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At least 19 recordsLinked to original sources
PubMed1992

Preferential cleavage of des-31,32-proinsulin over intact proinsulin by the insulin secretory granule type II endopeptidase. Implication of a favored route for prohormone processing.

Two Ca(2+)-dependent endopeptidase activities are involved in proinsulin to insulin conversion: type I cleaves COOH-terminal to proinsulin Arg31-Arg32 (B-chain/C-peptide junction); and type II preferentially cleaves at the Lys64-Arg65 site (C-peptide/A-chain junction). To further understand the mechanism of proinsulin processing, we have investigated types I and II endopeptidase processing of intact proinsulin in parallel to that of the conversion intermediates, des-31,32-proinsulin and des-64,65-proinsulin. The type I processed des-64,65-proinsulin and proinsulin at the same rate. In contrast, the type II endopeptidase processed des-31,32-proinsulin at a much faster rate (> 19-fold; p < 0.001) than it did intact proinsulin. Furthermore, unlabeled proinsulin concentrations required for competitive inhibition of 125I-labeled des-64,65-proinsulin and 125I-proinsulin processing by a purified insulin secretory granule lysate were similar (ID50 = 14-16 microM), whereas inhibition of 125I-labeled des-31,32-proinsulin processing required a higher nonradiolabeled proinsulin concentration (ID50 = 197 microM). Synthetic peptides corresponding to the sequences surrounding Lys64-Arg65 (AC-peptide/substrate) and Arg31-Arg32 (BC-peptide/substrate) of human proinsulin were synthesized for use as specific substrates or competitive inhibitors. Cleavage of the BC-substrate by type I and AC-substrate by type II was COOH-terminal of the dibasic sequence, with similar Ca(2+)-and pH requirements previously observed for proinsulin cleavage. Apparent Km and Vmax for type I processing of the BC-substrate was Km = 20 microM; Vmax = 22.8 pmol/min, and for type II processing of the AC-substrate was Km = 68 microM; Vmax = 97 pmol/min. In competitive inhibition assays, the BC-peptide similarly blocked insulin secretory granule lysate processing of des-64,65-proinsulin and proinsulin (ID50 = 45-55 microM), but did not inhibit des-31,32-proinsulin processing. However, the AC-peptide preferentially inhibited insulin secretory granule lysate processing of des-31,32-proinsulin (ID50 = microM) compared to proinsulin (ID50 = 330 microM), and not des-64,65-proinsulin. We conclude that the type I endopeptidase recognized des-64,65-proinsulin and proinsulin as similar substrates, whereas the type II endopeptidase has a stronger preference for des-31,32-proinsulin compared to intact proinsulin. Furthermore, we suggest that in intact proinsulin there exists a constraint to efficient processing that is relieved following type I processing. Structural flexibility, in addition to the presence of Lys64-Arg65, therefore appears to be important for type II endopeptidase specificity and may provide a molecular basis for a preferential route of proinsulin conversion via des-31,32-proinsulin.

Amino Acid Sequence↗
PubMed1989

Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split and 32-33 split proinsulins.

Monoclonal antibody-based two-site immunoradiometric assays are described for human insulin, proinsulin, 65-66 split and 32-33 split proinsulin. The detection limits of the assays lie in the range 0.8-2.5 pM. The assays for 65-66 and 32-33 split proinsulins do not distinguish between these substances and their respective C-terminal di-desamino derivatives. The assay of 65-66 split proinsulin does not cross-react with insulin, proinsulin or 32-33 split proinsulin. This material was undetectable (less than 1.0 pM) in plasma taken after an overnight fast in eight normal male subjects and the maximum individual concentration reached in plasma taken during an oral glucose tolerance test of these subjects was 3.8 pM. The proinsulin assay cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. The 32-33 split proinsulin assay cross-reacted 84 and 60% with proinsulin and 65-66 split proinsulin respectively. The insulin assay cross-reacted 5.3, 62 and 5.0% with intact proinsulin, 65-66 split proinsulin and 32-33 split proinsulin respectively. The very low concentration of 65-66 split proinsulin meant that this derivative did not interfere significantly with the specificity of the assays of proinsulin and insulin. The concentration of 32-33 split proinsulin could be calculated by subtracting the cross-reactivity of the measured proinsulin. The mean concentrations of insulin, proinsulin and 32-33 split proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 0.3, 2.3 +/- 0.3 and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4 and 19.7 +/- 6.0 respectively.

Adult↗
PubMed1989

Immunoradiometric assay of human proinsulin and partially processed proinsulin with use of monoclonal antibody and streptavidin-biotin labeling.

The sensitive and specific immunoradiometric assay is described for human proinsulin and its intermediate peptides (65-66 split and 32-33 split proinsulin). We developed a monoclonal antibody-based two-site immunoradiometric assay with use of streptavidin-biotin labeling. The detection limits of the assays lie in the range of 0.5-2.0 pM. In the proinsulin assay proinsulin cross-reacted 66% with 65-66 split proinsulin but not with insulin or 32-33 split proinsulin. In the assay of 65-66 split proinsulin it does not cross-react with insulin, proinsulin or 32-33 split proinsulin. In the 32-33 split proinsulin assay it cross-reacted 84% with proinsulin and 60% with 65-66 split proinsulin. The precision (C.V.) of the assays was less than 15% over the various concentration. The mean concentrations of insulin, proinsulin, 65-66 split proinsulin and 32-33 proinsulin in eight young male subjects in the fasting state were (pM +/- S.E.M.) 20 +/- 3.6, 2.3 +/- 0.3, undetectable (less than 1.0) and 2.1 +/- 0.7 and at the maximum reached during an oral glucose tolerance test, 150 +/- 26, 9.9 +/- 1.4, 3.8 +/- 0.6 and 19.7 +/- 6.0 respectively.

Adult↗
PubMed1985

Measurement of circulating human proinsulin concentrations using a proinsulin-specific antiserum.

Antibodies have been raised against biosynthetic human proinsulin that show less than 1% cross-reactivity with human insulin and C-peptide. A sensitive (IC50 0.16 pmol/ml; minimum detectable concentration 0.004 pmol/ml) radioimmunoassay has been developed using this antiserum and 125I-proinsulin that will measure proinsulin-like immunoreactivity in human serum without the need for prior separation of insulin or C-peptide. In healthy, fasted subjects (N = 23), the serum proinsulin concentration was 0.015 +/- 0.001 pmol/ml (mean +/- SEM). In six healthy subjects, serum proinsulin rose to 250% of basal after 120 min in response to 100 g oral carbohydrate, but to only 130% after 60 min following 25 g oral carbohydrate. The proinsulin/total immunoreactive insulin ratio and the proinsulin/C-peptide ratio fell sharply after both high and low carbohydrate loads. Endogenous human serum proinsulin-like immunoreactivity released into the circulation after 100 g carbohydrate was eluted from a Mono Q high-performance, ion-exchange column with the same retention time as biosynthetic human proinsulin. Treatment of biosynthetic proinsulin with trypsin under mild conditions led to a decrease in proinsulin-like immunoreactivity concomitant with an increase in C-peptide and insulin-like immunoreactivity, indicating that the proinsulin-specific antiserum did not preferentially recognize intermediates of proinsulin cleavage.

Adult↗
PubMed1986

The effects of proinsulin pretreatment on the combined actions of insulin and proinsulin in normal man.

With the recent availability of biosynthetic human proinsulin there has been a renewed interest in evaluating its metabolic effects, either alone or in combination with insulin. It has been suggested that pretreatment with proinsulin enhances the hypoglycemic response to subsequently administered insulin. On the other hand, the simultaneous administration of proinsulin and insulin has additive, not synergistic, effects. To clarify this question we used the euglycemic glucose clamp technique in 10 normal subjects to compare the steady state effects on glucose disposal of combined infusions of insulin (0.54 microgram/M2 . min, equivalent to 15 mU/M2 . min) and proinsulin (2.75 micrograms/M2 . min) given both simultaneously and sequentially. The mean +/- SEM steady state glucose disposal rates were similar whether the two hormones were given simultaneously (7.2 +/- 0.7 mg/min . kg), after proinsulin pretreatment (7.7 +/- 0.7 mg/min . kg), or after insulin pretreatment (7.1 +/- 0.7 mg/min . kg). The serum proinsulin concentration of 5.39 +/- 0.3 pmol/ml during the infusion of proinsulin alone was unchanged by the simultaneous infusion of insulin, suggesting that in the doses used, insulin did not affect proinsulin clearance. We conclude that in normal subjects there is no enhancement of the combined action of insulin and proinsulin to stimulate glucose disposal by pretreatment with proinsulin or insulin.

Adult↗
PubMed1988

Binding of proinsulin and proinsulin conversion intermediates to human placental insulin-like growth factor I receptors.

Insulin-like growth factor I (IGF-I) and proinsulin share similarities in both primary and tertiary structure. Proinsulin, endogenously secreted or exogenously administered, would, therefore, be expected to interact with IGF-I receptors. We determined the relative activities of IGF-I, insulin, proinsulin, and the proinsulin conversion intermediates in IGF-I radioreceptor assays using term human placental membranes. Insulin was approximately 0.5% as potent as IGF-I, and proinsulin was only 2% as potent as insulin. The six major proinsulin conversion intermediates were studied; all had activities intermediate between those of insulin and proinsulin. We conclude that the binding of proinsulin and the proinsulin conversion intermediates to IGF-I receptors is not of physiological significance at the concentrations occurring endogenously or after exogenous administration of proinsulin.

Binding, Competitive↗
PubMed1991

Differential rates of conversion of rat proinsulins I and II. Evidence for slow cleavage at the B-chain/C-peptide junction of proinsulin II.

Rat proinsulin I is converted into insulin more rapidly than is proinsulin II. To study this further, rat islets were labelled (10 min) and conversion kinetics of the labelled proinsulins were monitored during a 120 min chase. Proinsulins, conversion intermediates and both insulins were separated by h.p.l.c. The accumulation of des-64,65-(split proinsulin II) during the chase suggests that the B-chain/C-peptide junction of proinsulin II is cleaved more slowly than the equivalent site on proinsulin I. This accounts for the differential kinetics of conversion of proinsulins I and II, and is presumed to be caused by one (or more) of the amino acid replacements which distinguish the two proinsulins.

Animals↗
PubMed1988

Biosynthetic human proinsulin, a new therapeutic compound for diabetics? A comparative study of biosynthetic human proinsulin with biosynthetic human insulin.

The biological activities of proinsulin, the endogenous precursor of insulin are different in comparison to insulin. Proinsulin has a longer biological half-life in serum and a much lower hypoglycemic potency. Biosynthetic human proinsulin has been suggested as an intermediate acting "insulin" in the treatment of diabetes mellitus, having the advantage of not including a retardation substance. For this reason it is important to establish a dose relationship in comparison to human insulin. Human proinsulin and human insulin were tested in 2 groups of healthy volunteers after i.v. application on a unit equivalent basis. 0.025, 0.05 and 0.1 units/kg of either compound were applied intravenously in a randomised order. Following human proinsulin application, the nadir of blood glucose showed a delay of 5-10 min. in comparison to insulin, however, the increase of blood glucose following the nadir was much more retarded after human proinsulin application. The antilipolytic effect of proinsulin was significantly stronger in comparison to insulin. No significant differences could be observed in the kinetics of beta-hydroxybutyrate levels and the counterregulatory response. Human proinsulin, based on a unit equivalent dosage, exerts a more pronounced hypoglycemic and antilipolytic effect, which is probably due to the longer biological half-life in comparison to insulin.

3-Hydroxybutyric Acid↗
PubMed1978

Measurement of serum proinsulin-like material: cross-reactivity of porcine and human proinsulin in the insulin radioimmunoassay.

The variation in cross-reactivity of human and porcine insulins and proinsulins was determined with a number of insulin antisera. The relative immunoreactivity of insulin and proinsulin with these antisera ranged from a ratio of 1.08 to 5.7 on a molar basis. The displacement curves of human insulin and proinsulin were not parallel, and the relationship of porcine and human proinsulin varied with different antisera. Sera with varying concentrations of PLM (proinsulin and its intermediate components) and serum PLM and insulin fractions (separated by gel filtration) were measured in two assays using the antisera which reacted most differently with human insulin and proinsulin standards. With the use of these two antisera, measurement of serum PLM against a human insulin standard gave different results, which were only partially corrected by reading the values from a human proinsulin standard. The magnitude of the serum IRI differences with these antisera was related to the proinsulin/insulin ratio in each sample. These results indicate the necessity for each laboratory to critically evaluate the reaction of their insulin antiserum with human proinsulin. In addition, measurement of serum PLM in terms of a insulin standard will give different results depending upon the particular antiserum used.

Animals↗
PubMed1992

Immunoradiometric assay of insulin, intact proinsulin and 32-33 split proinsulin and radioimmunoassay of insulin in diet-treated type 2 (non-insulin-dependent) diabetic subjects.

Plasma insulin, intact proinsulin and 32-33 split proinsulin measured by specific immunoradiometric assays and insulin and C-peptide measured by radioimmunoassay were measured during a constant infusion of glucose test in ten diet-treated subjects with a history of Type 2 (non-insulin-dependent) diabetes (termed diabetic subjects), mean fasting plasma glucose 6.0 +/- 1.0 mmol/l (mean +/- SD), and 12 non-diabetic control subjects. Immunoreactive insulin concentrations measured by radioimmunoassay were 33% higher than insulin and 16% higher than the sum of insulin and its precursors by immunoradiometric assay. The diabetic and non-diabetic subjects had similar fasting concentrations of insulin, intact proinsulin and 32-33 split proinsulin. The ratio of fasting intact proinsulin to total insulin was greater in the diabetic than the non-diabetic group 12.0% (6.8-21.0%, 1 SD range) and 6.3% (4.0-9.8%), respectively, p less than 0.01), though the groups overlapped substantially. After glucose infusion, diabetic and non-diabetic subjects had similar intact proinsulin concentrations (geometric mean 4.9 and 5.2 pmol/l, respectively), but the diabetic group had impaired insulin secretion by immunoradiometric assay (geometric means 55 and 101 pmol/l, p less than 0.05) or by radioimmunoassay C-peptide (geometric means 935 and 1410 pmol/l, p less than 0.05), though not by radioimmunoassay insulin (87 and 144 pmol/l, p = 0.12), respectively. Individual immunoradiometric assay insulin responses to glucose expressed in terms of obesity were subnormal in nine of ten diabetic subjects. Radioimmunoassay insulin and C-peptide gave less complete discrimination (subnormal responses in six of ten and eight of ten, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Glucose↗
PubMed1991

Separation and quantitation of serum proinsulin and proinsulin intermediates in humans.

Two reversed-phase high-performance liquid chromatographic (RP-HPLC) systems were developed for the separation of human insulin, proinsulin and the major proinsulin intermediates. The individual components were quantified using two enzyme-linked immunosorbent assays for insulin and proinsulin immunore-active material (PIM) after (passive) evaporation of the organic modifier. Serum samples from normal subjects and patients with non-insulin-dependent diabetes mellitus were immunopurified and analysed in one of the RP-HPLC systems. The proportion of PIM relative to insulin immunoreactive material was higher in the diabetic patient compared with that in the normal subject. In both, PIM was heterogeneous, consisting of intact proinsulin and des-proinsulin intermediates.

Chromatography, High Pressure Liquid↗
PubMed1989

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells.

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.

Amino Acid Sequence↗
PubMed1977

[Human proinsulin. C-peptide radioimmunoassay method. 125I labeling of human proinsulin. C-petide].

125I-labelled human-C-peptide was prepared by chloramin T method, enzymic method and active ester method, respectively. Using respective 125I-labelled human-C-peptides in human proinsulin-C-peptide RIA, we compared the binding (Bo/T%) to antibody, displacement by standard human-C-peptide, the recovery test and stability. The usable 125I-labelled antigen for human proinsulin-C-peptide RIA could be prepared by chloramin T method and enzymic method wich labelled 125I to tyrosyl human proinsulin connecting peptide, and active ester method which conjugates 125I-labelled active ester to human proinsulin connecting peptide. The differences among those 125I-labelled antigens was not observed in displacement (B/Bo%) by standard human-C-peptide and the recovery test. In the case of constant preparation of 125I-labelled antigen for RIA, the enzymic method was the best from the viewpoint the reaction ratio is stable and stability of Bo/T% is good.

C-Peptide↗
PubMed1990

The relationships of concentrations of insulin, intact proinsulin and 32-33 split proinsulin with cardiovascular risk factors in type 2 (non-insulin-dependent) diabetic subjects.

Standard radioimmunoassay for insulin may substantially overestimate levels of insulin because of cross-reaction with other insulin-like molecules. We have measured concentrations of insulin, intact proinsulin and 32-33 split proinsulin using two-site monoclonal antibody based immunoradiometric assays, and of insulin by a standard radioimmunoassay ("immunoreactive insulin") in 51 Type 2 (non-insulin-dependent) diabetic subjects in the fasting state. The relationships of these concentrations were sought with those of total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglyceride, plasminogen activator inhibitor, blood pressure, and indices of body fat distribution. Significant relationships were apparent between concentrations of "immunoreactive insulin" as measured by standard radioimmunoassay and triglyceride (rs = 0.42, p less than 0.001), total cholesterol (rs = 0.25, p = 0.038), high density lipoprotein cholesterol (rs = -0.30, p = 0.018) and body mass index (rs = 0.30, p = 0.017), but only the relationships with triglyceride (rs = 0.36, p = 0.006) and body mass index (rs = 0.26, p = 0.34) remained significant when concentrations of immunoradiometrically measured insulin were employed. Concentrations of 32-33 split proinsulin, which comprises the major insulin-like molecule in these subjects, correlated positively with triglyceride (rs = 0.33, p = 0.009), total cholesterol (rs = 0.23, p = 0.050), and plasminogen activator inhibitor (rs = 0.26, p = 0.049), and negatively with high density lipoprotein cholesterol (rs = -0.29, p = 0.021).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells.

Glucagon-like peptide-I(7-37) [GLP-I(7-37)] is an intestinal peptide hormone that is released in response to oral nutrients and that potently augments glucose-mediated insulin secretion. GLP-I(7-37) has potent insulin-releasing activities in vivo in response to oral nutrients, in situ in the isolated perfused pancreas, and in vitro in cultured pancreatic B-cells. As such GLP-I(7-37) is a potent hormonal mediator in the enteroinsular axis involved in the regulation of glucose homeostasis. We now show that in addition to stimulating the release of insulin, GLP-I(7-37) stimulates proinsulin gene expression at the levels of gene transcription and cellular levels of proinsulin messenger RNA as well as the translational biosynthesis of proinsulin. These findings of the positive anabolic actions of GLP-I(7-37) on the synthesis of insulin in B-cells support the notion that GLP-I(7-37) may be of therapeutic use in stimulating the production of insulin in patients with noninsulin-dependent diabetes mellitus and that overproduction of insulin with subsequent hypoglycemia will not occur in response to the administration of GLP-I(7-37). Furthermore, these positive actions of GLP-I(7-37) on insulin production obviate the possibility of B-cell exhaustion in response to such a potent secretagogue.

Animals↗
PubMed1989

Proinsulin endopeptidase substrate specificities defined by site-directed mutagenesis of proinsulin.

Two endopeptidases are involved in the conversion of proinsulin; a type I activity directed at the B chain, Arg31,Arg32, C-peptide junction, and type II which cleaves the C-peptide, Lys64,Arg65, A chain junction. To define further the substrate specificities of these enzymes, a series of mutant preproinsulin cDNAs were generated by site-directed and deletion mutagenesis. These were inserted into pT7 plasmids and capped cRNA transcripts synthesized, that were then microinjected into Xenopus oocytes. Oocytes were biosynthetically radiolabeled with [3H]leucine and the secreted peptides (greater than 95% present as unprocessed proinsulins) then incubated with types I and II endopeptidase activities prepared from isolated insulinoma secretory granules. The reaction products were analyzed by high performance liquid chromatography. Des-38-62-proinsulin, in which all but six amino acids of C-peptide were deleted was not processed by either enzyme. The mutant Lys64,Arg65 to Thr64,Arg65 was not cleaved by the type II enzyme but was still a substrate for the type I enzyme. The mutant Arg31,Arg32 to Arg31,Gly32 correspondingly was not cleaved by the type I enzyme; however, in this case it was not attacked by the type II enzyme. These results indicate that not only is the presence of a dibasic sequence essential, but also that the secondary structure of the protein is important in determining whether the prohormone is susceptible to proteolytic processing.

Animals↗