Islet isolation assessment in man and large animals

Recent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity and in vitro and in vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 μ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason, in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers. © 1990 Casa Editrice il Ponte

Expression of neurogenin3 reveals an islet cell precursor population in the pancreas

Differentiation of early gut endoderm cells into the endocrine cells forming the pancreatic islets of Langerhans depends on a cascade of gene activation events controlled by transcription factors including the basic helix-loop-helix (bHLH) proteins. To delineate this cascade, we began by establishing the position of neurogenin3, a bHLH factor found in the pancreas during fetal development. We detect neurogenin3 immunoreactivity transiently in scattered ductal cells in the fetal mouse pancreas, peaking at embryonic day 15.5. Although not detected in cells expressing islet hormones or the islet transcription factors Isl1, Brn4, Pax6 or PDX1, neurogenin3 is detected along with early islet differentiation factors Nkx6.1 and Nkx2.2, establishing that it is expressed in immature cells in the islet lineage. Analysis of transcription factor-deficient mice demonstrates that neurogenin3 expression is not dependent on neuroD1/BETA2, Mash1, Nkx2.2, Nkx6.1, or Pax6. Furthermore, early expression of neurogenin3 under control of the Pdx1 promoter is alone sufficient to drive early and ectopic differentiation of islet cells, a capability shared by the pancreatic bHLH factor, neuroD1/BETA2, but not by the muscle bHLH factor, MyoD. However, the islet cells produced in these transgenic experiments are overwhelmingly α cells, suggesting that factors other than the bHLH factors are required to deviate from a default α cell fate. These data support a model in which neurogenin3 acts upstream of other islet differentiation factors, initiating the differentiation of endocrine cells, but switching off prior to final differentiation. The ability to uniquely identify islet cell precursors by neurogenin3 expression allows us to determine the position of other islet transcription factors in the differentiation cascade and to propose a map for the islet cell differentiation pathway

Abnormal infant islet morphology precedes insulin resistance in PCOS-like monkeys.

Polycystic ovary syndrome (PCOS) is prevalent in reproductive-aged women and confounded by metabolic morbidities, including insulin resistance and type 2 diabetes. Although the etiology of PCOS is undefined, contribution of prenatal androgen (PA) exposure has been proposed in a rhesus monkey model as premenopausal PA female adults have PCOS-like phenotypes in addition to insulin resistance and decreased glucose tolerance. PA female infants exhibit relative hyperinsulinemia, suggesting prenatal sequelae of androgen excess on glucose metabolism and an antecedent to future metabolic disease. We assessed consequences of PA exposure on pancreatic islet morphology to identify evidence of programming on islet development. Islet counts and size were quantified and correlated with data from intravenous glucose tolerance tests (ivGTT) obtained from dams and their offspring. Average islet size was decreased in PA female infants along with corresponding increases in islet number, while islet fractional area was preserved. Infants also demonstrated an increase in both the proliferation marker Ki67 within islets and the beta to alpha cell ratio suggestive of enhanced beta cell expansion. PA adult females have reduced proportion of small islets without changes in proliferative or apoptotic markers, or in beta to alpha cell ratios. Together, these data suggest in utero androgen excess combined with mild maternal glucose intolerance alter infant and adult islet morphology, implicating deviant islet development. Marked infant, but subtle adult, morphological differences provide evidence of islet post-natal plasticity in adapting to changing physiologic demands: from insulin sensitivity and relative hypersecretion to insulin resistance and diminished insulin response to glucose in the mature PCOS-like phenotype

Human monoclonal islet specific autoantibodies share features of islet cell and 64 kDa antibodies

The first human monoclonal islet cell antibodies of the IgG class (MICA 1-6) obtained from an individual with Type 1 (insulin-dependent) diabetes mellitus were cytoplasmic islet cell antibodies selected by the indirect immunofluorescence test on pancreas sections. Surprisingly, they all recognized the 64 kDa autoantigen glutamate decarboxylase. In this study we investigated which typical features of cytoplasmic islet cell antibodies are represented by these monoclonals. We show by double immunofluorescence testing that MICA 1-6 stain pancreatic beta cells which is in agreement with the beta-cell specific expression of glutamate decarboxylase. In contrast an islet-reactive IgM monoclonal antibody obtained from a pre-diabetic individual stained all islet cells but lacked the tissue specificity of MICA 1-6 and must therefore be considered as a polyreactive IgM-antibody. We further demonstrate that MICA 1-6 revealed typical features of epitope sensitivity to biochemical treatment of the target tissue which has been demonstrated for islet cell antibodies, and which has been used to argue for a lipid rather than a protein nature of target antigens. Our results provide direct evidence that the epitopes recognized by the MICA are destroyed by methanol/chloroform treatment but reveal a high stability to Pronase digestion compared to proinsulin epitopes. Conformational protein epitopes in glutamate decarboxylase therefore show a sensitivity to biochemical treatment of sections such as ganglioside epitopes. MICA 1-6 share typical features of islet cell and 64 kDa antibodies and reveal that glutamate decarboxylase-reactive islet cell antibodies represent a subgroup of islet cell antibodies present in islet cell antibody-positive sera

The Diabetic Dog as a Translational Model for Human Islet Transplantation.

The dog model has served as the primary method for early development of many diabetes therapies, including pancreatic islet transplantation techniques and immunosuppressive protocols. Recent trends towards the use of monoclonal antibody therapies for immunosuppression in human islet transplantation have led to the increasing use of primate models with induced diabetes. In addition to induced-disease models in large animals, scientists in many fields are considering the use of naturally-occurring disease models in client-owned pets. This article will review the applicability of naturally-occurring diabetes in dogs as a translational model for developing islet transplantation in the human diabetic patient

Cyclic AMP signalling in pancreatic islets

Cyclic 3'5'AMP (cAMP) is an important physiological amplifier of glucose-induced insulin secretion by the pancreatic islet β-cell, where it is formed by the activity of adenylyl cyclases, which are stimulated by glucose, through elevation in intracellular calcium concentrations, and by the incretin hormones (GLP-1 and GIP). cAMP is rapidly degraded in the pancreatic islet β-cell by various cyclic nucleotide phosphodiesterase (PDE) enzymes. Many steps involved in glucose-induced insulin secretion are modulated by cAMP, which is also important in regulating pancreatic islet β-cell differentiation, growth and survival. This chapter discusses the formation, destruction and actions of cAMP in the islets with particular emphasis on the β-cell