Biosynthesis and release of thyrotropin-releasing hormone immunoreactivity in rat pancreatic islets in organ culture. Effects of age, glucose, and streptozotocin.

Thyrotropin-releasing hormone immunoreactivity (TRH-IR) was measured in isolated islets and in medium from rat pancreatic islets maintained in organ culture. TRH-IR in methanol extracts of both islets and culture medium was eluted in the same position as synthetic TRH by ion-exchange and gel chromatography and exhibited dilution curves parallel with synthetic TRH in radioimmunoassay. [3H]Histidine was incorporated into a component that reacted with TRH antiserum and had the same retention time as synthetic TRH on reversed-phase high-performance liquid chromatography. A continuous release of TRH-IR into the culture medium was observed from islets of both 5-d-old (newborn) and 30-d-old (adult) rats with a maximum on the second day of culture (28.7 +/- 7.0 and 13.3 +/- 3.6 fmol/islet per d, respectively). The content of TRH-IR was higher in freshly isolated islets from newborn rats (22.4 +/- 2.3 fmol/islet) than in adult rat islets, which, however, increased their content from 1.3 +/- 0.5 to 7.0 +/- 0.5 fmol/islet during the first 3 d of culture. Adult rat islets maintained in medium with 20 mM glucose released significantly more TRH-IR than islets in 3.3 mM glucose medium (13.0 +/- 0.7 vs. 4.3 +/- 0.3 fmol/islet per d). In contrast, the content of TRH-IR in the islets was reversed (1.4 +/- 0.3 vs. 4.7 +/- 1.6 fmol/islet). By exposing islets from newborn rats to streptozotocin 0.7 mg/ml for 30 min, a 50% reduction of TRH-IR content in the islets compared with the non-treated islets was seen after subsequent culture for 7 d. The insulin content was reduced by 80%, while glucagon was slightly elevated. In conclusion, these results indicate that TRH is synthesized in rat pancreatic islets, and that the release is stimulated by glucose.

Growth hormone is a growth factor for the differentiated pancreatic beta-cell.

The regulation of the growth of the pancreatic beta-cell is poorly understood. There are previous indications of a role of GH in the growth and insulin production of the pancreatic islets. In the present study we present evidence for a direct long-term effect of GH on proliferation and insulin biosynthesis of pancreatic beta-cells in monolayer culture. In culture medium RPMI 1640 supplemented with 2% normal human serum islets or dissociated islet cells from newborn rats maintained their insulin-producing capacity. When supplemented with 1-1000 ng/ml pituitary or recombinant human GH the islet cells attached, spread out, and proliferated into monolayers mainly consisting of insulin-containing cells. The number of beta-cells in S-phase was increased from 0.9-6.5% as determined by immunochemical staining of bromodeoxyuridine incorporated into insulin-positive cells. The increase in cell number was accompanied with a continuous increase in insulin release to the culture medium reaching a 10- 20-fold increase after 2-3 months with a half-maximal effect at about 10 ng/ml human GH. The biosynthesis of (pro)insulin was markedly increased with a normal rate of conversion of proinsulin to insulin. It is concluded that GH is a potent growth factor for the differentiated pancreatic beta-cell.

Chromogranin-B, a putative precursor of eight novel rat glucagonoma peptides through processing at mono-, di-, or tribasic residues.

Chromogranin-B (CgB), a secretory granule protein, is normally synthesized in a variety of neuroendocrine tissues, including the pancreatic islet alpha-cells. We have demonstrated that rat CgB is expressed and extensively processed by limited proteolysis in a transplantable glucagonoma tumor line. Eight peptides (fragments 1-8) purified by HPLC from acidic tumor extracts were partially sequenced and showed homology to CgB amino acid sequences deduced from rat, mouse, and human cDNA. Similar peptides were not found in insulinomas of common origin. The determined amino acid sequence represents approximately 35% of the rat precursor CgB. Ten of a total of 231 sequenced residues deviated from the published rat cDNA sequence. The differences were clustered in 3 fragments, suggesting allelic polymorphism. Five of the 8 peptides could be derived from the precursor by processing at paired basic amino acids, but processing N-terminally at a single basic residue was also seen. One peptide equivalent to the previously reported C-terminal CgB (CCB) is released by processing at a tribasic segment. Fragment 1 containing the N-terminal sequence Ala-Pro-Val-Asp represents the actual N-terminus of CgB after removal of the putative signal peptide sequence. All processing sites used in glucagonoma tissue to derive the 8 isolated fragments were conserved between murine and human CgB. At least 3 dibasic sites present in rat, but not human, CgB sequence were actually not used. A previously reported CgB-derived pituitary peptide, GAWK, was further processed at a conserved internal dibasic site to yield fragment 6, indicating alternative processing in different tissues. The small undecapeptide, fragment 7, is 100% conserved among murine and human CgB and, thus, may have an important biological function. We conclude that CgB is extensively processed in glucagonoma tissue by limited proteolysis as prohormones at conserved basic residues. The proglucagon-converting enzymes present in transformed alpha-cells are likely candidates to be involved in tissue-specific CgB processing. Distinct biological activities of any of the CgB-derived fragments remain to be identified.

The growth hormone (GH)-binding protein cloned from human IM-9 lymphocytes modulates the down-regulation of GH receptors by 22- and 20-kilodalton human GH in IM-9 lymphocytes and the biological effects of the hormone in Nb2 lymphoma cells.

The cDNA coding for the 246-amino acid long N-terminal extracellular portion of the human (h) GH receptor, corresponding to the circulating GH-binding protein (hGHBP), was cloned by polymerase chain reaction from human IM-9 lymphocytes. The cDNA sequence was identical to that reported for human liver and placenta and demonstrated alternative splicing of exon 3. The protein with the exon 3-encoded domain was expressed and secreted in glycosylated form from baby hamster kidney (BHK) cells, purified to homogeneity, and sequenced; the amino acid sequence was identical to that predicted from liver cDNA. The cloned hGHBP competed in a dose-dependent fashion for binding of 125I-labeled 22-kilodalton (kDa) hGH, and at higher concentrations for binding of 125I-labeled 20-kDa hGH, to IM-9 lymphocytes. hGHBP decreased the association rate of [125I]hGH to the cells without decreasing the dissociation rate. hGHBP blocked the down-regulation of GH receptor in IM-9 cells by both 22- and 20-kDa hGH. hGHBP also blocked the binding of [125I]hGH to PRL receptors on Nb2 lymphoma cells and the effect of the hormone on thymidine incorporation. Binding of both 22- and 20-kDa hGH to the binding protein was demonstrated directly by immunoprecipitation with monoclonal antibody 263. The present work thus establishes the identity of the IM-9 human GHBP from those of liver and placenta, and demonstrates its ability to bind both 22- and 20-kDa hGH with good affinity and to block their biological actions mediated though both somatogenic and lactogenic receptors. The modulation of receptor down-regulation by the BP may be a relevant facet of its physiological role.

Thyrotrophin-releasing hormone in human ejaculate.

Immunoassayable TRH in human ejaculate was eluted from a gel column in a form with a molecular weight larger than that of the native peptide. With reverse-phase high-performance liquid chromatography (HPLC) the same activity co-eluted with standard TRH. Incubation of ejaculates at room temperature for 8 h was associated with a time-related increase in the total immunoassayable TRH. Analysis by HPLC of ejaculates after 12 h of incubation at room temperature indicated that, whereas the levels of the peptide co-eluting with native TRH declined with time, there was a concomitant increase in the concentration of a molecular species which also cross-reacted with the TRH antiserum, but which was more hydrophobic. The latter species is presumably identical to the tetrapeptide recently described by others and which may arise from the proteolytic degradation of secretory macromolecules. Although immunological activity was present in all six fractions of split ejaculates, the bulk of the peptide was associated with the later portions, implying a major vesicular contribution. However, secretions isolated from surgical preparations of the seminal vesicles contained undetectable levels of peptide, suggesting that the ejaculation process may represent a stimulus for its appearance in the semen. This study is further support for a local involvement of TRH in male reproductive function.

Quantifying biosynthetic human growth hormone in Escherichia coli with capillary electrophoresis under hydrophobic conditions.

A method has been developed which is able to quantitate the content of precursor biosynthetic human growth hormone (Pre-bhGH) in the cytosol of E. coli cells containing the gene for human growth hormone (hGH). The method uses hydrophobic C18 coated capillaries with native biosynthetic human growth hormone (bhGH) as an internal standard. This allows for highly robust and precise determinations as well as the evaluation of the presence of deamidated forms in the cytosol samples. Furthermore, by modifying the running buffer with zwitterionic surfactants and an organic modifier, it is possible to detect a related form with a three sulfur atom Cys-Cys bridge (trisulfide Pre-bhGH). Thus, a strong tool for monitoring the effect of fermentation conditions on the biosynthesis of bhGH is obtained.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. II. Effect of the stationary phase.

The potential value of eight commercial available polymer-based reversed-phase (RP) columns for peptide and protein separations was evaluated using crude acetic acid extracts of normal and diabetic human pancreata and mixtures of pure polypeptides as samples. All columns were characterized with acetic acid gradients in water as mobile phase, and different chromatographic profiles were obtained depending on the type of polymer column (bare or derivatized) and the type of ligand. Some of the columns were virtually free from effects related to the polymer skeleton whereas in others the separation was influenced by both the ligand and the polymeric backbone. Two selected polymeric RP columns were, together with a silica-based C4 column, further characterized with acetonitrile gradients in trifluoroacetic acid (TFA), and the separation temperature was found to have a drastic effect on the separation efficiency for proteins with mol. wt. greater than 6000 dalton. No such effect was seen for polypeptides with mol. wt. less than 6000 dalton. Mixtures of pure peptides and proteins were separated using acetic acid gradients in water, acetonitrile or isopropanol, and normally the highest efficiency was found with the use of acetonitrile as mobile phase modifier. Isopropanol was less suitable as an organic modifier. The separation of the beta-lactoglobulin A- and B-chains may be used to give a rapid estimate of the chromatographic usability of polymer-based RP-columns for peptide and protein separations in acetic acid gradients in water and in acetonitrile gradients. Recoveries for insulin, proinsulin, growth hormone, ovalbumin and human serum albumin were measured for several polymer-based RP columns eluted with acetic acid gradients in water and with acetonitrile-based mobile phases. The highest recoveries of serum albumin and ovalbumin were found after elution with acetic acid gradients in water.

Use of polymeric reversed-phase columns for the characterization of polypeptides extracted from human pancreata. I. Effect of the mobile phase.

The high-performance liquid chromatographic (HPLC) behaviour of two different styrene-divinyl-benzene-based reversed-phase (RP) columns was evaluated using crude acetic acid extracts from normal and diabetic human pancreata as samples. Acetic acid gradients in water and acetonitrile gradients in triethylammonium phosphate (TEAP) and trifluoroacetic acid (TFA) were used as mobile phases, and comparisons were made with a silica-based C4 column. When two different polymeric RP columns were eluted with acetic acid gradients in water, surprisingly similar HPLC profiles of the pancreatic extracts were obtained. Elution of the polymer-based columns with acetonitrile gradients in TFA or TEAP resulted in changes in the polypeptide selectivity of these columns, in parallel with that of a silica-based C4 column eluted under similar conditions, indicating the general usability of polymeric columns for RP-HPLC of peptides and proteins. The pronounced difference in composition between normal and diabetic samples, which also was demonstrated after size-exclusion chromatography (SEC) on a silica-based and an agarose-based high-performance SEC column, was found to be related to the different ischaemia times for the two types of pancreata.

Silica versus polymer-based stationary phases for reversed-phase high-performance liquid chromatographic analyses of rat insulin biosynthesis. A comparison of resolution and recovery.

Because of the problems caused by the irreversible binding of insulins and proinsulins to several silica-based reversed-phase columns, the use of polymeric reversed-phase columns was investigated for the analysis of rat islet polypeptides involved in insulin biosynthesis. No irreversible binding of insulins and proinsulins was observed for the polymeric reversed-phase columns, probably due to the absence of silanol groups. The six polypeptides involved in insulin biosynthesis in rat islets were equally well resolved in shallow trifluoroacetic acid-acetonitrile gradients on the silica-based Nucleosil 300-5C4 column (45 degrees C), the polymer-based Asahipak C4P-50 (25 and 45 degrees C), and ODP-50 columns (45 degrees C). In shallow triethylammonium phosphate-acetonitrile gradients (25 degrees C) satisfactory resolution of the two rat proinsulins was only obtained on the polymer-based Asahipak C4P-50 and C8P-50 columns. Increasing the separation temperature to 45 degrees C improved the separation of the two insulins and the two proinsulins in all cases. The shifts in retention times for the individual islet polypeptides observed in relation to the increased separation temperature were found to be different for the silica C4 and the polymer C4 columns. Recoveries of rat islet polypeptides were comparably high from the silica- and the polymer-based C4 columns and linear load-response curves were obtained in the microgram to picogram mass range on both columns.

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins.

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.

Reversed-phase high-performance liquid chromatographic characterization of acetic acid extracts of the normal and the diabetic human pancreas.

The combination of a divinylbenzene-based reversed-phase (RP) column and acetic acid gradients in water as mobile phase described in the accompanying paper was used for characterizing the extractable polypeptides from the normal and the diabetic human pancreas. The pancreas was lyophilized, minced and extracted three times in 3 M acetic acid. After mechanical clarification, the raw extracts were applied directly to the RP column. Alternatively, the extracts were lyophilized and subjected to size-exclusion chromatography on Sephadex G-50 in 3 M acetic acid. Two fractions with mol. wt. greater than 6000 dalton (Peak I) or with mol. wt. less than or equal to 6000 dalton (Peak II) were obtained. The Sephadex G-50 size-exclusion chromatography and the RP-high-performance liquid chromatographic (HPLC) analyses of the crude extracts from a normal pancreas clearly demonstrated the weight distribution and differences between the exocrine pancreas (containing primarily the major digestive enzymes) and the endocrine pancreas (containing insulin, glucagon, etc.). RP-HPLC analyses of crude extracts from various normal pancreatic glands resulted in very similar UV profiles, whereas those from a number of individual diabetic glands differed. Chromatograms of acetic acid extracts from normal pancreata were similar when analysed before or after lyophilization, whereas lyophilization of acetic acid extracts of diabetic glands resulted in severely obscured chromatograms. RP-HPLC analyses clearly demonstrated several differences between the diabetic and the normal pancreas. In the crude extracts, the extractable proteins from the diabetic pancreas were shifted towards lower molecular weight and/or hydrophobicity. Further, a peak co-eluting with authentic, human insulin could be demonstrated in the raw extract and in the peak II material from the normal pancreas, whereas virtually no mass signal was seen in the UV-profiles of similar materials from the diabetic gland. This finding was further verified by insulin radioimmunoassay (RIA) performed on the isolated fractions after RP-HPLC of a crude extract from a normal and a diabetic pancreas. The insulin content in the diabetic pancreas was found to be ca. 1% of that in the normal pancreas. When authentic glucagon was added to crude extracts from a diabetic pancreas, a single component was found after immediate analysis, but after several hours at room temperature the glucagon was found to be degraded. Added insulin was stable under these conditions. Similar RP analyses were performed on a silica C4 column eluted with an acetonitrile gradient in trifluoroacetic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of the haemoglobin-A1c-band in isoelectric focussing.

A double HbA1c-band was demonstrated in isoelectric focussing of blood from diabetics with a fasting blood glucose above 11 mmol/l. The same band-doubling was also demonstrated after incubation of erythrocytes in glucose/saline solutions. This finding may reflect the presence of the initial condensation product between haemoglobin and glucose, the Schiff-base intermediate.

Non-ideal behaviour of silica-based stationary phases in trifluoroacetic acid-acetonitrile-based reversed-phase high-performance liquid chromatographic separations of insulins and proinsulins.

Several C18 stationary phases were found to behave non-ideally when insulins and proinsulins were eluted with shallow acetonitrile gradients in 0.1% trifluoroacetic acid, resulting in poor peak shapes or no elution at all. With triethylammonium phosphate or ammonium sulphate as buffer components, the insulins and proinsulins were eluted with excellent peak shapes, presumably owing to better masking of residual silanol groups on the stationary phases. Similar use of trifluoroacetic acid-acetonitrile gradients on the less hydrophobic C4 or C3 stationary phases resulted in excellent peak shapes. The difficult separation of rat proinsulin I and II, which are important for the study of rat insulin biosynthesis, was only achieved with two different stationary-mobile phase combinations.

Analysis of proinsulin and its conversion products by reversed-phase high-performance liquid chromatography.

Proinsulin is synthesized in the beta-cells of the endocrine pancreas, one of the four cell types found in the islets of Langerhans. Specific enzymatic cleavage of proinsulin results in the formation of equimolar amounts of insulin and C-peptide, via several intermediate split-proinsulin forms. Most mammals produce a single insulin, but in rodents two non-allelic insulin genes are expressed. There is an inverse ratio between the two insulins in rats and mice, the reason for this being unknown. It has been suggested that differences in transcription, translation (biosynthesis) and/or posttranslational processes (enzymatic conversion, intracellular degradation) could be possible explanations. Elevated amounts of proinsulin-immunoreactive material (PIM) have been described to occur in various conditions/diseases, suggesting alterations in beta-cell function, but the composition of the secreted PIM (intact proinsulin or its intermediates) has been incompletely determined. Studies of the biosynthesis of proinsulins and their conversion with the purpose of revealing some of these points depend on accessible reversed-phase high-performance liquid chromatographic (RP-HPLC) analyses capable of separating all the relevant, closely related polypeptides involved. This review will deal with the optimization of the RP-HPLC separations as well as sample preparation and recovery. Applications of the selected methods in the study of proinsulin biosynthesis and its conversion will also be presented.

Preparative reversed-phase high-performance liquid chromatography of iodinated insulin retaining full biological activity.

Insulin monoiodinated in Tyr A14, A19, B16 and B26 can be separated from insulin and diiodoinsulins using reversed-phase high-performance liquid chromatography on LiChrosorb RP-18 columns. Monoiodoinsulins with high and low specific activities were isolated from a number of buffer systems without any reduction in binding affinity and biological activity in isolated rat fat cells. The reason for the previously observed reduction in the binding affinity was probably column bleeding, i.e., chemical degradation of the column support.

Separation, isolation and characterization of the four monoiodinated insulin tracers using reversed-phase high-performance liquid chromatography.

Baseline separation between insulin and insulin monoiodinated in Tyr A14, A19, B16 and B26 can be obtained using isocratic elution from a C18 column with triethylammonium trifluoroacetate-acetonitrile and the iodinated insulin derivatives can be isolated by lyophilization. Compared with similar tracers purified and isolated by disc electrophoresis/ion-exchange chromatography, the reversed-phase high-performance liquid chromatographically purified tracers are more homogeneous but show reduced binding affinity to adipocytes.

Recovery of polypeptides after reversed-phase high-performance liquid chromatography.

Mass recovery of individual polypeptides may be estimated under various practical conditions. With the purpose of obtaining rapid and reliable standard procedures for recovery measurements, we have compared five individual methods utilizing a silica-based stationary phase [Nucleosil C18 (7 microns)/ammonium sulphate-perchlorate-acetonitrile, pH 3.0] and a resin-based stationary phase (TSK Phenyl 5 PW RP/ammonium phosphate-acetonitrile, pH 7.0). The recoveries of insulin (6 kilodaltons), human growth hormone (22 kilodaltons) and human serum albumin (68 kilodaltons) estimated under five different experimental conditions were found to be concordant. Variations in column load, flow-rate, gradient shape and column dwell time and addition of cyclame did not increase the (reduced) recovery of serum albumin and growth hormone.

High-performance liquid chromatographic separation of membrane proteins isolated from erythrocyte ghosts.

Membrane proteins extracted from erythrocyte ghosts with sodium dodecyl sulphate (SDS), 3-(3-cholamidopropyl)-dimethylamminopropane sulfonate (CHAPS) or octylglucoside have been analyzed in various reversed-phase high-performance liquid chromatographic systems. Only SDS was able to solubilize considerable amounts of membrane proteins with mol.wt. greater than 15,000 daltons, but these membrane proteins were recovered in poor yield from a silica-based C4 column eluted with an acetonitrile gradient in trifluoroacetic acid (TFA). A resin-based phenyl column eluted with a similar TFA-acetonitrile gradient was found to be a better choice with respect to the recovery of membrane proteins with mol.wt. greater than 15,000 daltons, and when this column was eluted with an acetic acid gradient with increasing amounts of acetonitrile, erythrocyte ghost membrane proteins solubilized in SDS (mol.wt. 10,000-200,000 daltons) were separated in six major and several minor components with satisfactory recovery.

High-performance liquid chromatofocusing and column affinity chromatography of in vitro 14C-glycated human serum albumin. Demonstration of a glycation-induced anionic heterogeneity.

High-performance liquid chromatofocusing of human serum albumin (HSA) after in vitro glycation with purified [14C]glucose has shown that with increasing glycation time a progressive increase in two major anionic fractions (pI 4.8 and 4.65) occurs, while the pI 4.9 fraction decreases in parallel. As early as after 5 days of glycation time, the [14C]glucose content in the anionic fractions was markedly higher than in the pI 4.9 fraction. After 10 and 15 days of glycation, a considerable heterogeneity of 10-15 components could be demonstrated. In addition, phenyl-boronic acid (PBA) affinity chromatography was applied and an enrichment of the more glycated species could be obtained using this method. We conclude that, in contrast to previous reports, glycation of HSA induces anionic heterogeneity (in accordance with the theoretically expected loss of positively charged amino groups) and, although the efficiency in separating non-glycated from monoglycated HSA was found to be very low, an enrichment of these anionic species can be achieved using PBA affinity chromatography.