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PubMed1984

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes.

The receptor binding characteristics and biologic protency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15 degrees C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 +/- 0.8 X 10(-7) M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 +/- 0.3 X 10(-9) M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37 degrees C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 +/- 0.4 X 10(-8) M, whereas the same response was observed at an insulin concentration of 5.2 +/- 1 X 10(-11) M. Proinsulin also demonstrated an antilipolytic potency that was less than 1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1996

Hyperinsulinemia in nondiabetic Asian subjects using specific assays for insulin, intact proinsulin, and des-31, 32-proinsulin.

OBJECTIVE: To investigate the contributions of intact proinsulin and of des-31,32-proinsulin to fasting concentrations of insulin-like molecules in nondiabetic subjects from two ethnic groups (Asian and white) and to see whether Asian subjects are hyperinsulinemic compared with white subjects using highly specific assays for insulin. RESEARCH DESIGN AND METHODS: We investigated subjects with normal glucose tolerance (NGT) (82 Asian and 67 white) and impaired glucose tolerance (IGT) (16 Asian and 13 white), diagnosed by using standard World Health Organization criteria. Highly specific monoclonal antibody-based assays were used to measure insulin, intact proinsulin, and des-31,32-proinsulin. An index of insulin secretion was derived as a ratio of incremental insulin to incremental glucose concentrations from 0 to 30 min during an oral glucose tolerance test. RESULTS: Asian subjects with NGT, despite being significantly thinner than whites (BMI 24.4 +/- 3.5 vs. 25.7 +/- 3.7 kg/m2, P = 0.04), had a more central distribution of obesity (subscapsular-to-triceps skinfold ratios 1.36 +/- 0.69 vs. 1.17 +/- 0.41, P = 0.047). Asian subjects with NGT showed significant hyperinsulinemia 2 h after oral glucose load (plasma insulin median 274 pmol/l [range 26-1,505] vs. 186 pmol/l [27-720], P < 0.005) compared with whites. Asian subjects with NGT also had significantly higher insulin increments (P < 0.02) compared with white subjects and significantly higher fasting concentrations of intact proinsulin (median 2.7 pmol/l [range 0.9-14.1] vs. 2.1 [0.8-7.9], P < 0.02) but not of des-31,32-proinsulin. The ratio of proinsulin-like molecules to the total sum of three insulin-like molecules, however, was similar between Asian and white subjects with NGT and IGT. CONCLUSIONS: These results indicate that when specific assays for insulin are used, Asian subjects show postglucose load hyperinsulinemia and fasting hyperproinsulinemia compared with white subjects, suggesting increased insulin secretion and/or the presence of underlying insulin resistance in this ethnic group. The contribution of proinsulin-like molecules to total insulin-like molecules was similar between Asian and white subjects with NGT and IGT, and there was no contribution to hyperinsulinemia in Asian subjects.

Adult↗

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↗

PubMed1996

Different binding and degradation of proinsulin, insulin and insulin-like growth factor-1 (IGF-1) in cultured renal proximal tubular cells. Implications for the prolonged serum half-life of proinsulin.

Recent studies have reported that elevated proinsulin levels are indicative of an increased cardiovascular risk. Renal proximal tubular cells represent a major site for the metabolism of insulin-like hormones after glomerular filtration into the tubular lumen. To determine the binding and degradation of proinsulin in comparison with insulin and insulin-like growth factor-1 (IGF-1), we have used a rabbit proximal tubular cell line (PT-1). As confirmed by electron microscopy. PT-1 cells exhibit bipolar differentiation, demonstrating apical microvilli and invaginations of the basolateral membrane. To allow selective incubation of both compartments, cells were grown on filter membranes. Performing equilibrium binding assays with 125I-labelled hormones, severalfold higher binding was found at the apical than at the basolateral cell membrane, with the capacity range IGF-1 > insulin > proinsulin. Half-maximal displacement of 125I-labelled insulin and IGF-1 was observed at 0.6 and 2 nM, respectively, while crossover binding to the alternate receptor occurred with a 10- to 100-fold lower affinity. Half-maximal displacement of 125I-proinsulin binding was obtained at approx. 8 nM proinsulin and insulin, whereas IGF-1 was 10-fold less potent. The relative degradation of specifically bound tracer was lowest for proinsulin (apical 10%, basolateral: 13%). IGF-1 was degraded by 20% at the apical cell membrane, and up to 78% at the basolateral membrane. In contrast, almost the total amount of insulin bound was degraded at both membrane sites (apical 99%, basolateral: 83%). These results suggest separate insulin and IGF-1 receptors while proinsulin binds with high affinity to a third insulin-like receptor on the apical membrane of PT-1 cells.

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↗

PubMed1995

The contribution of proinsulin and des-31,32 proinsulin to the hyperinsulinemia of diabetic and nondiabetic cirrhotic patients.

We used specific, monoclonal antibody-based, two-site immunoradiometric assays to test the hypothesis that serum levels of proinsulin and des-31,32 proinsulin would be increased in cirrhosis, particularly in those with overt diabetes. A 75-g oral glucose tolerance test was performed after an overnight fast in eight cirrhotic patients with diabetes (fasting blood glucose, 7.8 +/- 2.2 [SE] mmol/L), seven nondiabetic cirrhotic patients, and eight normal control subjects. Fasting serum immunoreactive insulin levels were approximately six times higher in cirrhotics than in controls, but were not different between diabetic and nondiabetic cirrhotic patients. After oral glucose, the incremental area under the serum insulin concentration curve was 3,475 +/- 1,009 pmol.L-1.h in nondiabetic cirrhotic patients, significantly higher than in controls (761 +/- 48, P < .001) or diabetic cirrhotic patients (881 +/- 186, P < .05). Fasting serum proinsulin levels in diabetic cirrhotic patients (24.0 +/- 5.7 pmol/L) were higher than in controls (2.3 +/- .05, P < .001) or nondiabetic cirrhotic patients (4.4 +/- 0.8, P < .005). Fasting serum levels of des-31,32 proinsulin were also much higher in diabetic cirrhotic patients than in nondiabetic cirrhotic patients or controls (P < .02 and P < .005, respectively). Fasting proinsulin plus des-31,32 proinsulin constituted 12.5% +/- 1.4% of serum immunoreactive insulin in diabetic cirrhotics, higher than in nondiabetic cirrhotics (3.7% +/- 0.5%, P < .001) and normal controls (7.8% +/- 1.5%, P = .035).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1996

Relation of cord plasma concentrations of proinsulin, 32-33 split proinsulin, insulin and C-peptide to placental weight and the baby's size and proportions at birth.

Small and disproportionate size at birth are associated with type 2 diabetes and coronary heart disease in adult life. Insulin has an important role in controlling growth in utero and we hypothesised that reduced fetal insulin secretion could be one factor underlying these associations. We therefore measured cord plasma concentrations of proinsulin, 32-33 split proinsulin, insulin and C-peptide in 391 babies born at term and related them to the weight of the placenta and to the babies' size and proportions at birth. Babies with a small placental weight and a lower birth weight had lower cord plasma concentrations of split proinsulin and insulin. Babies who were disproportionate, either having a high ratio of head to abdominal circumference or being thin, had lower concentrations of split proinsulin, split proinsulin and insulin. The relations with split proinsulin were especially strong, the geometric mean concentration (pmol/l) falling from 14.2 in babies with a head to abdominal circumference ratio of 101.6% or less to 7.2 in those with a ratio above 107.3% (P < 0.0001), and from 17.4 in babies with a ponderal index above 28.5 kg/m3 to 7.4 in those with a ponderal index of 25.5 kg/m3 or less (P < 0.0001). These findings support the hypothesis that reduced fetal insulin secretion may be one factor underlying the associations between reduced growth in utero and type 2 diabetes and coronary heart disease in adult life.

Anthropometry↗

PubMed1997

Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3.

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.

Adenoviridae↗

PubMed1998

Intact proinsulin, des 31,32 proinsulin, and specific insulin concentrations among nondiabetic and diabetic subjects in populations at varying risk of type 2 diabetes.

OBJECTIVE: To examine hyperinsulinemia, insulin secretion, and beta-cell function in Pima Indians, South Asians, and whites, populations at varying risk of diabetes. RESEARCH DESIGN AND METHODS: We investigated 136 Pima Indian, 98 Asian, and 80 white nondiabetic and 172 Pima Indian, 40 Asian, and 49 white diabetic subjects. Highly specific assays for insulin, intact proinsulin, and des 31,32 proinsulin were used. Insulin secretion was assessed using ratio of increment (0 to 30 min) in insulin to glucose concentrations during an oral glucose tolerance test (OGTT). RESULTS: Nondiabetic Pima Indians were significantly more obese than Asians and whites. Pima Indian subjects had significantly higher (P < 0.01) fasting insulin concentrations (median 109 pmol/l, range 40-250) than Asian (37 pmol/l, range 17-91) and white (30 pmol/l, range 10-82) subjects. These differences remained significant when controlled for obesity. Nondiabetic Pima Indians also had higher fasting C-peptide concentrations and higher early insulin secretion during an OGTT. Fasting concentrations of intact proinsulin and des 31,32 proinsulin were also significantly higher in Pima Indians (P < 0.01). However, the proportion of proinsulin-like molecules was significantly lower (P < 0.01) in Pima Indians (median 7.9% vs. 12.7% for South Asians and 12.2% for whites). Subjects with diabetes from the three ethnic groups showed significantly higher fasting insulin concentrations but lower 30-min insulin and lower ratios of increment (0-30 min) in insulin to glucose concentrations than did nondiabetic subjects. The proportion of proinsulin-like molecules was not significantly different in diabetic subjects from the three ethnic groups. CONCLUSIONS: These specific assays for insulin indicate that after adjusting for obesity nondiabetic Pima Indians are truly hyperinsulinemic, which is consistent with their insulin resistance as measured by other methods. Hyperinsulinemia in this population with a high risk of diabetes is likely to be due to enhanced insulin secretion. Furthermore, in Pima Indians, the predominant beta-cell secretory product is insulin and not its precursors. We conclude that the differences in the risk of diabetes among these three groups are not due to differences in insulin secretion or insulin processing. Subjects with type 2 diabetes have defective early insulin secretion during OGTTs but show fasting hyperinsulinemia even when specific assays for insulin are used.

Adult↗

PubMed1980

[Semisyntheses using proinsulin, II. Preparation and application of Nepsilon 29, Nepsilon 59-bis(methylsulfonylethoxycarbonyl)proinsulin (author's transl)].

Nepsilon 29, Nepsilon 59-Bis(methylsulfonylethoxycarbonyl)proinsulin was prepared from native beef proinsulin for its possible use in the semisynthesis of preproinsulin. The modification was made possible by first protecting the alpha-amino group of proinsulin either with the tert-butoxycarbonyl group, after reaction with bis(tert-butoxycarbonyl)oxide, or with the citraconyl group, after reaction with citraconic anhydride. Whereas Nalpha 1-tert-butoxycarbonylproinsulin was achieved by direct alkoxycarbonylation, Nalpha 1-citraconylproinsulin was obtained from partial deprotection of the quantitatively acylated proinsulin. The Nalpha 1-protected proinsulin derivatives were further quantitatively reacted with N-(methylsulphonylethoxycarbonyloxy)succinimide. The tert-butoxycarbonyl group was next removed by treatment with trifluoroacetic acid, or the citraconyl group removed by mild acid (pH 2.2) treatment. The resulting Nepsilon 29, Nepsilon 59-bis(methylsulphonylethoxycarbonyl)proinsulin was used in model reactions with the mixed anhydride of tert-butoxycarbonylmethionine with the aim of standardizing the coupling conditions to be used in the eventual synthesis of preproinsulin.

Animals↗

PubMed1969

A specific anti-proinsulin serum and the presence of proinsulin in calf serum.

A specific antibody against proinsulin has been obtained by adsorbing the original anti-proinsulin guinea pig serum with a solid immunosorbent of Sephadex-insulin. The specificity of the antibody against antigenic determinants of proinsulin was established by radioimmunoassay and by passive cutaneous anaphylaxis (PCA) tests. The presence of proinsulin in calf serum has been demonstrated by (a) gel filtration of an acid alcohol extract of the serum followed by immunoassays of the fractions using anti-insulin or the specific anti-proinsulin serum, and (b) direct assay of calf serum with the specific anti-proinsulin serum.

Animals↗

PubMed2005

Proinsulin levels and the proinsulin:c-peptide ratio complement autoantibody measurement for predicting type 1 diabetes.

AIMS/HYPOTHESIS: We investigated whether random proinsulin levels and proinsulin:C-peptide ratio (PI:C) complement immune and genetic markers for identifying relatives at high risk of type 1 diabetes. MATERIALS AND METHODS: During an initial sampling, random glycaemia, proinsulin, PI:C and HLA DQ genotype were determined in 561 non-diabetic first-degree relatives who had been positive for islet autoantibodies on one or more occasions and in 561 age- and sex-matched persistently antibody-negative relatives. RESULTS: During follow-up (median 62 months), 46 relatives with antibodies at entry developed type 1 diabetes. At baseline, antibody-positive relatives (n=338) had higher PI:C values (p<0.001) than antibody-negative subjects with (n=223) or subjects without (n=561) later seroconversion. Proinsulin and PI:C were graded according to risk of diabetes as expressed by positivity for (multiple) antibodies or IA-2 antibodies, especially in persons carrying the high-risk HLA DQ2/DQ8 genotype and in prediabetic relatives. In the presence of multiple or IA-2 antibodies, a PI:C ratio exceeding percentile 66 of all antibody-negative relatives at entry (n=784) conferred a 5-year diabetes risk of 50% and 68%, respectively (p<0.001 vs 13% for same antibody status with PI:C<percentile 66). Cox regression analysis confirmed random PI:C as an independent predictor of the risk of diabetes (p< or =0.001). CONCLUSIONS/INTERPRETATION: Random proinsulin and PI:C represent dynamic markers of the state of beta cell function that complement immune markers in identifying relatives who are at homogeneously high risk of contracting type 1 diabetes and are therefore eligible for secondary prevention trials.

Adult↗

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↗

PubMed2000

Proinsulin cDNAs from the leopard frog, Rana pipiens: evolution of proinsulin processing.

We have isolated a proinsulin cDNA from the Amphibian Rana pipiens. The predicted R. pipiens insulin A- and B-chain amino acid sequences differ from that deduced from the closely related Rana catesbeiana at one residue (Asp for Pro at B2). The R. pipiens and Xenopus laevis proinsulin precursor sequences are of identical length, with the amino acid sequences of the mature A- and B-chains being well conserved. The proinsulin C-peptide amino acid sequence is less well conserved between R. pipiens and X. laevis and also differs in length. The R. pipiens C-peptide is shorter than the homologous X. laevis sequence due to a two amino acid residue truncation. The truncation of the R. pipiens C-peptide compensates for a two amino acid residue extension observed at the N-terminal of the A-chains of insulins from Ranid frogs. A change in the site of proinsulin processing can explain both the C-peptide and A-chain length differences. The evolution of the new proinsulin processing site required two amino acid substitutions.

Amino Acid Sequence↗

PubMed1980

Human proinsulin, VIII: studies on the S-tritylation of reduced proinsulin, insulin A and B chains and their detritylation.

Reduced proinsulin and insulin A and B chains were selectively and quantitatively converted to hexa(S-trityl)proinsulin, tetra(S-trityl)A chain and di(S-trityl)B chain. These derivatives were used as models to investigate the quantitative removal of S-trityl groups. Amongst the various detritylation methods studied, the best procedure was found to be acidolytic cleavage with trifluoroacetic acid in the presence of thiophenol or benzylmercaptan as cation scavengers. The detritylated derivatives, upon oxidative sulphitolysis, yielded electrophoretically homogeneous hexa(S-sulphonate)proinsulin, tetra(S-sulphonate)A chain and di(S-sulphonate)B chain. The hexa(S-sulphonate)proinsulin was reduced and reoxidized to proinsulin.

Animals↗

PubMed2002

Effects of carboxypeptidase E overexpression on insulin mRNA levels, regulated insulin secretion, and proinsulin processing of pituitary GH3 cells transfected with a furin-cleavable human proinsulin cDNA.

We recently developed two rat pituitary GH3 cell clones engineered to secrete human insulin (InsGH3). InsGH3 cells convert proinsulin into mature insulin, which is partially stored into a readily releasable pool of secretory granules. The efficiency of these processes, however, is relatively low in these cells, either in vitro or in vivo. This study was aimed at determining whether carboxypeptidase E (Cpe) overexpression can increase proinsulin processing and regulated secretion by InsGH3 clones. Indeed, in its membrane-bound form Cpe works as sorting receptor for the regulated secretory pathway of many hormones while, in its soluble form, Cpe takes part to the late step of insulin maturation. We obtained two Cpe-overexpressing cell lines from two different InsGH3 clones (InsGH3/C1 and C7). In the Cpe-overexpressing cell lines, derived from InsGH3 of clone 1 (InsGH3/C1-HACpe), in which the membrane-bound form of exogenous Cpe is accounted for by 90% of total Cpe immunoreactivity, we observed an increase in proinsulin gene expression, and in basal and stimulated insulin secretion compared with the original clone. In contrast, in the Cpe-overexpressing cell line derived from InsGH3 of clone 7 (InsGH3/C7-HACpe), where the exogenous membrane-bound form was only 60% of total Cpe, we detected a decrease in basal insulin release and a modest, albeit significant, increase in intracellular proinsulin processing. In conclusion, Cpe overexpression can increase regulated insulin secretion and proinsulin processing in InsGH3 cells; however, such improvements appear quantitatively and qualitatively modest.

Animals↗

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↗