Distribution patterns of proinsulin and insulin in human insulinomas: an immunohistochemical analysis in 76 tumors.

The distribution of proinsulin and insulin immunoreactivity was studied in 76 human insulinomas and in normal pancreas. One trabecular and two solid insulinomas showed the staining pattern of normal beta cells. A "near normal" staining pattern (perinuclear proinsulin and diffuse or polarized insulin staining) existed in 10 of 27 trabecular and 11 of 44 solid insulinomas. An "intermediate" staining pattern (intense perinuclear as well as weaker diffuse proinsulin staining with diffuse or polarized insulin staining) was observed in 10 of 27 trabecular and 20 of 44 solid insulinomas. Different "abnormal" staining patterns were found in 6 of 27 trabecular and 6 of 44 solid insulinomas. Of the 5 glandular insulinomas, 4 exhibited a "near normal" and one an "abnormal" staining pattern. No correlation was found between any particular staining pattern and the multihormonality or malignancy of the insulinomas. The diffuse labeling for proinsulin in about 50% of the insulinomas is suggestive of abnormal prohormone processing.     

Localization of proinsulin and insulin in human insulinoma: preliminary immunohistochemical results

We have carried out an immunohistochemical investigation of 15 human insulinomas applying monoclonal antibodies specifically recognizing proinsulin and insulin. Our results demonstrate that the epitopes unique to proinsulin and insulin can be detected with the respective monoclonal antibodies using the protein A-gold technique after routine formaldehyde fixation and paraffin embedding of the tissues. The immunostaining pattern for proinsulin and insulin in the insulinomas was different from the observed in B cells of pancreatic islets present in the adjacent normal pancreas. Furthermore, the pattern of immunostaining was found to vary from tumor to tumor. These findings strongly suggest the possibility of a disturbed proinsulin to insulin conversion in human insulinomas.     

WHO international reference reagents for human proinsulin and human insulin C-peptide

Candidate preparations for international reference reagents for immunoassays of human proinsulin and human insulin C-peptide were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as international reference reagents: human proinsulin (84/611, nominal ampoule content 6 micrograms) and human insulin C-peptide (84/510, 10 micrograms). Each preparation is intended as a primary reference reagent for the calibration of immunoassays.     

Equilibrium denaturation of insulin and proinsulin

The guanidine hydrochloride induced equilibrium denaturation of insulin and proinsulin was studied by using near- and far-ultraviolet (UV) circular dichroism (CD). The denaturation transition of insulin is reversible, cooperative, symmetrical, and the same whether detected by near- or far-UV CD. These results are consistent with a two-state denaturation process without any appreciable equilibrium intermediates. Analysis of the insulin denaturation data yields a Gibbs free energy of unfolding of 4.5 +/- 0.5 kcal/mol. Denaturation of proinsulin detected by near-UV CD appears to be the same as for insulin, but if detected by far-UV CD appears different. The far-UV CD results demonstrate a multiphasic transition with the connecting peptide portion unfolding at lower concentrations of denaturant. Similar studies with the isolated C-peptide show that its conformation and susceptibility to denaturation are independent of the rest of the proinsulin molecule. After the proinsulin denaturation results were adjusted for the connecting peptide contribution, a denaturation transition identical with that of insulin was obtained. These results show that for proinsulin, the connecting peptide segment is not a random coil; it is an autonomous folding unit, and the portion corresponding to insulin is identical with insulin in terms of conformational stability.     

Bioactivity and pharmacokinetics of human proinsulin in comparison to human insulin after intravenous and subcutaneous injection

The hypoglycemic actions of human insulin (1 IU/kg b.w.) and biosynthetic human proinsulin in about equimolar amounts were studied after intravenous and subcutaneous injection in rabbits. Blood samples were taken up to four hours after injection for the determination of blood glucose and immunoreactive levels of both insulin and human C-peptide. For the determination of human C-peptide, serum taken after proinsulin injection was divided into two fractions. One was examined directly by the human C-peptide radioimmunoassay and the other after incubation with a protein-A-sepharose coupled insulin antibody to find "free human C-peptide". Proinsulin in amounts equimolar to 1 IU insulin/kg b.w., exerted a 34% stronger hypoglycemic action after subcutaneous injection than after intravenous administration (area under curve estimation). Proinsulin-induced hypoglycemia did not last longer after intravenous administration than that induced by intravenous insulin. Although subcutaneous proinsulin did not show the same maximum decrease of blood glucose compared to subcutaneous insulin, its action was significantly prolonged (up to 180 min). Specific measurement of free human C-peptide showed no evidence of conversion of proinsulin to insulin and C-peptide.     

Distribution of delta sleep-inducing peptide in the newborn and infant human hypothalamus: an immunohistochemical study

The distribution of delta sleep-inducing peptide immunoreactive cell bodies, fibers, and terminal-like structures was investigated in the normal human hypothalamus during the first postnatal year, using immunohistofluorescence and peroxidase anti-peroxidase techniques. Immunolabeled perikarya were relatively few and were mostly scattered through the anterior (preoptic) and mediobasal regions (infundibular nucleus) of the hypothalamus. DSIP-immunoreactive fibers and terminal-like fibers were observed throughout the entire rostrocaudal extent of the hypothalamus. They exhibit high densities in the preoptic region, the organum vasculosum of lamina terminalis, infundibular nucleus and median eminence. Moderate to low densities of DSIP-immunoreactive fibers were observed in the other hypothalamic structures, located in the anterior and mediobasal regions of hypothalamus, such as periventricular, paraventricular, suprachiasmatic, ventromedial, dorsomedial and parafornical nuclei. In the present study, the analysis of the immunohistochemical pattern of DSIP-immunoreactive neuronal elements in the human infant hypothalamus during the first postnatal year provided evidence of the presence of several differences. We have found qualitative age-related changes in the density of DSIP immunoreactivity in several hypothalamic structures such as the anterior region and the median eminence.     

基因工程人胰岛素原和胰岛素的分离纯化及性质研究

E. coli DH 5 alpha cells harboring a plasmid pWR 590-BCA 4 for fused human proinsulin production were cultured. The fused human proinsulin was isolated from the fermented cells and then subjected it to cleavage with BrCN. The cleaved product was then converted to crude proinsulin-S-sulfonate using oxidative sulfitolysis. The isolation of human proinsulin-S-sulfonate was accomplished by ion exchange chromatography on QAE-sephadex A-25, followed by gel filtration on sephadex G-50. The purified human proinsulin-S-sulfonate was folded using a disulfide interchange method. The folding mixture was then chromatographed on sephadex G-50 and purified proinsulin was obtained. The proinsulin was then converted to human insulin and C-peptide by a combination cleavage with trypsin and carboxypeptidase B. The total yield of human insulin was about 5 mg/L The Zinc insulin crystals were obtained with amorphous human insulin using citrate method. The amino acid composition N-terminal sequences as well as C-terminal amino acid residues are in agreement with expected results. The hypoglycemic activity of purified human insulin is 26-27 U/mg, as judged by mouse convulsion assay, and the RIA activity is about 99% of that of porcine insulin.     

Distribution of vitamin B12 R binder in normal human tissues: an immunohistochemical study

We studied the distribution of vitamin B12 R binder in various normal human tissues by use of an immunoperoxidase technique. Positive staining for R binder was observed in almost all glandular epithelia of digestive system, bronchial glands, renal proximal tubules, prostate, uterus, Fallopian tube, mammary gland, and sweat glands. The distribution of R binder was similar to that of lactoferrin and secretory component. These findings support the hypothesis that R binder plays a role in the local defense mechanism.     

(A-C-B) human proinsulin, a novel insulin agonist and intermediate in the synthesis of biosynthetic human insulin.

The hormone insulin is synthesized in the beta cell of the pancreas as the precursor, proinsulin, where the carboxyl terminus of the B-chain is connected to the amino terminus of the A-chain by a connecting or C-peptide. Proinsulin is a weak insulin agonist that possesses a longer in vivo half-life than does insulin. A form of proinsulin clipped at the Arg65-Gly66 bond has been shown to be more potent than the parent molecule with protracted in vivo activity, presumably as a result of freeing the amino terminal residue of the A-chain. To generate a more active proinsulin-like molecule, we have constructed an "inverted" proinsulin molecule where the carboxyl terminus of the A-chain is connected to the amino terminus of the B-chain by the C-peptide, leaving the critical Gly1 residue free. Transformation of Escherichia coli with a plasmid coding for A-C-B human proinsulin led to the stable production of the protein. By a process of cell disruption, sulfitolysis, anion-exchange chromatography, refolding, and reversed-phase high-performance liquid chromatography, two forms of the inverted proinsulin differing at their amino termini as Gly1 and Met0-Gly1 were identified and purified to homogeneity. Both proteins were shown by a number of analytical techniques to be of the inverted sequence, with insulin-like disulfide bonding. Biological analyses by in vitro techniques revealed A-C-B human proinsulin to be intermediate in potency when compared to human insulin and proinsulin. The time to maximal lowering of blood glucose in the fasted normal rat appeared comparable to that of proinsulin. Additionally, we were able to generate fully active, native insulin from A-C-B human proinsulin by proteolytic transformation. The results of this study lend themselves to the generation of novel insulin-like peptides while providing a simplified route to the biosynthetic production of insulin.     

A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.     

Oligomerization and insulin interactions of proinsulin C-peptide: Threefold relationships to properties of insulin

Three principally different sites of action have been reported for proinsulin C-peptide, at surface-mediated, intracellular, and extracellular locations. Following up on the latter, we now find that (i) mass spectrometric analyses reveal the presence of the C-peptide monomer in apparent equilibrium with a low-yield set of oligomers in weakly acidic or basic aqueous solutions, even at low peptide concentrations (sub-μM). It further shows not only C-peptide to interact with insulin oligomers (known before), but also the other way around. (ii) Polyacrylamide gel electrophoresis of C-peptide shows detectable oligomers upon Western blotting. Formation of thioflavin T positive material was also detected. (iii) Cleavage patterns of analogues are compatible with C-peptide as a substrate of insulin degrading enzyme. Combined, the results demonstrate three links with insulin properties, in a manner reminiscent of amyloidogenic peptides and their chaperons in other systems. If so, peripheral C-peptide/insulin interactions, absolute amounts of both peptides and their ratios may be relevant to consider in diabetic and associated diseases.     

Effects of beta-mercaptoethanol and hydrogen peroxide on enzymatic conversion of human proinsulin to insulin

Human insulin is a hormone well-known to regulate the blood glucose level. Recombinant preproinsulin, a precursor of authentic insulin, is typically produced in E. coli as an inactive inclusion body, the solubilization of which needs the addition of reducing agents such as beta-mercaptoethanol. To make authentic insulin, recombinant preproinsulin is modified enzymatically by trypsin and carboxypeptidase B. The effects of beta-mercaptoethanol on the formation of human insulin derivatives were investigated in the enzymatic modification by using commercially available human proinsulin as a substrate. Addition of 1 mM beta-mercaptoethanol induced the formation of various insulin derivatives. Among them, the second major one, impurity 3, was found to be identical to the insulin B chain fragment from Phe1 to Glu21. Minimization of the formation of insulin derivatives and concomitant improvement of the production yield of human insulin were achieved by the addition of hydrogen peroxide. Hydrogen peroxide bound with beta-mercaptoethanol and thereby reduced the negative effects of beta-mercaptoethanol considerably. Elimination of the impurity 3 and other derivatives by the addition of over 10 mM hydrogen peroxide in the presence of beta-mercaptoethanol led to a 1.3-fold increase in the recovery efficiency of insulin, compared with those for the case without hydrogen peroxide. The positive effects of hydrogen peroxide were also confirmed with recombinant human preproinsulin expressed in recombinant E. coli as an inclusion body.     

Comparisons of insulin and biosynthetic human proinsulin actions in cultured hepatocytes. Kinetics and biologic potencies

The binding and biologic potencies as well as kinetics of action of human biosynthetic proinsulin (HPro) were studied in primary cultures of rat hepatocytes. HPro had 3% (on a molar basis) of the potency of porcine insulin for displacing (125I)-TyrA14-insulin from receptors. Maximally effective concentrations of insulin and HPro caused similar stimulations of 14C-glucose incorporation into glycogen and glycogen synthase activity. However, the dose response curve for HPro stimulation of glycogen synthase was shifted far to the right (EC50 = 4.1 +/- 1.1 nM) of that for insulin (.09 +/- .01). The relative biologic potency of HPro was approximately 3%. Biologically equivalent maximal doses of insulin (8.3 nM) and HPro (53.2 nM) stimulated glycogen synthase activity with similar time courses; half maximal between 15-30 min with maximal effects at 60 min. Deactivation of glycogen synthase upon removal of the hormone was very rapid for both hormones. The relative binding and biologic potencies of HPro compared to insulin in liver (approximately 3%) were similar to values previously seen in adipocytes. This fact, together with the similarity of kinetics of action, suggest that the in vivo hepatoselectivity of HPro is not a property of the target cell itself.     

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation. Accepted: 10 June 1999     

The primary structure of ratfish insulin reveals an unusual mode of proinsulin processing

The primary structure of insulin from the Holocephalan fish, Hydrolagus colliei (the ratfish), has been established by automated Edman degradation as: (Formula: see text). The presence of a COOH-terminal extension to the B-chain is consistent with the occurrence of a single base mutation in the region of the gene encoding one of the dibasic residue processing sites [Arg31(AGA)----Ile* (AUA)] with the result that the ratfish has utilised an alternative cleavage site within the C-peptide region of proinsulin.