Human proinsulin production in the milk of transgenic cattle.

BACKGROUND: The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. METHODS AND RESULTS: Pseudo-lentivirus containing the bovine ß-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. CONCLUSION: The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Condensation of the ß-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain.

ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

Novel Human Insulin Isoforms and Cα-Peptide Product in Islets of Langerhans and Choroid Plexus.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells, where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains ß-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP, and its amyloid formation was inhibited in vitro more efficiently by Cα-peptide than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase-processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus autopsy donors. Intriguingly, 100 years after the discovery of insulin, we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

[Diagnostics value and regulatory functions of roinsulin.]

Proinsulin is one of the indicators reflecting the functional activity of the pancreas. In insulin-independent diabetes mellitus the ratio proinsulin / insulin is increased. The review examined the causes of hyperproinsulinemia and the diagnostic value of proinsulin in patients with diabetes mellitus type 1 and 2. The role of proinsulin in the regulation of metabolic pathways and the preservation of the functional activity of cells under physiological conditions, during aging and during pathological processes is discussed. Studies in these areas justify the inclusion of proinsulin in the superfamily of signaling factors. The neuroprotective activity of proinsulin and its potential as a therapeutic tool for neurodegenerative diseases and retinal dystrophy are considered.

Modified Western blotting for insulin and other diabetes-associated peptide hormones.

Now, the quantification of proinsulin/insulin contents within organisms tends to be evaluated only by enzyme-linked immunosorbent assay (ELISA), although assessing the adequacy of results by some quantification method is important. Remarkably, few scientific papers use detection by Western blotting (WB), another immunological assay, of proinsulin/insulin. We found two problems with quantification of insulin and proinsulin by general WB: the shape of an insulin band in gel electrophoresis is distorted, and the retention potency to a blotting membrane of the peptide hormones (mainly insulin) is low. We solved the first problem by optimizing the sodium dodecyl sulfate concentration in the sample buffer and the second problem by glutaraldehyde fixation following treatment with a blocking solution for a short time. The improvements were confirmed by quantification of proinsulin/insulin in standards, MIN6c4 cell lysates, and MIN6c4 culture supernatants. Furthermore, we showed that the modified WB is applicable to other diabetes-associated peptide hormones: insulin analogs, glucagon, GLP-1s, somatostatins, ghrelins, and pancreatic polypeptide. Our data showed that the modified WB can contribute to qualitative or quantitative analyses of diabetes-associated peptides by providing analytical information based on electrophoresis, although ELISA, which is an almost exclusive method in the quantification of peptide hormones, supplies only numerical data.

Reduced expression of ERp46 under diabetic conditions in ß-cells and the effect of liraglutide.

BACKGROUND: Diabetes mellitus is characterized by peripheral insulin resistance, hyperglycemia and defective insulin secretion. Insulin producing pancreatic ß-cells are equipped with a highly developed endoplasmic reticulum (ER) and thus are affected by ER stress under hyperglycemic conditions. We have previously studied the influence of high glucose on cultured ß-cells in vitro. Proteomic analysis revealed a number of proteins involved in glucose toxicity, while further biochemical analysis identified the endoplasmic reticulum protein ERp46 as a molecule with a possible role in insulin production at the post-translational level. In addition, the involvement of incretin hormone glucagon-like peptide 1 (GLP-1) in diabetes proposes that incretin-mimetic compounds may be among the optimal choices in future therapeutic interventions; therefore their effects on various aspects of the pathogenesis of diabetes mellitus should be explored in detail. Based on the above, we examined the possible involvement of ERp46 in insulin production and the effect of the GLP-1 analogue liraglutide on the expression of ERp46 in vitro, in ß-cells cultured under high glucose conditions and in vivo, in the mouse db/db diabetic model, where pronounced hyperglycemia is a key characteristic. RESULTS: Confocal microscopy revealed areas of co-localization of ERp46 and pro-insulin in pancreatic islets. In order to explore the possible interaction between ERp46 and insulin immunoprecipitation was used. In extracts from cultured ß-cells, antibodies against pro-insulin co-precipitated ERp46 and antibodies against ERp46 co-precipitated pro-insulin, as shown by Western blotting. Furthermore, data from a proximity ligation assay positioned these two molecules closer than 30nm in distance. When pancreatic ß-cells were cultured in high glucose conditions they exhibited a decrease in ERp46 expression, while treatment with the GLP-1 analogue liraglutide restored ERp46 levels, leading to a significant increase of ERp46 in comparison to hyperglycemic conditions. In the diabetic mouse model db(-)/db, ERp46 expression was reduced in pancreatic islets, as documented by morphological and biochemical techniques. This decrease was abolished after treatment with the GLP-1 analogue in a dose-dependent manner. In an attempt to understand the underlying mechanism, we examined the sequence of the promoter of ERp46 and found consensus motifs that can be recognized by transcription factors ATF6 and XBP1. Subsequently, we performed chromatin immunoprecipitation assay and demonstrated that treatment of ß-TC-6 cells with 25mmol/L glucose decreases gradually the binding enrichment of ATF6 and XBP1 in ERp46 gene promoter. CONCLUSIONS: We propose that since ERp46 is a member of the disulfide isomerases family, it is likely to play a key role in insulin biosynthesis and its reduction under high glucose conditions may be a novel contributor to the glucotoxicity of ß-cells. In addition, the GLP-1 analogue liraglutide seems to interfere in this process and may exert its beneficial effects in diabetes by affecting insulin production via restoration of ERp46 expression.

Detection of the single-chain precursor in the production and purification process of recombinant human insulin.

High quality recombinant insulin requires being free of single-chain precursor (proinsulin), a task that depends on the selectivity and sensitivity of the monitoring process for detecting proinsulin. In this study we developed an enzyme-linked immunosorbent assay (ELISA) system that was specifically tailored to detect recombinant proinsulin. The proinsulin consists of six components: an initiating methionine, 48 amino acids from human growth hormones (HGH, used as the protection peptide), first connecting Arg-residue, B-chain of insulin, and second connecting Arg-peptide and A-chain of insulin. This form of proinsulin is more stable and can be efficiently expressed by E. coli than insulin. Herein, we evaluated the specificity, precision, recovery, sensitivity, and detection range of the proinsulin ELISA kit. The results showed that the ELISA kit is a very useful tool for monitoring the proinsulin yield in early stages of insulin production as well as the residual proinsulin in the final product, insulin.

A replacement for islet equivalents with improved reliability and validity.

Islet equivalent (IE), the standard estimate of isolated islet volume, is an essential measure to determine the amount of transplanted islet tissue in the clinic and is used in research laboratories to normalize results, yet it is based on the false assumption that all islets are spherical. Here, we developed and tested a new easy-to-use method to quantify islet volume with greater accuracy. Isolated rat islets were dissociated into single cells, and the total cell number per islet was determined by using computer-assisted cytometry. Based on the cell number per islet, we created a regression model to convert islet diameter to cell number with a high R2 value (0.8) and good validity and reliability with the same model applicable to young and old rats and males or females. Conventional IE measurements overestimated the tissue volume of islets. To compare results obtained using IE or our new method, we compared Glut2 protein levels determined by Western Blot and proinsulin content via ELISA between small (diameter≤100 µm) and large (diameter≥200 µm) islets. When normalized by IE, large islets showed significantly lower Glut2 level and proinsulin content. However, when normalized by cell number, large and small islets had no difference in Glut2 levels, but large islets contained more proinsulin. In conclusion, normalizing islet volume by IE overestimated the tissue volume, which may lead to erroneous results. Normalizing by cell number is a more accurate method to quantify tissue amounts used in islet transplantation and research.

Distinguishing persistent insulin autoantibodies with differential risk: nonradioactive bivalent proinsulin/insulin autoantibody assay.

A subset of children develops persistent insulin autoantibodies (IAA; almost always as the only islet autoantibody) without evidence of progression to diabetes. The aim of the current study was the development and characterization of the performance of a nonradioactive fluid phase IAA assay in relation to standard IAA radioassay. We developed a nonradioactive IAA assay where bivalent IAA cross-link two insulin moieties in a fluid phase. The serum samples positive for anti-islet autoantibodies from 150 newly diagnosed patients with diabetes (Barbara Davis Center plus Diabetes Autoantibody Standardization Program [DASP] workshop) and 70 prediabetic subjects who were followed to diabetes were studied. In addition, sequential samples from 64 nondiabetic subjects who were persistently IAA(+) were analyzed. With 99th percentile of specificity, the new assay with the technology from Meso Scale Discovery Company (MSD-IAA) detects as positive 61% (61 of 100) of new-onset patients and 80% (56 of 70) of prediabetic patients compared with our current fluid phase micro-IAA radioassay (mIAA; 44 and 74%, respectively). In addition, MSD-IAA demonstrated better sensitivity than our mIAA from blinded DASP workshop (68 vs. 56% with the same 99% specificity). Of 64 IAA(+) nondiabetic subjects, 25% (8 of 32) who had only IAA and thus the low risk for progression to diabetes were positive with MSD-IAA assay. In contrast, 100% (32 of 32) high-risk children (IAA plus other islet autoantibodies) were positive with MSD-IAA. The IAA detectable by radioassay, but not MSD-IAA, were usually of lower affinity compared with the IAA of the high-risk children. These data suggest that a subset of IAA with current radioassay (not MSD-IAA) represents biologic false positives in terms of autoimmunity leading to diabetes. We hypothesize that factors related to the mechanism of loss of tolerance leading to diabetes determine high affinity and MSD-IAA reactivity.

Proinsulin, GLP-1, and glucagon are associated with partial remission in children and adolescents with newly diagnosed type 1 diabetes.

OBJECTIVE: Proinsulin is a marker of beta-cell distress and dysfunction in type 2 diabetes and transplanted islets. Proinsulin levels are elevated in patients newly diagnosed with type 1 diabetes. Our aim was to assess the relationship between proinsulin, insulin dose-adjusted haemoglobin A1c (IDAA1C), glucagon-like peptide-1 (GLP-1), glucagon, and remission status the first year after diagnosis of type 1 diabetes. METHODS: Juvenile patients (n = 275) were followed 1, 6, and 12 months after diagnosis. At each visit, partial remission was defined as IDAA1C ≤ 9%. The patients had a liquid meal test at the 1-, 6-, and 12-month visits, which included measurement of C-peptide, proinsulin, GLP-1, glucagon, and insulin antibodies (IA). RESULTS: Patients in remission at 6 and 12 months had significantly higher levels of proinsulin compared to non-remitting patients (p < 0.0001, p = 0.0002). An inverse association between proinsulin and IDAA1C was found at 1 and 6 months (p = 0.0008, p = 0.0022). Proinsulin was positively associated with C-peptide (p < 0.0001) and IA (p = 0.0024, p = 0.0068, p < 0.0001) at 1, 6, and 12 months. Glucagon (p < 0.0001 and p < 0.02) as well as GLP-1 (p = 0.0001 and p = 0.002) were significantly lower in remitters than in non-remitters at 6 and 12 months. Proinsulin associated positively with GLP-1 at 1 month (p = 0.004) and negatively at 6 (p = 0.002) and 12 months (p = 0.0002). CONCLUSIONS: In type 1 diabetes, patients in partial remission have higher levels of proinsulin together with lower levels of GLP-1 and glucagon compared to patients not in remission. In new onset type 1 diabetes proinsulin level may be a sign of better residual beta-cell function.

Concomitant enzyme-linked immunosorbent assay measurements of rat insulin, rat C-peptide, and rat proinsulin from rat pancreatic islets: effects of prolonged exposure to different glucose concentrations.

Until now, there have been few assays to measure C-peptide and proinsulin in the rat. We used a well-established rat insulin ELISA and validated two novel ELISAs for rat C-peptide and rat/mouse proinsulin to examine secretion and content of insulin, proinsulin, and C-peptide from rat islets cultured for 72 h at different glucose concentrations in culture medium. To examine long-term effects in vitro rather than short-term effects of exposure to low, normal, and high glucose, the exposure time to the different glucose concentrations was set to 72 h. The measurement uncertainty of the values obtainable from the ELISAs was determined by calculation of the variation pattern from the intraassay variation generated by unknown samples, and repeatability was determined by analysis of controls. The precision study and the analysis of controls confirm that the validated ELISAs for rat C-peptide and proinsulin would be useful for further studies on the effects of preculture in different glucose concentrations. The higher the glucose concentration used during the 72-h culture period of rat islets, the higher insulin, C-peptide and proinsulin values were obtained in a subsequent short-term glucose-challenge experiment. The proportion of proinsulin to insulin secreted increased, as did islet content, with increasing glucose concentration during preculture. We also observed a nonequimolar, glucose-dependent secretion and content of rat insulin over rat C-peptide after culture at 11.1 and 28 mM glucose.

Homogeneous insulin and C-Peptide sensors for rapid assessment of insulin and C-peptide secretion by the islets.

OBJECTIVE: Glucose-stimulated islet insulin or C-peptide secretion experiments are a fundamental tool for studying and assessing islet function. The goal of this work was to develop Ab-based fluorescent homogenous sensors that would allow rapid, inexpensive, near-instantaneous determinations of insulin and C-peptide levels in biological samples. RESEARCH DESIGN AND METHODS: Our approach was to use molecular pincer design (Heyduk et al., Anal Chem 2008;80:5152-5159) in which a pair of antibodies recognizing nonoverlapping epitopes of the target are modified with short fluorochrome-labeled complementary oligonucleotides and are used to generate a fluorescence energy transfer (FRET) signal in the presence of insulin or C-peptide. RESULTS: The sensors were capable of detecting insulin and C-peptide with high specificity and with picomolar concentration detection limits in times as short as 20 min. Insulin and C-peptide levels determined with the FRET sensors showed outstanding correlation with determinations performed under the same conditions with enzyme-linked immunosorbent assay. Most importantly, the sensors were capable of rapid and accurate determinations of insulin and C-peptide secreted from human or rodent islets, verifying their applicability for rapid assessment of islet function. CONCLUSIONS: The homogeneous, rapid, and uncomplicated nature of insulin and C-peptide FRET sensors allows rapid assessment of ß-cell function and could enable point-of-care determinations of insulin and C-peptide.

Pancreatic islets of bank vole show signs of dysfunction after prolonged exposure to high glucose concentrations in vitro.

Bank voles develop glucose intolerance/diabetes mellitus when kept in captivity. We have characterized beta-cell function of glucose intolerant/diabetic animals, and found that this animal model has features of both human type 1 and type 2 diabetes. The aim of this study was to study the functional alterations of islets isolated from glucose tolerant bank voles after a prolonged exposure to various glucose concentrations in vitro. For this purpose, pancreatic islets from normal (glucose tolerant) male and female bank voles were cultured at different glucose concentrations (5.6, 11.1 (control), or 28 mM) whereupon islet functions were examined. Overall, islet insulin output was lowered at 5.6 mM glucose, and similar to control, or enhanced after culture in 28 mM glucose. High glucose culture led to decreased insulin contents, but there was no change in islet DNA content and in morphological assessments of cell death, with the latter findings suggesting that the so-called glucotoxicity had not evolved. A slight gender difference was observed in that islets isolated from females exhibited a glucose-regulated (pro)insulin biosynthesis rate and insulin gene expression. In conclusion, we have found that islets isolated from female and male bank voles are affected by glucose concentrations in vitro in that some signs of dysfunction were observed upon high glucose exposure. A minor gender difference was observed suggesting that the islets of the females may more readily adapt to the elevated glucose concentration than islets of the male bank voles. It could be that these in vitro gender differences observed may represent a mechanism underlying the gender difference in diabetes development observed among bank voles.

Size-exclusion chromatography based on silica-diol for the analysis of the proinsulin fusion protein.

Size-exclusion chromatography based on silica-diol sorbent was employed to analyze the recombinant proinsulin fusion protein obtained during the process of refolding and the following ion-exchange purification. The assay was qualified as a control method estimating its accuracy, precision, linearity, limit of detection, limits of quantitation, specificity, and robustness. The results show the reliability for the intended use.

Expression of mutant Ins2C96Y results in enhanced tubule formation causing enlargement of pre-Golgi intermediates of CHO cells.

Misfolded proteins are recognized by the protein quality control and eventually degraded by the ubiquitin-proteasome system. Previously, we demonstrated accumulation of a misfolded non-glycosylated protein, namely proinsulin, in enlarged pre-Golgi intermediates and dilated rough endoplasmic reticulum (ER) domains in pancreatic beta-cells of Akita mice. In order to exclude effects possibly due to coexisting wild type and mutant proinsulin in pancreatic beta-cells, CHO cells expressing singly wild type or mutant C96Y proinsulin 2 were now analyzed by electron microscopic morphometry and immunogold labeling as well as serial section 3D analysis. We found a significant increase in volume density of pre-Golgi intermediates in CHO Ins2(C96Y) cells which was principally due to an increase of its tubular elements, and no significant changes of the ER. The average diameter of the pre-Golgi intermediates of CHO Ins2(C96Y) cells was about twice that of CHO Ins2(wt) cells. The enlarged pre-Golgi intermediates and the ER of CHO Ins2(C96Y) cells were positive for proinsulin, which was not detectable in the significantly enlarged Golgi cisternal stack. Treatment of CHO Ins2(C96Y) cells with proteasome inhibitors resulted in the formation of proinsulin-containing aggresomes. We conclude that misfolded proinsulin causes enlargement of pre-Golgi intermediates which indicates their involvement in protein quality control.

Effect of insufficient insulin treatment in streptozotocin-induced diabetes mellitus.

OBJECTIVES: In this study, we investigated clinically, pathologically, and immunohistochemically the effect of insufficient short-acting insulin treatment on streptozotocin (STZ)-induced diabetes mellitus in rats. METHODS: Three groups composed of 10 rats each were studied as follows: (1) a group that received only STZ (50 mg/kg) (STZ group); (2) a group that received 50 mg/kg STZ and, after 12 hours, 8 IU of short-acting insulin treatment (STZ + INS group), repeated every night for 5 days; and (3) a control group. Ketonuria and blood glucose levels were examined every day. Blood was obtained from 2 rats from each group, and necropsy was performed every day during the 5-day period. RESULTS: Hyperglycemia was observed in the STZ and STZ + INS groups 24 hours after, but levels were higher in the STS + INS group than those in the STZ-only group. Histopathology was similar in the STZ and STZ + INS groups, and degeneration was observed in both groups, but immunohistochemistry revealed a more severe reduction in insulin-secreting cells in the STZ + INS group than that in the STZ group. There were no hyperglycemia and histopathological or immunochemical alteration in the control group. CONCLUSIONS: This study showed that insufficient short-acting insulin treatment can increase the diabetogenic effect of STZ in rats.

Clinical features and morphological characterization of 10 patients with noninsulinoma pancreatogenous hypoglycaemia syndrome (NIPHS).

OBJECTIVE: Noninsulinoma pancreatogenous hypoglycaemia syndrome (NIPHS), characterized by postprandial neuroglycopaenia, negative prolonged fasts and negative perioperative localization studies for insulinoma, but positive selective arterial calcium stimulation tests and nesidioblastosis in the gradient-guided resected pancreas, is a rare hypoglycaemic disorder of undetermined aetiology. We analysed the clinical, morphological and immunohistological features to further clarify the aetiology and pathogenesis of this rare disease. PATIENTS: Ten consecutive patients with NIPHS (nine men and one woman, aged 29-78 years) were included in the study. Six of the 10 received a gradient-guided subtotal (70%) or distal (50%) pancreatectomy. In the remaining four patients, diazoxide treatment was initiated and the precise mechanism of its action was assessed by meal tests. RESULTS: All of the patients showed a combination of postprandial neuroglycopaenia, negative prolonged fasts (except one patient) and negative localization studies for insulinoma, but positive calcium stimulation tests and nesidioblastosis in the gradient-guided resected pancreas. Immunohistological studies of the resected pancreatic tissues revealed neither an increased rate of proliferation of beta-cells nor an abnormal synthesis and/or processing of either proinsulin or amylin. Evidence of overexpression of the two pancreatic differentiation factors, PDX-1 and Nkx-6.1, as well as the calcium sensing receptor (CaSR) was absent. Nevertheless, abnormal expression of islet neogenesis-associated protein (INGAP), a human cytokine expressed only in the presence of islet neogenesis, in ducts and/or islets, was identified in three of the five patients studied. All of the six patients who received a surgical operation were relieved of further neuroglycopaenic attacks, but one patient who received a subtotal pancreatectomy developed diabetes. In the remaining four patients who received diazoxide treatment, hypoglycaemic episodes were satisfactorily controlled with an attenuated response of beta-cell peptides to meal stimulation. CONCLUSIONS: Our results strengthen the existence of this unique clinical hypoglycaemic syndrome from beta-cell hyperfunction as well as the value of the selective arterial calcium stimulation test in its correct diagnosis and localization. The mechanisms underlying beta-cell hyperfunction and release of insulin to calcium, however, remain poorly characterized. Nevertheless, in a subset of patients with NIPHS, there exists some, as yet undefined, pancreatic humoral/paracrine factor(s) other than proinsulin, amylin, PDX-1, Nkx-6.1 and possibly glucagon-like peptide-1 (GLP-1) that are capable of inducing the INGAP gene and, if activated, will initiate ductal proliferation and islet neogenesis. As for the treatment, we recommend that diazoxide be tried first in each patient and, should it fail, a gradient-guided subtotal or distal pancreatectomy be attempted.

Sustained exposure to high glucose concentrations modifies glucose signaling and the mechanics of secretory vesicle fusion in primary rat pancreatic beta-cells.

The mechanism(s) by which chronic hyperglycemia impairs glucose-stimulated insulin secretion is poorly defined. Here, we compare the "nanomechanics" of single exocytotic events in primary rat pancreatic beta-cells cultured for 48 h at optimal (10 mmol/l) or elevated (30 mmol/l) glucose concentrations. Cargo release was imaged by total internal reflection fluorescence microscopy of lumen-targeted probes (neuropeptide Y [NPY]-pH-insensitive yellow fluorescent protein [NPY-Venus] or NPY-monomeric red fluorescent protein), while the fate of the vesicle membrane was reported simultaneously with phosphatase-on-the-granule-of-insulinoma-enhanced green fluorescent protein. Under all conditions studied, exocytosis proceeded via a "cavity recapture" mechanism in which the vesicle and plasma membranes fused transiently. While essentially complete release of NPY-Venus was observed in 24 +/- 1% of glucose-stimulated exocytotic events in cells maintained at 10 mmol/l glucose, this value was reduced reversibly to 5 +/- 2% of events by culture at 30 mmol/l glucose, in line with decreases in Glut2 and glucokinase gene expression, and attenuated glucose-stimulated increases in NADPH and intracellular [Ca2+]. Since vesicle release in response to cell depolarization with KCl was not affected by culture at 30 mmol/l glucose, we conclude that hyperglycemia causes the abnormal termination of individual insulin release events principally by inhibiting glucose signaling.

Developmental regulation of a proinsulin messenger RNA generated by intron retention.

Proinsulin gene expression regulation and function during early embryonic development differ remarkably from those found in postnatal organisms. The embryonic proinsulin protein content decreased from gastrulation to neurulation in contrast with the overall proinsulin messenger RNA increase. This is due to increasing levels of a proinsulin mRNA variant generated by intron 1 retention in the 5' untranslated region. Inclusion of intron 1 inhibited proinsulin translation almost completely without affecting nuclear export or cytoplasmic decay. The novel proinsulin mRNA isoform expression was developmentally regulated and tissue specific. The proportion of intron retention increased from gastrulation to organogenesis, was highest in the heart tube and presomitic region, and could not be detected in the pancreas. Notably, proinsulin addition induced cardiac marker gene expression in the early embryonic stages when the translationally active transcript was expressed. We propose that regulated unproductive splicing and translation is a mechanism that regulates proinsulin expression in accordance with specific requirements in developing vertebrates.