[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.

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.

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.

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.

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles.

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic beta cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6-phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by approximately 90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in beta cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595-608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261-1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR-ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.

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.

Peripheral metabolism of insulin, proinsulin, and C-peptide in the pregnant rat.

To clarify alterations in carbohydrate metabolism which occur in pregnancy, metabolic clearance rates of insulin, proinsulin, and C-peptide were measured by the constant infusion technique in term-pregnant rats and in virgin littermates. In addition, placental permeability to these peptides was evaluated by simultaneous determination of their concentration in fetal blood, amniotic fluid, and maternal arterial blood and the renal extraction and excretion of insulin and C-peptide were determined during simultaneous studies of renal hemodynamics. The metabolic clearance rate (MCR) of insulin was higher (P less than 0.005) in pregnant animals (61.5+/-1.7 ml/min per kg nonconceptus body weight) than in virgin littermates (51.5+/-2.2 ml/min per kg). Insulin disappearance from the circulation after both single injection and discontinuance of a constant infusion was also faster in gravid animals. In contrast, the MCR of proinsulin and C-peptide, and the disappearance of C-peptide from the circulation were similar in pregnant and control rats. The placenta was virtually impermeable to each of the three polypeptides since their mean levels in both fetal blood and amniotic fluid did not exceed 2.5 ng/ml and were only minimally influenced by pharmacological concentrations as high as 60 ng/ml in the maternal circulation. The renal clearance of insulin (renal arteriovenous insulin difference X renal plasma flow) was lower, and its contribution to insulin MCR was less in pregnant animals than in controls (19.4+/-1.5% vs. 28.7+/-3.7%, P less than 0.05), whereas the renal clearance and renal clearance/MCR of C-peptide were similar in pregnant rats and virgin littermates. These results indicate that the peripheral metabolism of insulin is accelerated in pregnancy, while that of pro-insulin and C-peptide is unaffected. Since transplacental passage of insulin is negligible and its renal clearance is not increased, the enhanced MCR of insulin in pregnancy is due to increased metabolism at an extrarenal site probably within the placenta itself.

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.

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.

Fingerprint analysis of insulin and proinsulins.

Proteolysis of insulin or (pre)proinsulin with S. aureus protease V8 in Tris buffer at neutral pH yields a characteristic pattern of peptide fragments that is resolved using high-performance liquid chromatography. Identification of the fragments of interest was achieved by comparison of insulins of different species, of modified insulins and of proinsulin and N-extended proinsulin, and by amino acid analysis. The fingerprint method allows, for example, the simultaneous analysis of porcine and human insulin, the identification of a modified insulin generated in dosing devices, as well as the individual analysis of the two disulfide linkages between the A- and B-chain in refolded insulins.

Three-dimensional imaging of intact isolated islets of Langerhans with confocal microscopy.

We present a new technique for analyzing the three-dimensional structure of intact isolated islets of Langerhans. Adult rat and human islets were stained with whole-mount immunofluorescence techniques and optically sectioned with a confocal microscope. This has several advantages over traditional methods: 1) the technical difficulties in serial sectioning and handling the large numbers of sections are avoided, 2) optical sectioning by confocal microscopy gives improved resolution and strongly suppresses light from out-of-focus structures, and 3) entire islets can be rapidly imaged for the presence of positive staining. This new technique should facilitate the study of the three-dimensional structure of islets of Langerhans.

Evidence for presence of proinsulin-immunoreactive glycoprotein(s) in fetal bovine pancreatic extracts.

A large-molecular-weight proinsulin-immunoreactive protein fraction was obtained from an extract of fetal bovine pancreases by gel filtration in 6 M guanidine-1 M acetic acid. Concanavalin A-Sepharose-affinity column chromatography of the large-molecular-weight fraction yielded a discrete alpha-methyl-mannoside-displaceable immunoreactive peak that also displayed N-acetylglucosamine-specific binding to wheat germ lectin-Sepharose. Chemically tritiated and radioiodinated lectin-reactive proteins interacted specifically with antibodies to insulin and bovine proinsulin. Immunochemically purified (reaction with antibodies followed by separation of antigen-antibody complexes on protein A-Sepharose) radiolabeled lectin-reactive proteins were analyzed by gel filtration in guanidine-acetic acid and by sodium dodecyl sulfate polyacrylamide gel electrophoresis after disulfide bond-cleavage treatments. Results from these studies suggest the existence of an approximately 67,000-Mr glycoprotein that contains antigenic domains common to proinsulin and insulin.

Measurement of proinsulin and intermediates. Validation of immunoassay methods by high-performance liquid chromatography.

Human proinsulin and 32-33 split proinsulin have been measured in the peripheral circulation by immunoradiometric assays (IRMAs) and have been shown to be elevated in impaired glucose tolerance and non-insulin-dependent diabetes mellitus (NIDDM). The IRMA for 32-33 split proinsulin did not discriminate between this molecule and des-32 or des-31,32 split proinsulin. We describe the comparison of IRMA for human plasma proinsulin and 32-33 split proinsulins with assays combined with high-performance liquid chromatography (HPLC), which can discriminate between 32-33 split, des-32 split, and des-31,32 split proinsulin. Subjects were those with normal glucose tolerance (n = 8) and those with NIDDM (n = 17), who were studied while fasting and 30 min after a glucose load. After collection, blood was centrifuged promptly, and the serum/plasma was stored frozen until assay. Both IRMA and HPLC methods were calibrated against synthetic peptides. Interassay coefficients of variation for the IRMA for proinsulin and 32-33 split proinsulin were < 13% over the ranges 3.8-65 pmol/l and 6.4-65 pmol/l, respectively. The following regression lines were obtained: proinsulin IRMA = -0.143 + 1.066 HPLC, r = 0.860; 32-33 split proinsulin IRMA = 0.048 + 1.051 HPLC; and des-31,32 split proinsulin, r = 0.814. For both analytes, there was no significant difference in the relationship of IRMA to HPLC results between the various subject groups and various time points. Thus, the IRMA for proinsulin has been validated by an independent method.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucose on proinsulin messenger RNA in rats in vivo.

The purpose of these studies was to determine whether glucose, the principal regulator of insulin biosynthesis in mammals, controls synthesis through alterations in levels of proinsulin mRNA in whole animals. Rats were starved for 3 days and then either refed or injected with glucose or saline for 24 h. Glucose injection raised plasma glucose levels equivalent to levels seen with refeeding but provided less than 20% of caloric replacement. Pancreatic RNA was extracted and the relative concentration of proinsulin mRNA was determined by blot hybridization with a cloned rat proinsulin cDNA probe. In starved animals proinsulin mRNA levels were 15-20% that of fed controls. Glucose injection produced a specific three- to fourfold increase in proinsulin mRNA levels relative to total pancreatic RNA, within 24 h. The effect was measurable 2 h after glucose injection and appeared largely complete by 12 h. Actinomycin D blocked the glucose-induced increase in proinsulin mRNA. These studies demonstrate effects of changes of plasma glucose on levels of proinsulin mRNA. Their rapidity of onset and large magnitude are comparable to effects of glucose on rates of insulin biosynthesis in isolated islets and suggest that insulin biosynthesis is regulated at least in part by levels of proinsulin mRNA.

Compensatory responses in mice carrying a null mutation for Ins1 or Ins2.

Intrauterine growth retardation and postnatal acute diabetes result from insulin deficiency in double homozygous null mutants for Ins1 and Ins2 (Duvillié B, et al., Proc. Natl. Acad. Sci. USA 94:5137-5140, 1997). The characterization of single homozygous null mutants for Ins1 or Ins2 is described here. Neither kind of mutant mice was diabetic. Immunocytochemical analysis of the islets showed normal distribution of the endocrine cells producing insulin, glucagon, somatostatin, or pancreatic polypeptide. Analysis of the expression of the functional insulin gene in Ins1-/- or Ins2-/- mice revealed a dramatic increase of Ins1 transcripts in Ins2-/- mutants. This compensatory response was quantitatively reflected by total pancreatic insulin content similar for both types of mutants and wild-type mice. Moreover, both mutants had normal plasma insulin levels and normal glucose tolerance tests. The determination of beta-cell mass by morphometry indicated beta-cell hyperplasia in the mutant mice. The beta-cell mass in Ins2-/- mice was increased almost threefold, which accounts for the increase of Ins1 transcripts in Ins2-/-mutants. This study thus contributes to evaluate the potential of increasing the beta-cell mass to compensate for low insulin production.

Dexamethasone-induced neuropeptide Y expression in rat islet endocrine cells. Rapid reversibility and partial prevention by insulin.

Neuropeptide Y (NPY) is a widely distributed neurotransmitter in the central and peripheral nervous system. In the normal rat pancreas, NPY is confined to neuronal elements, including fibers penetrating the islets. However, treatment of rats with the glucocorticoid dexamethasone (DEX) induces NPY expression also in islet cells. Previously performed double immunocytochemistry (ICC) and in situ hybridization (ISH) combined with ICC revealed that the majority of NPY-expressing islet cells are beta-cells. The present study, using ICC, ISH, and Northern blot, addressed the question whether the islet cell expression of NPY induced by DEX is affected by concomitant insulin (60 U/kg body wt daily for 12 days) treatment. Further, the time course of NPY expression in the islet cells after DEX withdrawal was examined. Treatment with DEX (2 mg/kg body wt daily for 12 days) confirmed the induction of NPY expression in numerous cells, most of which were beta-cells, dispersed within the islets. Northern blot analysis of RNA extracted from isolated islets of DEX-treated rats revealed a strong signal for NPY. Furthermore, DEX also induced NPY expression in isolated rat islets during a 5-day culture period in DEX (100 nmol/l). In vivo, the DEX-induced islet cell expression of NPY mRNA was rapidly reversed after cessation of DEX, being nondetectable 5 days post-treatment; NPY peptide was nondetectable 10 days post-treatment, indicating a slower turnover of the formed peptide. After combined treatment with DEX and insulin, the frequency of islet cells expressing NPY was markedly lower than after treatment with DEX alone. The vast majority of the NPY-expressing cells were beta-cells. In conclusion, DEX-induced NPY expression in rat islet cells is dependent on continuous DEX treatment and is partly prevented by exogenous insulin. The results suggest that the DEX-induced islet NPY expression is regulated by insulin.

Functional and morphologic characterization of human insulinomas.

Circulating levels of insulin, proinsulin-like component, glucagon, growth hormone, and pancreatic polypeptide were measured in 12 patients with functioning insulinomas, and the suppressibility of serum insulin by somatostatin and diazoxide was assessed before surgical removal of the tumors. The hormone content of the tumors was evaluated by radioimmunoassay and by immunofluorescence and the structure of the tumor cells by electron microscopy. Based on these findings, we propose a new classification of insulinomas in two groups: group A is characterized morphologically by abundant well-granulated typical B-cells, trabecular arrangement of tumor cells, and uniform insulin immunofluorescence; functionally, these tumors are associated with a moderate elevation of proinsulin-like component and with an almost complete suppressibility of serum insulin by somatostatin and diazoxide. In contrast, tumors of group B are characterized by scarce well-granulated typical B-cells, a medullary-type histologic structure, and irregular insulin immunofluorescence; functionally these tumors show elevated circulating levels of proinsulin-like component and a marked resistance of insulin secretion to somatostatin and diazoxide inhibition. This way of separating human insulinomas in groups A and B represents a simplification of existing classifications and emphasizes the quantitative ultrastructure in relationship to suppressibility of insulin secretion. The proposed classification of human insulinomas in groups A and B, however, does not allow the assessment of the clinical or histopathologic malignancy of the tumors.