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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Considerable progress has been made in our understanding of islet-cell function and its relationship to regulation of whole body glucose metabolism. At the genetic level, the regulatory regions in islet-specific genes are being characterised. Transcription factors that interact with these regions have been cloned and these will be instructive in elucidating how islet-specific genes are regulated during development and regeneration. Identification of the enzymes responsible for proteolytic conversion of proinsulin to insulin represents a major advance in understanding prohormone processing. Cleavage of proinsulin is mediated by at least two prohormone convertases (PC3/PC1 and PC2). Their activity is regulated by an acidic gradient between the Golgi and secretory granules and by calcium ions. It is not yet clear how insulin or the PC's are specifically diverted into the regulated secretory pathway. Regulation at this step may be defective in some diabetic patients resulting in relatively elevated circulating proinsulin levels. Specific features of GLUT 2 and glucokinase (GK), proteins that regulate Beta-cell glucose transport and phosphorylation, indicate that these may be key components of the glucose sensor. GLUT 2 is necessary to reconstitute glucose-sensitive insulin secretion in pituitary tumour cells expressing a proinsulin cDNA. Furthermore, the expression of GLUT 2 in Beta cells, but not in hepatocytes, is decreased in diabetes mellitus. However, under normal circumstances GK is probably rate limiting for Beta-cell glucose utilisation. Thus, it is likely that both GLUT 2 and GK determine the set point for glucose-stimulated insulin secretion. Elucidation of distal effectors that regulate insulin secretion is also crucial to our understanding of Beta-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cellular and molecular biology of the beta cell. 147 77

The enzymology of proinsulin conversion was studied in COS cells by cotransfection of three species of proinsulin and each of three conversion endoproteases (furin, PC2, and PC3). In addition to the parts of basic residues linking the B-chain to C-peptide (Arg31-Arg32) and C-peptide to the A-chain (Lys64-Arg65), which were present in all three proinsulins studied, human proinsulin presents a P4 basic residue (four residues NH2-terminal to the point of cleavage) only at the former junction (Lys29) and rat proinsulin II only at the latter (Arg62). Human proinsulin Arg62 (prepared by site-directed mutagenesis of human proinsulin) contains a P4 basic residue at both junctions. Transfected cells were incubated for four successive 2-h periods. The media were pooled, and pro-insulin, conversion intermediates, and insulin were separated by reverse-phase high-performance liquid chromatography to monitor conversion activity. There was little conversion of any proinsulin in COS cells without cotransfection of an exogenous endoprotease. When furin or PC3 was cotransfected with any of the three proinsulins, there was extensive processing, with insulin as the major conversion product. PC2, by contrast, failed to cleave human proinsulin but was able to cleave both human proinsulin Arg62 and rat proinsulin II. Cleavage by PC2 of these proinsulins was predominantly at the C-peptide-A-chain junction, generating the conversion intermediate des-64,65-split proinsulin as the major product and only very small amounts of insulin itself.
Diabetes 1995 Sep
PMID:Processing of proinsulin by furin, PC2, and PC3 in (co) transfected COS (monkey kidney) cells. 765 31

Hyperproinsulinemia in non-insulin-dependent diabetes mellitus (NIDDM) is due to an increased release of proinsulin from pancreatic beta cells. This could reside in increased secretory demand placed on the beta cell by hyperglycemia or in the proinsulin conversion mechanism. In this study, biosynthesis of the proinsulin conversion enzymes (PC2, PC3, and carboxypeptidase-H [CP-H]) and proinsulin, were examined in islets isolated from 48-h infused rats with 50% (wt/vol) glucose (hyperglycemic, hyperinsulinemic, and increased pancreatic proinsulin to insulin ratio), 20% (wt/vol) glucose (normoglycemic but hyperinsulinemic), and 0.45% (wt/vol) saline (controls). A decrease in the islet content of PC2, PC3, and CP-H from hyperglycemic rats was observed. This reduction did not correlate with any deficiency in mRNA levels or biosynthesis of PC2, PC3, CP-H, or proinsulin. Furthermore, proinsulin conversion rate was comparable in islets from hyperglycemic and control rats. However, in islets from hyperglycemic rats an abnormal increased proportion of proinsulin was secreted, that was accompanied by an augmented release of PC2, PC3 and CP-H. Stimulation of the beta cell's secretory pathway by hyperglycemia, resulted in proinsulin being prematurely secreted from islets before its conversion could be completed. Thus, hyperproinsulinemia induced by chronic hyperglycemia likely results from increased beta cell secretory demand, rather than a defect in the proinsulin processing enzymes per se.
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PMID:Increased secretory demand rather than a defect in the proinsulin conversion mechanism causes hyperproinsulinemia in a glucose-infusion rat model of non-insulin-dependent diabetes mellitus. 788 51

Pancreatic beta-cell dysfunction is a characteristic of non-insulin-dependent diabetes mellitus (NIDDM). An aspect of this dysfunction is that an increased proportion of proinsulin is secreted, but an actual beta-cell defect that leads to hyperproinsulinemia is unknown. Nevertheless, an impairment in beta-cell proinsulin conversion mechanism has been suggested as the most likely cause. Insulin is produced from its precursor molecule, proinsulin, by limited proteolytic cleavage at two dibasic sequences (Arg31, Arg32 and Lys64, Arg65). Two endopeptidase activities catalyze this cleavage: PC2 and PC3. PC2 endopeptidase cleaves predominately at Lys64, Arg65, and PC3 endopeptidase cleaves at Arg31, Arg32. The recent identification and characterization of these endopeptidases has enabled a better understanding of the human proinsulin-processing mechanism. In particular, experimental evidence suggests that the majority of human proinsulin processing is sequential. PC3 cleaves proinsulin first to generate a proinsulin conversion intermediate that is the preferred substrate of PC2. Both PC2 and PC3 activities are influenced by Ca2+ and pH, but the more stringent Ca2+ and pH requirements of PC3 suggest it as the most likely enzyme to regulate proinsulin conversion, as well as initiate it. When an increased demand is placed on the proinsulin-processing mechanism by a glucose-stimulated increase in proinsulin biosynthesis, there is a coordinate increase in PC3 biosynthesis (but not in PC2). This supports PC3 as the key endopeptidase that regulates proinsulin processing. In this perspective, the current concepts of the enzymology and regulation of proinsulin conversion at a molecular level are reviewed.(ABSTRACT TRUNCATED AT 250 WORDS)
Diabetes 1994 Apr
PMID:What beta-cell defect could lead to hyperproinsulinemia in NIDDM? Some clues from recent advances made in understanding the proinsulin-processing mechanism. 813 54

In the short term (< 2 h), proinsulin biosynthesis is predominately glucose regulated at the translational level; however, the details at the molecular level behind this mechanism are not well defined. One of the major hindrances for gaining a better understanding of the proinsulin biosynthetic mechanism has been a lack of an abundant source of beta-cells that express a phenotype of regulated proinsulin biosynthesis in the appropriate 2.8-16.7 mmol/l glucose range as defined in normal pancreatic islets. In this study, we demonstrate that in the MIN6 cell line, specific glucose-regulated translational control of proinsulin biosynthesis is present in the appropriate glucose concentration range. In addition to that of proinsulin, the biosynthesis of the two proinsulin conversion endopeptidases, PC2 and PC3, was coordinately glucose regulated in MIN6 cells, whereas that of the exopeptidase, carboxypeptidase H, was unaffected by glucose. Proinsulin, PC2 and PC3 biosynthesis was specifically stimulated over that of total MIN6 cell protein synthesis above a threshold of 4 mmol/l glucose that reached a maximum rate between 8 and 10 mmol/l glucose. Glucose-induced proinsulin, PC2, and PC3 biosynthesis was rapid (occurring after a 20-min lag period but reaching a maximum by 60 min), unaffected by the presence of actinomycin D; and in parallel experiments, stimulatory glucose concentrations did not alter MIN6 cell total preproinsulin, PC2, or PC3 mRNA levels. Thus, short-term (< 2 h) glucose stimulation of proinsulin, PC2 and PC3 biosynthesis in MIN6 cells, like that in isolated islets, was mediated at the translational level. Intracellular signals for mediating glucose-stimulated proinsulin PC2 and PC3 biosynthesis translation in MIN6 cells also appeared to be similar to those in pancreatic islets, requiring glucose metabolism and a supporting role for protein kinase A. However, protein kinase C or a Ca(2+)-dependent protein kinase did not appear to be required for glucose-regulated proinsulin biosynthesis in MIN6 cells, as in islets. MIN6 cells are the first beta-cell line that indicate glucose-regulated proinsulin biosynthesis translation essentially identical to that in differentiated islet beta-cells and will be an important experimental model to better define the mechanism of proinsulin biosynthesis in detail.
Diabetes 1996 Jan
PMID:Glucose-regulated translational control of proinsulin biosynthesis with that of the proinsulin endopeptidases PC2 and PC3 in the insulin-producing MIN6 cell line. 852 57

Islet amyloid polypeptide (IAPP), 'amylin', is the component peptide of islet amyloid formed in Type 2 diabetes. IAPP is expressed in islet beta-cells and is derived from a larger precursor, proIAPP, by proteolysis. An in vitro translation/translocation system was used to separately examine processing of human proIAPP by the beta-cell endopeptidases PC2, PC3 or furin. ProIAPP was converted to mature IAPP by PC2 but there was little conversion by furin or PC3. These data are consistent with processing of proIAPP in beta-cell secretory granules. Abnormal cellular proteolysis associated with type 2 diabetes could contribute to IAPP amyloidosis.
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PMID:Processing of pro-islet amyloid polypeptide (proIAPP) by the prohormone convertase PC2. 855 6

The hormone insulin remains the cornerstone of diabetic therapy since it is required for almost all cases of Type 1 and many cases of Type 2 diabetes. Since the discovery of insulin in 1921, much has been learned about its chemistry, structure and action as well as its production in the beta cell. Insulin is formed through a series of precursors, beginning with preproinsulin, the protein encoded in the insulin gene. These precursors direct the prohormone into the secretory pathway and ultimately into the secretory granules where it is converted into insulin and C-peptide. These products are stored and secreted together in a highly regulated manner in response to glucose and other stimuli. This review focuses on the recently discovered prohormone convertases, PC2 and PC3 (PC1), the enzymes responsible for the endoproteolytic processing of proinsulin to insulin and C-peptide in the beta cell as well as for the selective processing of proglucagon to glucagon in the alpha cell or GLP1 in intestinal L-cells. PC2 and PC3 are calcium-dependent serine proteases related to the bacterial enzyme subtilisin. They cleave selectively at Lys-Arg or Arg-Arg sites in precursors, generating products with C-terminal basic residues that are then removed by carboxypeptidase E, an exopeptidase. All 3 enzymes are expressed mainly in secretory granules of neuroendocrine cells throughout the body and in the brain. Inherited defects affecting the prohormone-processing enzymes have recently been found in association with unusual syndromes of obesity and other metabolic disorders.
Diabetes Metab 1996 Apr
PMID:The role of prohormone convertases in insulin biosynthesis: evidence for inherited defects in their action in man and experimental animals. 879 89

Proinsulin conversion to insulin occurs in secretory granules of pancreatic beta-cells. This processing has been suggested to require both the endoproteases PC2 and PC3 with each cleaving at only one of the two sites linking the insulin A- and B-chains with C-peptide. To evaluate this in an appropriate cellular setting, conversion of human proinsulin was followed in GH3 (rat pituitary) cells normally unable to convert this prohormone but equipped with the regulated secretory pathway. For this purpose, human proinsulin was expressed in GH3 cells, alone or in combination with PC2 and/or PC3, using recombinant adenoviruses. Cells were infected with the given adenoviruses and 24 h later were pulse-chased. Kinetics of proinsulin conversion were monitored by reverse-phase high-performance liquid chromatography. It was observed that while the two endoproteases do display a preference for a single site of cleavage (PC2 at the A-chain/C-peptide junction; PC3 at the B-chain/C-peptide junction) and act in a synergistic manner to promote proinsulin conversion, either PC2 or PC3 alone can cleave at both sites to fully convert proinsulin to insulin. These results also show that a cell can be successfully infected by three different recombinant adenoviruses.
Diabetes 1997 Jun
PMID:Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3. 916 68

We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.
Diabetes 1997 Aug
PMID:Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells. 923 54

The production of insulin from proinsulin involves cleavage of intact proinsulin into proinsulin conversion intermediates by the processing of enzymes PC2 and PC3 before fully processed insulin is produced. Intact proinsulin and these conversion intermediates are measured in many immunoreactive insulin (IRI) assays, and therefore contribute to the absolute IRI measurement. The proportion of basal IRI made up of proinsulin (PI)-like molecules (PI/IRI) is increased in NIDDM. Whether stimulated IRI levels are similarly made up of disproportionately increased PI/IRI or whether the relative proportions of proinsulin and its conversion intermediates are altered has not been evaluated. An index of the efficiency of proinsulin processing within the pancreatic beta-cell can be achieved by measuring PI/IRI immediately following acute stimulation of beta-cell secretion, and then determining the proportion of intact proinsulin and proinsulin conversion intermediates contributing to circulating proinsulin-like molecules. In this study, we determined the PI/IRI levels under basal and arginine-stimulated conditions in 17 healthy and 16 NIDDM subjects; high-performance liquid chromatography (HPLC) was also performed in a subset of these subjects to measure the relative contribution of intact proinsulin and its conversion intermediates to total proinsulin-like molecules. In NIDDM subjects, levels of both basal (44.6 +/- 9.6 vs. 9.3 +/- 1.5 pmol/l; P = 0.0007) and stimulated (64.0 +/- 12.7 vs. 19.8 +/- 2.8 pmol/l; P = 0.001) proinsulin-like molecules were higher than in healthy subjects. Although IRI was higher in NIDDM than in control subjects under basal conditions (106 +/- 19 vs. 65.1 +/- 8.1 pmol/l; P = 0.05), it was lower in NIDDM than in control subjects following stimulation (increment: 257 +/- 46 vs. 416 +/- 51 pmol/l; P = 0.03). PI/IRI ratios were increased in NIDDM subjects under both basal (43.3 +/- 5.0 vs. 14.0 +/- 1.3%; P < 0.0001) and stimulated (increment: 10.1 +/- 2.1 vs. 2.5 +/- 0.2%; P = 0.0006) conditions, compatible with the release of a disproportionately increased amount of proinsulin-like products. HPLC analysis revealed that, in the stimulated state, intact proinsulin made up 40.1 +/- 6.7% of proinsulin-like molecules in NIDDM individuals (n = 9) and 30.1 +/- 5.6% in healthy subjects (n = 7; NS). The remainder of the proinsulin-like molecules comprised the des-31,32-split proinsulin conversion intermediate. The increase in PI/IRI in NIDDM under basal and especially under stimulated conditions suggests that proinsulin conversion is indeed perturbed in this disorder. Because the relative proportions of intact and des-31,32-split proinsulin are similar in both healthy and NIDDM subjects, the orderly cleavage of proinsulin at its two junctions appears preserved. However, at the time of exocytosis, the secretory granule in the islet of NIDDM subjects contains an increased proportion of incompletely processed proinsulin, presumably reflecting a slower rate of conversion or granules' reduced time of residence in beta-cells.
Diabetes 1997 Nov
PMID:Release of incompletely processed proinsulin is the cause of the disproportionate proinsulinemia of NIDDM. 935 18


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