Proinsulin Research Articles

Plasma leptin is not associated with insulin resistance and proinsulin in non‐diabetic South Asian Indians

In an earlier study, we observed only a weak association between plasma insulin (non-specific assay) and leptin in South Asian Indians. This was in contrast to the observations in many other ethnic groups. With the availability of measurements of specific insulin (SI) and proinsulin (PI) in the same study group, we have reanalysed the data to look for possible correlation of leptin with proinsulin and with insulin resistance calculated from the fasting values of specific insulin and glucose using the HOMA model. Subjects with normoglycaemia (n = 117) and impaired glucose tolerance (n = 27, WHO criteria) were included in the analysis. Leptin values were higher in women. Multiple linear regression analysis showed that the variations in leptin concentrations in men were associated with BMI, WHR, and 2 h SI values (R2 = 56.2 %) while fasting SI and proinsulin concentrations had no significant association. In women BMI and age showed a significant association with serum leptin values (R2 = 40.1 %). Univariate and multivariate analyses using insulin resistance as the dependent variable showed that it had no association with leptin in both genders. Leptin had no correlation with proinsulin also. This study confirmed that in Asian Indians the association between plasma leptin and insulin concentrations is weak and that leptin has no influence on insulin resistance. Proinsulin and leptin are also not correlated in this population. Insulin resistance shows correlation with the β-cell function both in men and women. © 1998 John Wiley & Sons, Ltd.

Dissimilar association of conventional immuno-reactive versus specific insulin with cardiovascular risk factors: a consequence of proinsulinaemia?

In this study involving 365 non-diabetic elderly Caucasians, we examined the relationship of immuno-specific insulin (ISI), total immuno-reactive insulin (IRI), proinsulin (PI) and proinsulin–insulin ratio (PI:ISI) to serum high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), systolic blood pressure (SBP) and diastolic blood pressure (DBP), mean blood pressure (MBP) and pulse pressure. In a multiple regression analysis, adjusted for age, sex and obesity, a 1.3-fold stronger inverse association with HDL-C levels was found for IRI than for ISI, with a 1.6-fold better fit of the regression equation. The positive association of insulin with TG was 1.6-fold stronger for IRI compared to ISI, with a 2.5-fold better fit. In contrast, the positive association of IRI with the various blood pressure parameters was 1.5–1.9-fold weaker than for ISI, with a 2.1–3.8-fold worse fit. Both PI:ISI ratio and PI were independently associated with TG levels, but not with HDL-C. The PI:ISI ratio, but not PI, was associated with blood pressure, but dependent on glycaemia. In conclusion, compared to ISI, IRI overestimates the association of insulin with serum lipids and underestimates the association of insulin with blood pressure. The use of non-specific insulin assays may explain the inconsistencies in the findings of previous epidemiological studies.

T cell epitopes of insulin defined in HLA-DR4 transgenic mice are derived from preproinsulin and proinsulin.

Approximately one-half of Caucasians with newly diagnosed insulin-dependent diabetes mellitus (IDDM) have autoantibodies to insulin, and the majority of those express the HLA-DR4 genotype [Ziegler, R., Alper, C. A., Awdeh, Z. L., Castano, L., Brink, S. J., Soeldner, J. S., Jackson, R. A. & Eisenbarth, G. S. (1991) Diabetes 40, 709-714]. However, it has been difficult to demonstrate T cell proliferative responses to human insulin in IDDM patients [Durinovic-Bello, I., Hummel, M. & Ziegler, A. G. (1996) Diabetes 45, 795-800]. We have immunized transgenic mice expressing the susceptible HLA-DR (alpha1*0101,beta1*0401) (hereafter called DRB1*0401) and human CD4 molecules on a murine major histocompatibility complex class II null background, with human preproinsulin (PPI), proinsulin (PI), and insulin and derived large panels of T cell hybridomas to determine the immunogenic epitopes of these proteins. These results show that the prohormones PI or PPI carry the major immunogenic T cell epitope in the DRB1*0401 transgenic mice. The PPI/PI immunodominant epitope LALEGSLQK was localized at the C-peptide/A-chain junction. This T cell epitope PPI/PI LALEGSLQK is unusual because, normally, it is proteolytically destroyed during the maturation of the insulin molecule. Additionally, this T cell epitope is both processed and presented by human DRB1*0401-positive Epstein-Barr virus transformed B cells, and it can also stimulate T cells from the peripheral blood of HLA-DR4-positive patients with type 1 diabetes. These findings may partly explain why susceptibility to type 1 diabetes is associated with HLA-DR4-positive individuals and why T cell responses to the mature insulin protein are rarely detected in IDDM patients.

Specific insulin and proinsulin concentrations in nondiabetic South Indians

The study was performed to determine plasma levels of proinsulin (PI) and specific insulin (SI) in normoglycemic (NGT) Asian Indians and to assess the effect of obesity and impaired glucose tolerance (IGT) on these concentrations. Blood samples from 151 adult nondiabetic South Indian subjects were collected during an epidemiological survey of diabetes. Plasma SI and PI levels were measured in fasting and 30-minute and 120-minute samples of a glucose tolerance test (World Health Organization criteria) using monospecific antibodies. The total insulin (TI) level was also measured by the nonspecific assay. The molar ratio of PI to SI ( PI SI ) was calculated. Correlations of the peptides with anthropometry, serum lipids, and blood pressure (BP) were studied by univariate and multivariate analyses. Comparisons were also made in NGT versus IGT groups. As expected, TI values were higher than SI values, but the patterns of response were similar for both. SI and PI responses in NGT were similar to the values found in Mexican-Americans who had a higher body mass index (BMI). Asian Indians were thus found to have a high SI response despite a low BMI. Obesity and IGT produced an increased response of both PI and SI, with normal PI SI ratios thus showing an absence of hyperproinsulinemia in either condition. Fasting PI showed a strong association with serum triglycerides, and proinsulin at 120 minutes was associated with cholesterol. None of the peptides showed a correlation with BP. Using specific assays for insulin and PI, it is shown that Asian Indians with NGT have a hyperinsulinemic response despite a low BMI. Obesity and mild hyperglycemia in IGT produce a simultaneous increase in PI and SI with no alteration in the PI SI ratio.

Disproportionately elevated proinsulin levels reflect the degree of impaired B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus.

An increased proportion of fasting proinsulin (PI) relative to immunoreactive insulin (IRI; increased PI/IRI) occurs in noninsulin-dependent diabetes mellitus (NIDDM). To determine whether the magnitude of the increase in PI/IRI is an indicator of the degree of reduced B cell secretory capacity, we measured fasting plasma glucose, PI, IRI, and PI/IRI and related them to maximal B cell secretory capacity (AIRmax) in 9 subjects with NIDDM [age, 61 +/- 3 yr; body mass index (BMI), 27.5 +/- 1.3 kg/m2; duration of NIDDM, 10.8 +/- 1.8 yr; mean +/- SEM] and in 10 healthy subjects matched for age and BMI (age, 61 +/- 6 yr; BMI, 27.9 +/- 1.5 kg/m2). AIRmax was quantified as the incremental insulin response to i.v. arginine at maximal glycemic potentiation (plasma glucose > 25 mmol/L). Mean fasting plasma glucose was 13.7 +/- 1.4 mmol/L (range, 7.5-18.3 mmol/L) in NIDDM subjects and 5.0 +/- 0.1 mmol/L in the controls. Fasting PI was higher in NIDDM (33.1 +/- 5.2) than in controls (9.4 +/- 2.5 pmol/L; P < 0.01), but IRI levels were similar (93.4 +/- 10.9 vs. 82.8 +/- 23.4 pmol/L; P = NS). The PI/IRI ratio was significantly elevated in NIDDM compared to control subjects (35.9 +/- 4.1% vs. 12.8 +/- 0.8%; P < 0.01). After elevation of the glucose level to 30.3 +/- 0.4 mmol/L (NIDDM) and 30.3 +/- 0.5 mmol/L (controls), AIRmax was quantified as 622 +/- 71 pmol/L in NIDDM and 1997 +/- 315 pmol/L in controls, (P < 0.001). The PI/IRI ratio correlated inversely with AIRmax in the NIDDM patients (r = -0.76; P < 0.01). We conclude that the magnitude of the elevation in fasting PI/IRI is related to the reduction in AIRmax. Thus, the fasting PI/IRI ratio appears to be a marker of the degree of reduced AIRmax in NIDDM.

Genetic engineering of glucose-stimulated insulin secretion in Chinese hamster ovary cells.

To engineer an a non-islet cell capable of glucose-stimulated insulin secretion, a chinese hamster ovary cell line (CHO) was transfected with a mammalian expression vector carrying the human insulin cDNA (pCB/hINS). More proinsulin than insulin was released daily by the stably transformed cell line (CHO-INS). Examination of acid-ethanol extracts confirmed that both insulin and proinsulin were stored. Immunohistochemical analysis of the cells also showed that (pro)insulin was stored. Unlike beta cells, CHO-INS cells did not secrete insulin in response to glucose. To investigate this lack of effect, we examined whether transfection of GLUT2 cDNA, which is ordinarily not expressed in CHO-INS cells, would confer glucose-stimulated insulin secretion. Consequently, we have demonstrated that glucose regulated insulin release occurs in the CHO-INS-GLUT2 cell line and that glucose potentiates the insulin secretory response to non-glucose secretagogues.

Release of incompletely processed proinsulin is the cause of the disproportionate proinsulinemia of NIDDM.

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.

Release of incompletely processed proinsulin is the cause of the disproportionate proinsulinemia of NIDDM

Release of incompletely processed proinsulin is the cause of the disproportionate proinsulinemia of NIDDM

Is insulin or its precursor independently associated with hypertension? An epidemiological study in Japanese-Brazilians.

Japanese individuals living outside Japan are more susceptible to chronic diseases included in the insulin resistance syndrome. Hyperinsulinemia and hypertension are associated, but large studies adjusting for confounders are still required. The present evaluated if insulin (I) or proinsulin (PI) was associated with hypertension after adjustment for other risk factors, in first (n=238) and second (n=292) generation Japanese-Brazilians, aged 40 to 79 years, living in a developed city in Brazil. Blood pressure (BP) was measured by random-zero sphygmomanometry. People with mean systolic/diastolic BP >140/90 mm Hg or taking antihypertensive drugs were considered hypertensive. Diagnosis of diabetes was based on results of an oral glucose tolerance test using WHO criteria. I and PI after fasting and 2 hours after glucose load were determined by specific immunofluorimetric assays. The first generation was older than the second (65.6+/-9.2 versus 53.6+/-8.4 years, P<.01) and male/female ratios were 1.14 and 0.87, respectively. The age-adjusted prevalence of hypertension was 29.2% with no difference between sexes or generations. Higher body mass index (25.2+/-4.3 versus 23.8+/-3.3 kg/m2), waist-to-hip ratio (0.939+/-0.067 versus 0.919+/-0.073), plasma glucose (6.3+/-2.3 versus 5.6+/-1.8 mmol/L), cholesterol (5.74+/-1.19 versus 5.48+/-1.08 mmol/L), and creatinine (74+/-26 versus 83+/-36 micromol/L) were found among the hypertensives (P<.05). Univariate analyses showed associations of obesity, diabetes, and dyslipidemia with hypertension. Logistic regression analyses demonstrated that 2-hour I (OR, 1.22; 95% CI, 1.02 to 1.46) and fasting PI (OR, 1.14; 95% CI, 1.00 to 1.31) remained significantly associated with hypertension, after adjustment for age, sex, generation, family history of hypertension, smoking habits, waist-to-hip ratio, serum creatinine, glucose intolerance, and dyslipidemia. Japanese-Brazilians have a higher prevalence of hypertension than the general population in Brazil. High levels of 2-hour I, seen in hypertensives, may be interpreted as independent risk factors for hypertension in this population. Our findings suggest that fasting PI should be useful, in addition to insulin, to assess risk factors for hypertension in epidemiological studies.

High proinsulin levels in late PRE-IDDM stage

Aim: To analyse the fasting proinsulin levels in first degree relatives of patients with insulin-dependent diabetes mellitus (IDDM) with different risk for developing the disease. Patients and methods: Non siblings first degree relatives, 33, of IDDM patients were separated into three groups with different risk for developing IDDM: Group 1, 14 first degree relatives (eight male/six female), aged from 18 to 57 years, normal first phase insulin release (FPIR) in the intravenous glucose tolerance test, negative ICA; Group 2, 11 first degree relatives (six male/five female), aged from 16 to 62 years, normal FPIR and ICA < 20 JDF U; Group 3, eight first degree (six male/two female), from 16 to 52 years, FPIR diminished and ICA > 20 JDF U. All patients had normal oral glucose tolerance test at the initiation of the study. We tested fasting proinsulin (PRO) and insulin (IRI) levels by radioimmunoassay (RIA) and the PRO IRI ratio. Results: Four first degree from the group 3 developed IDDM after 2–32 months. No differences were observed in fasting PRO levels and PRO IRI ratio between the groups. However, the PRO (21.7 ± 5.8 pmol/1) and PRO IRI ratio (0.29 ± 0.10) levels of the subjects who developed IDDM were significantly higher ( P < 0.05) than those values obtained in subjects who did not developed the disease. Conclusion: these data indicate that fasting PRO levels and the PRO IRI ratio may be an additional marker in post-puberty first-degree relatives of IDDM patients with immunological and metabolic evidence of high risk for developing the disease.

Processing and secretion of insulin-related peptides in an insulinoma cell line

Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line betaTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of betaTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed-phase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucose-rich growth medium the cells turned over their (pro)insulin inventory (90 +/- 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 microM) and isobutylmethylxanthine (1 mM), approximately 80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 +/- 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 microM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only approximately 50% converted to mature insulin, but 85 +/- 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion.

&lt;b&gt;lmmunoreactive proinsulin detected by enzyme-linked immunosorbent &lt;/b&gt;&lt;b&gt;assay &lt;/b&gt;

&lt;b&gt;lmmunoreactive proinsulin detected by enzyme-linked immunosorbent &lt;/b&gt;&lt;b&gt;assay &lt;/b&gt;

Postprandial lipid metabolism and beta-cell function in non-insulin-dependent (type 2) diabetes mellitus after a mixed meal with a high fat content.

Postprandial lipid profiles and release of insulin (INS), intact proinsulin (PI), and 32-33 split proinsulin (SPI) in response to a mixed meal with a high fat content were determined over a 12 h period in non-obese control subjects (n = 10) and non-insulin-dependent (Type 2) diabetic (NIDDM) patients with normotriglyceridaemia (NTG; n = 11) and hypertriglyceridaemia (HTG; n = 10), by calculation of the 'areas under the curves' (AUC). The postprandial triglyceride-AUC was significantly greater in HTG-NIDDM patients (p < 0.05) than in NTG-NIDDM or control subjects. Chylomicron clearance was impaired only in HTG-NIDDM patients (p < 0.05). Chylomicron-remnant clearance was impaired in both groups of NIDDM patients (p < 0.05). The postprandial suppression of plasma non-esterified fatty acid (NEFA) content was impaired in HTG-NIDDM patients (p < 0.05). The postprandial INS-, PI- and SPI-AUCS were significantly greater than in the control subjects (p < 0.05). In NIDDM, triglyceride-AUC correlated significantly with PI and SPI release (triglyceride-AUC vs PI, p < 0.05; triglyceride-AUC vs SPI, p < 0.01). Chylomicron AUC was unrelated to the fasting plasma INS, PI or SPI content, unlike chylomicron-remnant-AUC (Chylomicron-remnant-AUC vs INS, p = NS; chylomicron-remnant-AUC vs PI, p < 0.01; chylomicron-remnant-AUC vs SPI, p < 0.01). The NEFA response was associated with fasting plasma SPI content (NEFA-AUC vs SPI, p < 0.05). Postprandial chylomicron AUC was not related to the overall secretion of INS, PI or SPI. However, triglyceride-, chylomicron-remnant- and NEFA-AUCs were all associated positively with the release of PI and SPI (p < 0.05). In multivariate analyses, chylomicron-remnant clearance had the major relationship with the release of insulin precursors, accounting for 23% of the variability (p < 0.01). Inclusion of overall response of free fatty acids improved the model, with both parameters together accounting for 30% of the variability (p < 0.01). The output of the beta-cell over the postprandial period differed between the NIDDM patients and the control subjects in that when glycaemic stimulation was moderate, the proportion of insulin-like molecules as a percentage of the total output was greater than in control subjects but this was not the situation when glycaemia was greatest. We conclude that abnormal postprandial lipaemia in NIDDM is associated with beta-cell output, possibly mediated by the availability of free fatty acids.

Postprandial Lipid Metabolism and β-cell Function in Non‐insulin‐dependent (Type 2) Diabetes Mellitus After a Mixed Meal with a High Fat Content

Postprandial lipid profiles and release of insulin (INS), intact proinsulin (PI), and 32-33 split proinsulin (SPI) in response to a mixed meal with a high fat content were determined over a 12 h period in non-obese control subjects (n = 10) and non-insulin-dependent (Type 2) diabetic (NIDDM) patients with normotriglyceridaemia (NTG; n = 11) and hypertriglyceridaemia (HTG; n = 10), by calculation of the ‘areas under the curves’ (AUC). The postprandial triglyceride-AUC was significantly greater in HTG-NIDDM patients (p < 0.05) than in NTG-NIDDM or control subjects. Chylomicron clearance was impaired only in HTG-NIDDM patients (p < 0.05). Chylomicron-remnant clearance was impaired in both groups of NIDDM patients (p < 0.05). The postprandial suppression of plasma non-esterified fatty acid (NEFA) content was impaired in HTG-NIDDM patients (p < 0.05). The postprandial INS-, PI- and SPI-AUCS were significantly greater than in the control subjects (p < 0.05). In NIDDM, triglyceride-AUC correlated significantly with PI and SPI release (triglyceride-AUC vs PI, p < 0.05; triglyceride-AUC vs SPI, p < 0.01). Chylomicron AUC was unrelated to the fasting plasma INS, PI or SPI content, unlike chylomicron-remnant-AUC (Chylomicron-remnant-AUC vs INS, p = NS; chylomicron-remnant-AUC vs PI, p < 0.01; chylomicron-remnant-AUC vs SPI, p < 0.01). The NEFA response was associated with fasting plasma SPI content (NEFA-AUC vs SPI, p < 0.05). Postprandial chylomicron AUC was not related to the overall secretion of INS, PI or SPI. However, triglyceride-, chylomicron-remnant- and NEFA-AUCs were all associated positively with the release of PI and SPI (p < 0.05). In multivariate analyses, chylomicron-remnant clearance had the major relationship with the release of insulin precursors, accounting for 23 % of the variability (p < 0.01). Inclusion of overall response of free fatty acids improved the model, with both parameters together accounting for 30 % of the variability (p < 0.01). The output of the β-cell over the postprandial period differed between the NIDDM patients and the control subjects in that when glycaemic stimulation was moderate, the proportion of insulin-like molecules as a percentage of the total output was greater than in control subjects but this was not the situation when glycaemia was greatest. We conclude that abnormal postprandial lipaemia in NIDDM is associated with β-cell output, possibly mediated by the availability of free fatty acids.

Effect of sulfonylurea withdrawal on proinsulin levels, B cell function, and glucose disposal in subjects with noninsulin-dependent diabetes mellitus.

To examine the effect of sulfonylurea withdrawal on the proinsulin (PI) to immunoreactive insulin (IRI) ratio in subjects with noninsulin dependent diabetes mellitus (NIDDM), we measured fasting and arginine-stimulated PI and IRI levels in 15 subjects with NIDDM (mean age, 64.4 yr; body mass index, 27.3 kg/m2) during chronic treatment with glyburide (n = 12) or other sulfonylureas (n = 3) and after withdrawal from the medication for 2-4 weeks. Additionally, we performed iv glucose tolerance tests to measure the insulin sensitivity index, glucose effectiveness at zero insulin, iv glucose tolerance, and the acute insulin response to glucose. Discontinuation of sulfonylurea therapy resulted in an increase in fasting plasma glucose from 10.5 +/- 0.8 to 13.1 +/- 0.9 mmol/L (P < 0.001). This was associated with a decrease in the fasting IRI concentration (120 +/- 21 to 92 +/- 21 pmol/L; P < 0.005) and the fasting PI concentration (58 +/- 10 to 41 +/- 7 pmol/L; P < 0.01); however, the PI/IRI ratio did not differ (50 +/- 6% during medication and 48 +/- 5% after withdrawal; P = 0.43). Similarly, the acute PI/IRI ratio did not change (8.6 +/- 2.4% on therapy; 8.4 +/- 1.2% off therapy; P = 0.91). No change was observed in other metabolic parameters, including insulin sensitivity index (0.76 +/- 0.21 x 10(-5) min-1/pM on therapy; 0.76 +/- 0.19 x 10(-5) min-1/pM off therapy), acute insulin response to arginine (225 +/- 37 pmol/L on therapy; 225 +/- 40 pmol/L off therapy), acute insulin response to glucose (10 +/- 6 pmol/L on therapy; 5 +/- 4 pmol/L off therapy), glucose effectiveness at zero insulin (0.0127 +/- 0.0007 min-1 on therapy; 0.0119 +/- 0.0009 min-1 off therapy), and iv glucose tolerance (0.85 +/- 0.05%/min on therapy; 0.71 +/- 0.07%/min off therapy). We conclude that sulfonylurea therapy does not correct the elevated PI/IRI ratio or absent first phase insulin response of NIDDM and does not have an effect on parameters of peripheral tissue glucose uptake.

True insulin and intact proinsulin levels in acromegalic patients.

To determine whether proinsulin (PI) contributes significantly to the immunoreactive insulin (IRI) concentrations in acromegalics, we measure PI, "true insulin" and IRI in a group of acromegalics compared with a control group. Serum PI was determined by the immunofluorimetric assay (IFMA). Insulin was also determined by an IFMA that measures true insulin and by a radioimmunoassay (RIA). We performed an oral glucose tolerance test (OGTT) in a total group of 46 subjects: 10 controls with normal OGTT and body mass index < 25 kg/m2 (control group I), 10 controls with normal OGTT and body mass index > 25 kg/m2 (control group II), 15 patients with active acromegaly and normal OGTT and 11 patients with active acromegaly and IGT. Plasma glucose, serum GH, insulin and proinsulin were measured in all OGTT samples. Basal levels of insulin-like growth factor I (IGF-I) were measured in acromegalics. Mean body mass index in acromegalics with normal and impaired glucose tolerance were significantly higher compared with control group I and similar when compared with control group II. Proinsulin increased during OGTT in acromegalics with impaired glucose tolerance compared to control group I, and only fasting proinsulin compared to control group II. In normal OGTT acromegalics, only fasting proinsulin was increased. The RIA insulin during OGTT was significantly higher for both acromegalic groups compared to control group I and only at fasting when compared with control group II. This difference was not evident when insulin was measured by IFMA. These results suggest that in acromegalics, hyperinsulinism measured by RIA was at least in part due to hyperproinsulinism.

Synthesis and Processing of Genetically Modified Human Proinsulin by Rat Myoblast Primary Cultures

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.

Constitutive (pro)insulin release from pancreas of transgenic mice expressing monomeric insulin.

To evaluate the role of protein aggregation and calcium in the sorting of insulin for regulated vs. constitutive release from the intact pancreas, we targeted the expression of a monomeric mutant form of human (pro)insulin (B9/B27) to the pancreatic beta-cells of transgenic mice. This mutant insulin does not form dimers or hexamers, but can aggregate at high concentration in the presence of calcium. A homozygous line (171) was produced that expressed 55% of the total (pro)insulin message in their beta-cells as the mutant form and had normal pancreatic total (pro)insulin content [measured as immunoreactive insulin (IRI)]. Fasting glucose levels in these transgenics and in homozygous control mice expressing native human (pro)insulin were normal, although levels were abnormally elevated during ip glucose tolerance testing. In the presence of extracellular calcium, regulated IRI release from the isolated perfused pancreas of the transgenic mice was undetectable in the absence of secretagogues and responded with normal phasic kinetics when stimulated with increasing steps of glucose, with glucose plus isobutylmethylxanthine, or with arginine. Without extracellular calcium (0 calcium plus EGTA), normal pancreas did not release IRI in either the presence or absence of secretagogues. In contrast, without calcium or secretagogues, transgenic pancreas spontaneously and constitutively released IRI at high levels equivalent to those elicited by glucose (22 mM) plus calcium from normal pancreas. This release was partially inhibited by glucose or arginine. Constitutive secretion was acutely sensitive to calcium; inhibition occurred within minutes after the addition of calcium and quickly returned to its characteristic level (with overshoot) when calcium was subsequently removed. Somatostatin, at a concentration that caused 50% inhibition of normal glucose-stimulated secretion, did not affect constitutive release. Control pancreas from the transgenic mice, expressing native human (pro)insulin, responded normally to secretagogues and did not constitutively release hormone in the absence of calcium. It is concluded that expression of monomeric human insulin in pancreatic beta-cells from transgenic mice did not interfere with normal phenotypic insulin secretion, indicating that the functional secretory apparatus was not impaired. Constitutive secretion of IRI from the intact pancreas requires both the expression of a monomeric form of insulin and the absence of extracellular calcium, two conditions that reduce aggregation. These results are consistent with the hypothesis that protein aggregation favors sorting to the regulated pathway, whereas suppressed aggregation causes sorting for constitutive release.

Constitutive (pro)insulin release from pancreas of transgenic mice expressing monomeric insulin

Constitutive (pro)insulin release from pancreas of transgenic mice expressing monomeric insulin

Relationship of proinsulin and insulin with noninsulin-dependent diabetes mellitus and coronary heart disease in Japanese-American men: impact of obesity--clinical research center study.

Obesity is associated with noninsulin-dependent diabetes mellitus (NIDDM) and coronary heart disease (CHD), and these interactions have usually been related to changes in immunoreactive insulin (IRI) levels. A role of proinsulin (PI) in this association has been suggested. We, therefore, examined IRI, PI, and true insulin levels and the PI/IRI ratio by glucose tolerance or CHD status in a cross-sectional study of 170 Japanese-American men (45-74 yr old) in whom 2 measures of adiposity (body mass index and intraabdominal fat) were made to assess potential associations in this population with a high prevalence of both NIDDM and CHD. Subjects were classified as having normal glucose tolerance (n = 58), impaired glucose tolerance (IGT; n = 55), or NIDDM (n = 57) or were classified by CHD status (without CHD, n = 127; with CHD, n = 43). A positive linear relationship existed between obesity, determined either as the body mass index or intraabdominal fat, and IRI, PI, and true insulin, but not the PI/IRI ratio. In the NIDDM subjects, PI levels were disproportionately greater than those in subjects with normal glucose tolerance or IGT, so the PI/IRI ratio was significantly greater in the NIDDM group [mean (95% confidence interval): normal glucose tolerance, 11.8% (range, 10.4-13.5); IGT, 12.8% (range, 10.8-15.1); NIDDM, 19.2% (range, 15.4-24.0); P = 0.0002] even when adjusted for obesity (P = 0.0004). In subjects with CHD compared to subjects without CHD, IRI (P = 0.0026) and true insulin levels (P = 0.0043) were increased, but PI levels were not. However, these differences were not present after adjustment for obesity. In contrast, when intraabdominal fat was adjusted for IRI or true insulin, a significant effect of intraabdominal fat on CHD risk was maintained (P = 0.045 and P = 0.029, respectively), suggesting that another factor(s) associated with central obesity may be involved in CHD risk. Thus, in Japanese-American men, elevated PI and PI/IRI ratio are markers of B-cell dysfunction, and these are not the result of obesity. An elevated true insulin level is present in those with CHD, but this appears to be the result of obesity. In contrast, central adiposity confers an additional risk for CHD independent of insulin.