas. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. Specific immunostaining for each of these three proteins was found localized to the islets of Langerhans. Both RBP and CRBP were localized in cells that were peripherally distributed within the islets, with an anatomic distribution that resembled that of the glucagon-containing A cells. Immunoreactive TTR was localized in cells that were more centrally distributed in the islets, with an anatomic distribution that resembled that of the insulin-containing B cells. These findings were confirmed by radioimmunoassay of a homogenate of isolated rat islets. By using sensitive and specific radioimmunoassays for each antigen, unusually high levels of CRBP, RBP, TTR, and cellular retinoic acid-binding protein (CRABP) were found in rat islets. The physiological significance of the localization of RBP, CRBP, CRABP, and TTR in the islets is not known. The findings suggest that retinoids and their binding proteins may play important metabolic roles within islet cells, and hence that they may be involved in some way in the biological, endocrine, function of the islets.     

Molecular and cellular heterogeneity in the Rheumatoid Arthritis synovium: Clinical correlates of synovitis

Rheumatoid Arthritis is characterized by autoimmune-mediated attack of the joint synovial lining resulting in destruction of bone and cartilage, and is a clinically and biologically heterogenous disease with respect to both course of disease and outcome to therapy. The current armamentarium of approved therapies does not result in complete clinical response in all patients. Improved techniques for imaging and performing biopsies on the rheumatoid synovium have facilitated multiple studies of the dysregulated cellular and molecular pathways in disease, and have provided evidence for a spectrum of pathogenic phenotypes across RA patients. These phenotypes are differentially affected by targeted therapies such as anti-TNFα and anti-CD20, and their presence prior to treatment impacts upon subsequent clinical outcomes. Ongoing histologic and molecular assessment of these synovial phenotypes through the implementation of routine synovial biopsy or using systemic biomarkers will improve targeting of therapies to specific patient subsets in both clinical trials and practice.     

Alteration of the Langerhans islets in pancreatic cancer patients.

An abnormal glucose metabolism occurs in up to 80% of pancreatic cancer patients shortly or a few months before the first clinical admission. Reasons for this abnormality are obscure. We investigated immunohistochemically the pattern of islets in 14 pancreatic cancer specimens and used 14 chronic pancreatitis samples and 10 normal pancreata as controls. To study the topographical relationship of these islets to the cancer, islets in four different arbitrary zones within and around the cancer were evaluated. Ten out of 14 cancer specimens showed a significant loss of beta cells (p < 0.005) and eight of them also showed a significant increase of alpha cells (p < 0.005), all of them from hyperglycemic patients. Most affected islets were found within zone 1 (intratumoral) and zone 2 (peritumoral), to a lesser extent in zone 3 (acini close to tumor) and none in zone 4 (acini remote from tumor). No comparable changes were found in chronic pancreatitis patients. The incidence of 72% with alteration of islets in our material correlates with the frequency of abnormal glucose levels in human pancreatic cancer patients. Our findings support the notion that islet cell abnormalities is likely caused by substances released from cancer cells.     

One thousand faces of Langerhans islets.

Studies with different strains of Syrian hamsters and Syrian golden hamsters have revealed the remarkable potential of islet cells to undergo orthotopic and heterotopic metaplasia. The most common spontaneous change included the development of hepatocytes in aged and malnourished hamsters. Of the many other alterations that occurred during carcinogenesis, most of the metaplastic changes originated within the islet periphery and progressed inside and outside the islets. The development of ductular structures within islets and their progression either to structures identical to human serous cystadenoma or to highly invasive adenocarcinomas were the most common alterations. The remarkably greater invasive potential of cancer cells arising within the islets contrasted sharply with the slow growth of the tumors developing within ducts (intraductal tumors). Studies in human tissue also showed development of malignant cells within islets, and, in some cases, transition of islet cells to malignant cells was suggested. The overall results, along with recent findings in other studies in cultured human and hamster islets, indicate the enormous potential of islet cells to differentiate and undergo malignant transformation. Whether the metaplastic and malignant cells derive from stem cells embedded within islets or from transdifferentiated islet cells remains to be seen.     

Regulation of tubulin synthesis in islets of Langerhans.

Tubulin represents a major protein in rat pancreatic islets, which averages 0.5% of the total protein content and 6% of the noninsulin protein synthesized under basal metabolic conditions. Glucose increases the synthesis of tubulin twofold to threefold. Tubulin synthesis is also stimulated by adenosine 3':5'-cyclic monophosphate in both the absence and presence of glucose; this effect of adenosine 3':5'-cyclic monophosphate occurs preferential to noninsulin protein synthesis at physiological glucose concentrations. Tubulin synthesis was decreased more than 75% by fasting, an effect prevented by maintaining animals on glucose exclusively. The fasting-induced reduction in tubulin synthesis is corrected in vitro by increasing adenosine 3':5'-cyclic monophosphate levels. These findings parallel changes previously reported in insulin release and suggest that physiological agents can exert their insulin secretory action through an effect upon the rate of tubulin synthesis.     

Proinsulin processing in electrically permeabilized rat islets of Langerhans

Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3.5-7.4. Conversion was optimum at pH 4.5-5.5 and was reduced, but not abolished, at pH 7.4. Ca2+ (10 microM) and 4 beta-phorbol 12-myristate 13-acetate (500 nM), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.     

Transplantation of islets of Langerhans: new developments

The clinical results recently reported by the Edmonton group in recipients of allogeneic islet grafts, all of whom achieved at least temporary insulin independence, has rekindled interest in transplantation of islets of Langerhans as a means to cure diabetes. Long-term islet graft survival has been achieved in a non-human primate pre-clinical model with a protocol of T-cell signaling blockade using a new monoclonal antibody. Islet xenotransplantation (namely the use of animal islets, with the aim of transplanting them into humans), or stem cell technology (the controlled differentiation of stem cells to obtain specialised cells for the treatment of diabetes) are other procedures currently being evaluated in animal models. The recent clinical success suggests that, in the near future, diabetes might be treated by islet transplantation early in the clinical course of the disease before the development of complications, and without the risks associated with conventional immunosuppression.     

Reproducible high yields of rat islets of Langerhans

Isolation of islets of Langerhans from the pancreas by action of collagenase, a major breakthrough for physiological studies in vitro, has long appeared empirical, and the results were sometimes unpredictable. Isolation yields (number of islets obtained per pancreas) and their reproducibility, purification from exocrine remnants and vitality of the islets obtained, can improve owing to precise techniques, adapted to the architecture of collagen in the pancreas. We have tested four isolation-purification techniques in the rat pancreas. The best results were obtained by combining intra-ductal collagenase injection, with complete but moderate distension of the gland, avoiding leakage, multistep digestion (in situ and then in vitro), followed by purification on a discontinuous bovine serum albumin (BSA) gradient. Average yields were 670 +/- 40 islets per pancreas (range 570-800), versus 170 +/- 9 (range 40-350) with the technique used initially. The use of BSA discontinuous gradient improved the purification yield: 90-96% of islets obtained were concentrated in the 26/29% BSA interface. Furthermore, this technique shortened the duration of purification step: 1 hr (centrifugation gradient) vs 3 hrs (handpicked). It was verified that islets were morphologically free of exocrine tissue. Islet structure was well preserved either in conventional histology or insulin and glucagon immunoperoxidase staining. Islet vitality, as assessed by trypan blue exclusion test, was 100% of freshly isolated islets, and 89% after 24 hrs culture. Insulin secretory responses to a given stimulus were stronger than in the case of islets isolated by former techniques: 10-12 times the basal release (vs 5 times) with clear dose-response proportionality.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptides of the islets of Langerhans: a peptidomics study

Neuropeptides from the endocrine pancreas (the islets of Langerhans) play an important role in the regulation of blood glucose levels. Therefore, our aim is to identify the "peptidome" (the in vivo peptide profile at a certain time) of the pancreatic islets, which is beneficial for medical progress related to the treatment of diabetes. So far, there are few neuropeptides isolated and sequenced from the endocrine pancreas and mainly in situ hybridisation and immunocytochemical techniques have been used to demonstrate the occurrence of peptides in the pancreas. These techniques do not allow for unequivocal identification of peptides. In contrary, mass spectrometry identifies peptides unambiguously. We have analysed the peptidome of the islets using peptidomics, i.e. a combination of liquid chromatography, mass spectrometry and bioinformatics. We are able to identify the peptidome of islets extracts. We not only confirm the presence of peptides with a well-known effect on blood glucose levels, but also identify new peptides, which are unknown to affect blood glucose levels.     

Corticotropin-releasing factor: its action on the islets of Langerhans

Synthetic rat and ovine CRF were tested in an in vitro isolated pancreatic islet system for their ability to influence insulin and glucagon release. Acute exposure of islets to both rat and ovine CRF resulted in a significant increase in glucagon release but only over a narrow range of concentration (50-200 pg/ml). Neither peptide had a significant effect on insulin release. Our results raise the possibility that release of glucagon may be stimulated by CRF as part of the overall response to stress.