Insulin, the principal secretory product of the /3-ce31s of the islets of Langerhans, occupies a central role in mammalian metabolism. The biosynthesis of this hormone involves two distinct proteolytic steps. Initially the N-terminal "leader" sequence is cleaved from preproinsulin (the primary translation product of the insulin gene) by a signal peptidase present in the membrane of the endoplasmic reticulum. Subsequently the product, proinsulin, is transported via the Golgi to nascent secretory granules, and is converted to insulin by the excision of the connectiong (C) peptide. This thesis describes a study of the enzymes involved in the conversion of proinsulin to insulin. Chapter 1 comprises a review of the literature relating to the biosynthesis of insulin in the pancreatic /?-cell, and of post-translational proteolysis in other secretory tissues. The tissue source for the majority of the experiments conducted in the present study was a transplantable rat isnulinoma. In chapter 2 the biosynthesis of insulin in this tissue is examined. It is demonstrated that the conversion of proinsulin to insulin follows a molecular pathway indistinguishable from that of pancreatic islet cells. This chapter also examines the subcellular location of acidic "carboxypeptidase B-like" enzymes in the tumour. Previously such an activity has been identified in insulinoma secretory granules. The present study identifies 2 activities capable of hy-drolysing a synthetic carboxypeptidase B substrate which are distinguished on the basis of subcellular location and inhibitor profiles. One activity is localized to lysosomes and the other identified as carboxypeptidase H (EC 3.4.17.10), and shown to be a component of the insulin secretory granule. Chapter 3 describes a procedure for the purification of the insulinoma caboxypeplidase H, and demonstrates that the purified enzyme is capable of converting the putative proinsulin conversion intermediates [seco 32/33]-proinsulin and [seco 65/66]-proinsulin to their respective desdibasic forms, and diarginyl insulin to insulin. In chapter 4 the subcellular and tissue distribution of immunoreactive carboxypeptidase H in the rat is described. Immunoreactive species were detected in the a and /?- cells of the islets of Langerhans, and in the pituitary. However immunoreactivity was not demonstrated in the adrenal medulla, although this tissue does contain enzymatic activity and was the tissue used for the purification of this enzyme by other workers. The possibility that multiple forms of the enzyme are present is discussed with regard to this and other data. Chapter 4 also describes the results of a study of the biosynthesis of carboxypeptidase H in insulinoma cells. It is shown that the enzyme is initially synthesized as a precursor of apparent molecular weight 56000 which is subsequently converted to the mature 54000 molecular weight form. It is proposed that the observed change in molecular weight results from post-translational modification of the enzyme's N-linked oligosaccharide chains. The results presented in chapter 5 demonstrate the presence of a novel Ca2+ -dependent acidic endopeptidase in insulin secretory granules which, in conjunction with carboxypeptidase H, is capable of converting proinsulin to insulin in vitro. The relationship of this activity to the enzymes previously implicated in proinsulin processing, and the compatibility of its molecular properties with the intragranular environment, are discussed. In chapter 6 procedures for the partial purification of the proinsulin processing endopeptidase are described. It is shown that there are actually two endopeptidases which each cleave proinsulin at one of the two processing sites. Preliminary characterization of these enzymes is presented, and the implications of these observations on granule biogenesis discussed. Chapter 7 comprises a general discussion of the work presented in this thesis. Attention is focussed on the contributions made by the present study to our understanding of secretory granule biogenesis, and on possible methods which might enable the successful purification of the proinsulin endopeptidases.

Type 1 diabetes (T1D) is a chronic autoimmune disease targeted against the pancreatic β-cells. Proinflammatory cytokines are considered to play a major role in the destruction of the insulin-producing β-cells. This thesis studied strategies to counteract proinflammatory cytokines in experimental T1D. Both animal models for T1D as well as β-cell preparations exposed in vitro to putative noxious conditions were examined. In the first study we observed that cytokine treatment of mouse pancreatic islets lacking inducible nitric oxide synthase (iNOS) induced a prolongation of the early stimulatory phase of glucose stimulated insulin secretion. Various experiments led to the conclusion that this prolonged stimulatory effect may involve the DAG/PLD/PKC pathway. Next, we transplanted mouse islets deficient in iNOS to spontaneously diabetic NOD mice. We observed a normalization of hyperglycemia but not a delayed allograft rejection compared to transplanted wild type islets. Thus, absence of iNOS in the graft was not sufficient to prolong allograft survival. In paper III we found that sustained glucose stimulation of rat pancreatic islets was coupled to a decreased conversion of proinsulin to insulin. Islet treatment with IL-1β was also coupled to a decreased proinsulin conversion. Islet proconvertase activity may be a target in islet damage. In paper IV prolactin (PRL) was administered to mice in the multiple low dose streptozotocin model and we observed that PRL enhanced a Th2 response. This may contribute to the protective action by PRL in this model of autoimmune T1D. Finally, by examining β-cells overexpressing Suppressor of cytokine signalling 3 (SOCS-3) it was found that this could inhibit IL-1β induced signalling through the NF-κB and MAPK pathways. SOCS-3 overexpression also inhibited apoptosis induced by cytokines in primary β-cells. Lastly, we demonstrated that SOCS-3 transgenic islets were protected in an allogeneic transplantation model.