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.

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No description available.

The effect of Biosynthetic Human Proinsulin on glucose uptake by skeletal muscle was studied in the isolated perfused hindquarter of fasted rats. Animals were randomly assigned to the control group, insulin-perfused or proinsulin-perfused group. Glucose disappearance from the perfusate and muscle glycogen levels before and after 2 hours perfusion were measured. Perfusate glucose concentration showed the greatest decline in the insulin group, which was significantly lower (p < .01) than control from 60 to 120 min. Proinsulin perfusion resulted in a smaller and delayed decrease in perfusate glucose. The proinsulin perfusate glucose levels were significantly higher (p < .05) than the insulin glucose values during the second hour of perfusion. After the first hour of perfusion, insulin infusion resulted in higher rates of glucose uptake than control (p < .005) or proinsulin infusion (p < .05). The glucose uptake by muscles perfused with proinsulin was significantly different from control values only at the 2 hour time point (p < .05). Glycogen concentration following insulin infusion increased significantly in the oxidative muscles, i.e. soleus (p < .05) and red vastus (p < .002). These increases in glycogen were significantly different from the changes observed in muscles of control animals. The plantaris and white vastus muscles, which have fast twitch fibers, did not show a significant response to insulin. Proinsulin perfusion decreased glycogen levels regardless of the muscle type. This decline was significantly different from the glycogen changes in soleus (p < .025), plantaris (p < .001) and white vastus (p < .05) muscles of control animals. The proinsulin glycogen fall was also significantly different from the insulin induced response in soleus, plantaris and red vastus muscles (p < .001). These results show that proinsulin has 8.6 % of the biologic potency of insulin for glucose uptake in rat skeletal muscle. Insulin induced an increase in glycogen concentration in oxidative muscles, but proinsulin elicted a drop in glycogen level regardless of the muscle type.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Hyperproinsulinemia is associated with type 2 diabetes (T2DM) and obesity and is a predictor for future coronary heart disease. This thesis examines the effect of bariatric surgery on glucometabolic status including insulin and proinsulin responses after meal. Further we explored longitudinally the effects of bariatric surgery on glucose, insulin and proinsulin secretion as well as lipids, liver enzymes and magnesium concentrations. We explored by a standardised meal test the postprandial dynamics of proinsulin and insulin and effects on glucose and lipids in patients treated with gastric bypass (RYGBP) surgery and in patients treated with bileopancreatic diversion with duodenal switch surgery (BPD-DS). Comparisons were made to morbidly obese patients and normal weight controls (NW). RYGBP surgery markedly lowers fasting and postprandial proinsulin concentrations although BMI was higher compared to NW-controls. BPD-DS surgery induces a large weight loss and normalises postprandial responses of glucose, proinsulin and insulin and markedly lowers triglycerides. We evaluated non-diabetic morbidly obese patients who underwent bariatric surgery followed-up for up to four years after surgery. Long-term follow-up showed that RYGBP surgery is not only characterized by markedly and sustained lowered BMI but also lowered concentrations of proinsulin, insulin, ALT and increased HDL-C possibly via reduced hepatic insulin resistance. We also examined how magnesium status is affected by bariatric surgery as magnesium has been shown to be inversely related to glucose and to insulin resistance. The serum magnesium concentrations increased by 6% after RYGBP and 10% after BPD-DS. In summary, RYGBP and BPD-DS surgery results in marked weight loss, alterations in insulin and proinsulin dynamics, lowered fasting and postprandial proinsulin concentrations and improved glucometabolic and magnesium status.

The finding that more than 152 tissue-restricted antigens are expressed by thymic medullary epithelial cells is redefining the importance of thymic central tolerance induction in the prevention of autoimmune diseases. One of the tissue-restricted antigens in the thymus is proinsulin, and in both mice and humans, reduced thymic proinsulin levels have been shown to predispose to Type 1 diabetes. Using transgenic mice expressing a functional beta-Galactosidase gene under the regulation of the Ins2 promoter we have determined that between 1-3% of all medullary thymic epithelial cells express proinsulin and that these cells are frequently part of the Hassall's Corpuscles like structures in mice. Using a cross between the beta-Galactosidase expressing mice and Immortomice (expressing SV40 large T Antigen under the regulation of the MHC I promoter), we have isolated and cultured two proinsulin and two non-proinsulin producing medullary epithelial cell lines. Microarray analysis and RT-PCR analysis of the cell lines revealed the over-expression of approximately 50 genes (>4 fold or more) in the proinsulin producing lineage, versus the non proinsulin producing lineage, and approximately half the over-expressed genes can be considered tissue-restricted antigens. We do not find any evidence for chromosomal clustering of the over-expressed genes nor do we report the expression of any other pancreatic n-cell antigens or specific pancreatic proinsulin regulatory proteins (Pdx-1, Glut-2 or GCK) within the proinsulin producing cell lines but we do detect their expression in whole thymus. Our results suggest that chromosomal clustering is not a phenomenon associated with thymic tissue-restricted antigen expression and that the mechanisms allowing for thymic tissue-restricted antigen expression are not related to the expression mechanisms of such antigens in peripheral tissues.

The two non-allelic preproinsulin genes of the mouse have been cloned and their nucleotide sequences determined. The mouse preproinsulin I gene, like its rat counterpart, has only one intron. Homology between the two mouse genes extends in the 5' direction to about position -500. Homology 3' of the coding sequence terminates shortly after the polyadenylation signal with a dA rich region found in gene I. The coding sequences of the two genes have been compared. The deduced amino acid sequences of the mature hormones are identical to the published protein sequences and to the corresponding sequences of rat insulins I and II. The prepeptides of mouse insulin I and II differ at six positions. However, they maintain hydrophobic cores that are required for transport of the nacent peptide across microsomal membranes. The B-peptide of mouse insulin I differs from insulin II at two positions: at position B9 a proline has replaced a serine, and at position B29 a lysine has replaced a methionine, compared to the sequence of insulin II. The A-peptides of the two hormones are identical. The C-peptide of mouse proinsulin I has a deletion eliminating amino acids C17 (Gly) and C18 (Ala) compared to the sequence of proinsulin II. The presence of this deletion in mature RNA was confirmed through an S1 nuclease assay. The transcriptional start sites for the preproinsulin genes were determined with S1 and Mung Bean nuclease assays, and with a primer extension assay. The data indicate that transcription of the mouse preproinsulin genes starts 6 bp 5' of the site reported for the rat II gene. Single-stranded DNA probes were used to determine the structure of the 3' ends of the preproinsulin mRNAs. Hybridization conditions were used which only allowed each probe to detect its cognate mRNA. Digestion of the resulting DNA-RNA duplex molecules with S1 nuclease followed by gel electrophoresis demonstrated that transcription of mouse preproinsulin I mRNA terminates 18 bases after the polyadenylation signal. Transcription of preproinsulin II mRNA terminates 43 bases after the polyadenylation signal, thus extending 25 bases past the last point of homology between the two genes. The 3' end-specific probes were used in experiments designed to determine the ratio of preproinsulin I and II mRNA in pancreatic extracts of normal, fasted and fasted and refed mice. In all cases the amount of preproinsulin I mRNA exceeded preproinsulin II by about 2.3:1. These results were extended to include an analysis of preproinsulin mRNA from freshly isolated islets and islets incubated for 48 hours in the presence of 2.8 mM or 16.7 mM glucose. With both high and low glucose concentrations, the amount of preproinsulin I mRNA exceeded preproinsulin II by about 2.3:1. The mouse islets were also incubated with 3H-leucine and the ratio of insulin I and II determined after fractionation by HPLC. Unlike the mRNA results, the level of insulin II, within the islets and secreted into the media, exceeded insulin I by about 2:1 under all conditions. The available preproinsulin prepeptide amino acid sequences have been compared. The sequence of mouse I prepeptide differs from most other insulin prepeptides at amino acid position 4. At that position a tryptophan residue that is conserved in most insulin prepeptides has been replaced by a leucine in the mouse I prepeptide. This change causes a shift in the hydropathy profile in that region of the mouse insulin I prepeptide making it more hydrophobic. Every other insulin prepeptide is relatively hydrophilic at that position. This difference is postulated to interfere with signal recognition particle mediated regulation of translation and/or transport of nacent mouse preproinsulin I to microsomal membranes, and nay account for the discordant mRNA-peptide ratios. The structure of the insulin genes in a number of myomorph rodents has been examined. The data indicate that only members of the sub-family Murinae have two insulin genes.

No description available.