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Biomedical subjects

B H Frank

Publications and source records attributed to B H Frank.

At least 73 records · Page 4Linked to original sources
PubMed1987

Insulin secretion and clearance. Comparison after oral and intravenous glucose.

Insulin secretion and clearance in response to the administration of oral and intravenous glucose was investigated in nine normal men. C-peptide metabolic kinetics were calculated by analysis of individual C-peptide decay curves after the bolus injection of biosynthetic human C-peptide. Glucose was administered to the subjects on three occasions: as a 75-g oral dose, a 75-g i.v. infusion, and an intravenous glucose infusion at a variable rate adjusted to mimic the peripheral glucose levels obtained after the oral glucose load (matching experiment). Glucose, insulin, and C-peptide concentrations were measured for the subsequent 5 h. The glucose level after the oral glucose load (115.9 +/- 2.6 mg/dl, mean +/- SE) closely approximated that after the matching experiment (120.5 +/- 2.5 mg/dl) but was significantly lower than after 75 g i.v. glucose (127.7 +/- 3.4 mg/dl, P less than .05). Analysis of the areas under the peripheral concentration curves (60-360 min) showed that the responses of both insulin (52.7 +/- 5.6 and 46.5 +/- 4.5 pmol.ml-1.300 min-1) and C-peptide (252.7 +/- 27.5 and 267.0 +/- 21.6 pmol.ml-1.300 min-1) were not significantly different after the oral and 75-g i.v. glucose studies, respectively, whereas in the matching experiment, both the insulin (26.1 +/- 3.9 pmol.ml-1.300 min-1) and C-peptide (178.0 +/- 18.9 pmol.ml-1.300 min-1) responses were lower (P less than .05) than in the other two studies. Insulin secretory rates were derived from peripheral C-peptide concentrations with an open two-compartment model and individually derived model parameters. (ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1987

Characterization and use in immunoradiometric assay of monoclonal antibodies directed against human proinsulin.

Proinsulin in human serum is heterogenous. Existing assay methods do not distinguish between the various forms intermediate on the pathway of processing from proinsulin to insulin. We report the production of mouse monoclonal antibodies against human proinsulin with biosynthetic human proinsulin (produced by recombinant DNA technology) as immunogen. Four monoclonal antibody-producing cell lines were obtained from two separate fusions. Two of the antibodies had affinity constants for intact proinsulin of 3.1 X 10(10) and 5.6 X 10(10) L/mol and bound all forms of proinsulin and insulin. Cross-reactivity with intact proinsulin was greater than with 65-66 and 32-33 split proinsulins and des 64-65 and des 31-32 proinsulins. The third antibody had an affinity constant for intact proinsulin of 6.3 X 10(10) L/mol and reacted only with intact proinsulin, 65-66 split proinsulin, and des 64-65 proinsulin; there was no reaction with 32-33 split proinsulin, des 31-32 proinsulin, or C-peptide. The fourth antibody reacted only with intact proinsulin and had an affinity constant of 4.1 X 10(8) L/mol. We report the use of two antibodies in a sensitive two-site immunoradiometric assay for intact proinsulin. Insulin, C-peptide, and 32-33 split proinsulin did not react in the assay up to a concentration of 100 pM. The 65-66 split proinsulin and des 64-65 proinsulin reacted with a potency of approximately 55% relative to intact proinsulin. Serum proinsulin concentrations measured with this assay were compared with those determined by an alternative method that detects only split proinsulins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1986

Proinsulin radioimmunoassay in the evaluation of insulinomas and familial hyperproinsulinemia.

Two new radioimmunoassays for human proinsulin (hPI) have been developed and used to study patients with islet cell tumors and familial hyperproinsulinemia. Both antisera were adsorbed against human C-peptide conjugated to Sepharose, following which cross-reactivity to insulin and C-peptide was less than 0.001%. Antiserum 18D recognized the junction between the insulin B-chain and C-peptide and provided fivefold greater sensitivity than our previously reported hPI assay. Antiserum 11E recognized a determinant which includes or is adjacent to the A-chain-C-peptide junction or which is specified by the tertiary structure. In all 20 patients studied with surgically confirmed islet cell tumors, fasting plasma proinsulinlike material (PLM) was abnormal (greater than 3 SD from the mean measured in either lean or obese subjects) in both assays. This provided better discrimination than has been reported for PLM measured by gel filtration (abnormal in 13 of 14 of the present samples) with a considerably less laborious procedure. Samples from two families in which a mutant proinsulin is present in the circulation have immunoreactivity in the two assays consistent with previous identification of the molecule as an A-chain-C-peptide-linked intermediate of proinsulin conversion. The immunoreactivity of a sample from another family in which large amounts of proinsulin circulate are consistent with an intact molecule being the predominant form. This assay will be useful for confirming the diagnosis of insulin-secreting tumor in patients suspected of recurrent fasting hypoglycemia and in physiologic studies of proinsulin secretion.

Adenoma, Islet Cell↗
PubMed1986

Effect of experimental diabetes on insulin binding by renal basolateral membranes.

Removal of insulin from the peritubular vessels involves binding of insulin to specific receptors in the basolateral membranes (BLM); this is followed by phosphorylation of the receptor which may mediate the actions of the hormone. In most tissues receptor number is regulated by plasma insulin levels and is increased in insulinopenic diabetics. To determine whether cortical BLM insulin receptors are similarly regulated, we studied insulin binding to receptors in BLM from normal control rats and rats with streptozotocin diabetes of varying severity. Specific binding of insulin did not differ between control and modestly insulinopenic diabetics but was increased significantly in the severely insulinopenic diabetics. Insulin treatment returned binding to normal. Scatchard analysis suggested an increase in the binding capacity of the severe diabetic BLM rather than an increase in affinity for insulin. This latter was confirmed by competitive experiments in which similar displacement curves were obtained with control and diabetic membranes. Insulin removed by glomerular filtration binds to specific receptors in the luminal membranes but unlike BLM receptors, phosphorylation of these luminal receptors has not been observed. To determine whether luminal and BLM receptors differ structurally, binding sites in both membranes were affinity labelled with 125I-insulin and the cross linking agent, disuccinimidyl suberate, and subjected to SDS-polyacrylamide gel electrophoresis in the presence of a reducing agent. Autoradiograms revealed that the major specifically labelled subunit in both membranes is a 135,000 Mr species which is more abundant in the BLM. We conclude that insulin receptors in cortical BLM respond to severe insulinopenic diabetes as do receptors in most other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1986

Intestinal mucosa is a target tissue for pancreatic polypeptide.

Studies were carried out to identify mammalian tissues capable of specifically binding mammalian pancreatic polypeptide (PP). Bovine PP (bPP) radiolabeled with 125I was purified by HPLC to yield [125I]iodo-(Tyr-27) bPP. The label was injected into three pairs of fasted littermate dogs and allowed to circulate for 5 min. One of the dogs was a control which received an excess of unlabeled porcine PP to provide competition for receptor binding. Unbound bPP was removed by perfusion with Krebs-Ringer bicarbonate and the tissue fixed in situ with Karnovsky's fixative. Tissue samples from various organs were removed, weighed, and counted. The entire gastrointestinal tract demonstrated high levels of 125I after injection of the labeled peptide. The duodenum, jejunum, ileum, and colon were the only tissues to exhibit specific binding of bPP. These tissues (mucosal and muscle layers) from experimental animals exhibited 31-76% higher binding than the corresponding tissues from the control animals. Sections of the gastrointestinal tract were scraped to separate the mucosal layer from the underlying muscle layer. The mucosal layer of the duodenum, jejunum, and ileum exhibited 145-162% increases in binding compared to the control animals. The muscle layer of these tissues demonstrated no significant increase. These findings demonstrate that mucosal layer of the small intestine is a target tissue for mammalian PP.

Animals↗
PubMed1986

The effects of proinsulin pretreatment on the combined actions of insulin and proinsulin in normal man.

With the recent availability of biosynthetic human proinsulin there has been a renewed interest in evaluating its metabolic effects, either alone or in combination with insulin. It has been suggested that pretreatment with proinsulin enhances the hypoglycemic response to subsequently administered insulin. On the other hand, the simultaneous administration of proinsulin and insulin has additive, not synergistic, effects. To clarify this question we used the euglycemic glucose clamp technique in 10 normal subjects to compare the steady state effects on glucose disposal of combined infusions of insulin (0.54 microgram/M2 . min, equivalent to 15 mU/M2 . min) and proinsulin (2.75 micrograms/M2 . min) given both simultaneously and sequentially. The mean +/- SEM steady state glucose disposal rates were similar whether the two hormones were given simultaneously (7.2 +/- 0.7 mg/min . kg), after proinsulin pretreatment (7.7 +/- 0.7 mg/min . kg), or after insulin pretreatment (7.1 +/- 0.7 mg/min . kg). The serum proinsulin concentration of 5.39 +/- 0.3 pmol/ml during the infusion of proinsulin alone was unchanged by the simultaneous infusion of insulin, suggesting that in the doses used, insulin did not affect proinsulin clearance. We conclude that in normal subjects there is no enhancement of the combined action of insulin and proinsulin to stimulate glucose disposal by pretreatment with proinsulin or insulin.

Adult↗
PubMed1986

In vivo deactivation of proinsulin action on glucose disposal and hepatic glucose production in normal man.

We have studied the deactivation of the in vivo actions of insulin and biosynthetic human proinsulin (recombinant DNA) to stimulate the glucose disposal rate (GDR) and to inhibit hepatic glucose output (HGO) in man. Twelve healthy, lean, young subjects were studied using a modification of the euglycemic glucose clamp technique. Subjects received 4-h infusions on separate occasions of insulin (15 mU/m2/min equivalent to 0.54 microgram/m2/min) or proinsulin (2.75 micrograms/m2/min), achieving steady-state serum levels of 32 +/- 3 microU/ml (equivalent to 0.23 +/- 0.02 pmol/ml) and 3.7 +/- 0.2 pmol/ml, respectively. Suppression of HGO was similar (83-84%) with proinsulin and insulin, but stimulation of GDR above basal was greater with insulin (3.41 +/- 0.43 versus 1.98 +/- 0.28 mg/kg/min, P less than 0.001). Following cessation of the hormone infusions, serum proinsulin concentration fell in a biphasic fashion with half-times of 25 and 146 min for the two phases. Serum half-disappearance time for insulin was 5 min. Deactivation of the hormone's effects to stimulate GDR was 50% complete by 35 min after insulin and 71 min after proinsulin. In contrast, 50% of the recovery times for the effect on suppression of HGO were 55 min after insulin and 188 min after proinsulin. Serum glucagon levels did not differ significantly after the insulin and proinsulin infusions. In summary: (1) Deactivation of proinsulin and insulin's effects to suppress HGO proceeds more slowly than deactivation of their effects to stimulate GDR; and (2) There is a markedly prolonged and disproportionately delayed deactivation of proinsulin's effects on suppression of HGO. This later finding may prove of therapeutic value in the treatment of diabetes mellitus.

Adult↗
PubMed1986

A sensitive radioimmunoassay for human proinsulin, with sequential use of antisera to C-peptide and insulin.

In this assay for immunoreactive human proinsulin (IRPI), it first is separated from plasma by use of an antiserum to human C-peptide. An immunoprecipitate is then formed by using a precipitating antiserum and polyethylene glycol, after which IRPI is dissociated from the antiserum by incubation in warm HCl, pH 2.0. The resulting mixture is assayed for insulin immunoreactivity by a double-antibody tracer-competition method involving incubation for four days with a high-affinity anti-insulin antiserum. Human proinsulin of recombinant-DNA origin is used as the standard. Added C-peptide at supraphysiological concentrations did not interfere with or react in the assay. Human insulin cross reacted by 1.5%. The detection limit for IRPI (2 SD from zero-dose binding) is 3 pmol/L. Proinsulin conversion intermediates are measured nearly as well as intact proinsulin. IRPI concentrations in 10 nondiabetic human subjects averaged 12.0 (SEM 1.6) pmol/L. The ratio of proinsulin to immunoreactive insulin averaged 14.3 (SEM 2.2)%. After intravenous arginine, the increase in proinsulin was less than that of insulin, and it declined more slowly.

Arginine↗
PubMed1985

Developing rat brain binds monoiodinated insulin isomers similarly to other extrahepatic target tissues.

We recently reported a series of binding and metabolic studies which led to the conclusion that the developing rat brain is a target tissue for insulin. Since insulin target tissues (extrahepatic) are capable of differentiating between various monoiodoinsulin isomers, we measured the binding of the B26 monoiodoinsulin isomer compared to the A14 in newborn rat brain preparations to determine if the developing rat brain shared the same relative binding of these isomers (viz. B26 greater than A14) with other extrahepatic tissues. The B26 isomer bound 1.57, 1.50 and 1.34 times as much as did the A14 to brain membranes, glia and neurons, respectively, whereas both isomers were bound equally by liver plasma membranes. Competition-inhibition curves were generated using homologous unlabeled (127I) insulin isomers. Binding of the B26 isomer was greater than the A14 at all concentrations. Scatchard plots showed that the receptor concentrations for the two isomers were similar, and affinity profiles showed that the differences in binding could be accounted for by the greater affinity of the receptors for the B26 isomer. The results indicate that the developing rat brain shares with other extrahepatic insulin target tissues a greater affinity for B26 monoiodoinsulin isomer compared to A14. Future studies of insulin binding should avoid using mixtures of iodinated insulins so that a uniform interpretation of data is made possible.

Animals↗
PubMed1985

Receptor binding and biological potency of several split forms (conversion intermediates) of human proinsulin. Studies in cultured IM-9 lymphocytes and in vivo and in vitro in rats.

The biological activities of several derivatives of human proinsulin (HPI) containing peptide bond cleavages or peptide deletions in the connecting peptide region were examined in vivo in rats and in several in vitro systems. The two derivatives which were tested in vivo, split (32-33)HPI and des-(64,65)HPI, both demonstrated greater potency in lowering blood glucose than did intact HPI. The receptor binding affinities of split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, des-(33-56)HPI, des-(31,32)HPI, split (32-33)HPI, and split (56-57)HPI were examined in cultured IM-9 lymphocytes, freshly isolated rat adipocytes, and purified rat liver membranes and were compared to the binding of intact HPI and insulin. In these systems, HPI averaged approximately 1% of the activity of insulin. Modification of proinsulin in the connecting peptide region near the A-chain of insulin to form split (65-66)HPI, des-(57-65)HPI, des-(64,65)HPI, or des-(33-56)HPI resulted in an increase in affinity for receptor binding ranging from 11 to 27-fold over that of intact HPI. In contrast, modifications near the B-chain of insulin to form either des-(31,32)HPI or split (32-33)HPI resulted in roughly a 5-fold increase in affinity, whereas a cleavage within the connecting peptide to form split (56-57)HPI showed only a 2-fold increase in affinity as compared to intact HPI. The biological potencies of these materials were examined in isolated rat adipocytes. At high concentrations (10(-7) M), each derivative produced the same maximal response. At lower concentrations, differences in the relative potencies paralleled the differences in receptor binding affinity previously noted.

Adipose Tissue↗
PubMed1985

The preparation and characterization of mono-iodinated photoreactive analogs of insulin.

Photoreactive derivatives of insulin (B29-(p-azidobenzoyl-insulin) iodinated primarily in either the B26 or A14 tyrosine of insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The binding affinities and photoaffinity labeling characteristics of these derivatives were studied in isolated rat adipocytes. Under nonreducing conditions, three forms of the insulin receptor were labeled equally by the B26-derivative, the A14-derivative, and the mixture of the iodinated derivatives. When dithiothreitol was used to reduce the radiolabeled receptors, the radioactivity associated with the binding subunit was much less than that in the intact receptor and the magnitude of the decrease was proportional to the amount of iodine in the A chain of the photoderivatives. Use of the photoreactive derivative iodinated primarily in the B26 position resulted in greater labeling of insulin receptor subunits since most of the radioactivity (80%) remained associated with the receptor upon reduction.

Adipose Tissue↗
PubMed1985

Biochemical and clinical implications of proinsulin conversion intermediates.

Since a complete map of insulin-related peptides in humans requires consideration of proinsulin, Arg32/Glu33-split proinsulin, Arg65/Gly66-split proinsulin, des-Arg31,Arg32-proinsulin, des-Lys64, Arg65-proinsulin, and insulin, we applied high performance liquid chromatography coupled with radioimmunoassay to investigate the formation of proinsulin conversion intermediates in vitro and in vivo. Kinetic analysis of proinsulin processing by a mixture of trypsin and carboxypeptidase B (to stimulate in vivo processes) revealed (a) a rapid decline in proinsulin concommitant with formation of conversion intermediates, (b) formation of des-Arg31, Arg32-proinsulin and des-Lys64,Arg65-proinsulin in the ratio 3.3:1 at steady state, and (c) complete conversion of the precursor to insulin during extended incubation. Studies on normal human pancreas identified a similar ratio of des-Arg31,Arg32-proinsulin to des-Lys64,Arg65-proinsulin (approximately 3:1), whereas two insulinomas contained sizable amounts of des-Arg31,Arg32-proinsulin, but barely detectable amounts of des-Lys64,Arg65-proinsulin. None of the tissues contained measurable quantities of Arg32/Glu33- or Arg65/Gly66-split proinsulin. Analysis of plasma from three diabetic subjects managed by the intravenous infusion of human proinsulin revealed less than 1% processing of the circulating precursor to conversion intermediates and no processing of the precursor to human insulin. Nevertheless, analysis of plasma from the same subjects managed by the subcutaneous infusion of proinsulin revealed 4-11% processing of the precursor to intermediates that had the properties of des-Arg31,Arg32-proinsulin and Arg65/Gly66-split proinsulin. We conclude that (a) processing of proinsulin to insulin in vivo as in vitro likely occurs by preferential cleavage at the Arg32-Glu33 peptide bond in proinsulin, (b) proinsulin is inefficiently processed in the vascular compartment, and (c) subcutaneous administration of the precursor can result in the formation of conversion intermediates with the potential for contributing to biological activity.

Carboxypeptidases↗
PubMed1985

A radioimmunoassay for circulating human proinsulin.

A radioimmunoassay for human proinsulin (hPl) has been developed using biosynthetic hPl prepared by recombinant DNA technology as immunogen, standard, and tracer. The antiserum was raised in a guinea pig and then adsorbed against insulin and C-peptide conjugated to Sepharose to improve its specificity. After adsorption of the antiserum, the cross-reactivities to insulin and C-peptide were each less than 0.2%. Competition studies using in vitro enzymatically split forms of proinsulin demonstrated that the major antigenic determinant recognized was the junctional region between the B-chain of insulin and the C-peptide. The range of the assay extended from 10 to 150 fmol/tube, with a 50% displacement of 45-55 fmol/tube. This sensitivity proved suitable for measurements of serum hPl concentrations during infusion of biosynthetic hPl into normal subjects and type I diabetic subjects. Eighty-five of 89 serum samples from the normal subjects and each of 20 samples from diabetic subjects diluted in parallel with the hPl standard. Since the direct assay sensitivity was not sufficient for measurement of endogenous hPl levels, a simple procedure for quantitative extraction of proinsulin-like material (PLM) from up to 40 ml of plasma on insulin antibody-Sepharose columns was developed. Logit-log slopes were calculated for dilutions of extracts of samples collected in the fasting state and 60 min after 75 g or oral glucose from eight healthy subjects. The slopes of 15 of the 16 samples did not differ significantly from the slope of the hPl standard.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Human proinsulin standards.

Two new batches of pancreatic human proinsulin have been compared with biosynthetic human proinsulin. Standards of these three proinsulin preparations were made on the basis of quantitative amino-acid analyses and compared in two proinsulin radioimmunoassays with a proinsulin standard prepared 14 years ago. The curves of the new standards were superimposable. However, they differed considerably from the curve of the old standard which proved to be only one-third of the strength of the new standards, thereby leading to a threefold over-estimation of proinsulin concentrations when the old standard is used. We conclude that the new standards should replace previously used standards.

Amino Acids↗
PubMed1984

Proinsulin in human serum: problems in measurement and interpretation.

The immunoreactivity of an extracted pancreatic human proinsulin standard in an indirect immunoradiometric assay was found to be at least one hundred times higher than that of biosynthetic human proinsulin. Limited tryptic digestion of the biosynthetic proinsulin increased its immunoreactivity in the assay and this was attributed to the production of partially cleaved proinsulin molecules which still retained the C-peptide moeity. This inference was confirmed by the finding that pure samples of 65/A1 and 32/33 split proinsulins reacted in the assay very similarly to the pancreatic proinsulin standard. The implications of these results are that the immunoassays of proinsulin using antisera to C-peptide may recognise the intact proinsulin molecule very poorly, if at all, and that the 'proinsulin' measured by such assays of human serum may be largely if not entirely intermediates of proinsulin cleavage.

C-Peptide↗
PubMed1984

Isolation and characterization of a genetic variant of bovine proinsulin.

A genetic variant of bovine proinsulin has been isolated using preparative reverse-phase HPLC. The new proinsulin (bovine proinsulin II) differs from the known proinsulin (bovine proinsulin I) by a single amino acid residue at position C-48 in the connecting peptide. The amino acid replacement is a leucine substitution for proline. The two proinsulins were found in a ratio of approximately 9:1, proinsulin I: proinsulin II. No chemical or biological differences were observed for the two proinsulins other than their different elution times on reverse-phase HPLC.

Amino Acid Sequence↗
PubMed1984

Receptor binding and biological activity of specifically labeled [125I]- and [127I]monoiodoinsulin isomers in isolated rat adipocytes.

The receptor binding characteristics and biological activity of single site monoiodinated insulin was investigated using isolated rat adipocytes. Pork insulin was iodinated by the lactoperoxidase method, and each of the four monoiodo derivatives (iodotyr-A14, iodotyr-A19, iodotyr-B16, and iodotyr-B26) was isolated by high performance liquid chromatography. Both radioactive 125I-labeled and nonradioactive 127I-labeled iodoinsulins were prepared. In competitive binding experiments in which each 125I-labeled iodoisomer competed for receptor binding at 15 C with its homologous 127I-labeled iodoisomer, the concentrations of homolog required for 50% inhibition of tracer binding were 1.67 +/- 0.06, 2.14 +/- 0.23, and 2.35 +/- 0.27 X 10(-9) M for the B26, A14, and B16 iodoisomers, respectively (mean +/- SEM; n = 3). Scatchard analysis of the homologous competition curves using a two-site model indicated that there was no difference in the total number of specific sites to which each isomer could bind or in their affinity for binding to the low affinity site. However, a significantly (P less than 0.05) higher affinity for (B26)iodoinsulin binding to the high affinity site was observed compared to either the (A14)- or (B16)iodoisomers (Kd values of 1.09 +/- 0.18, 2.09 +/- 0.40, and 2.24 +/- 0.15 X 10(-9) M for B26, A14, and B16, respectively). Degradation of the isomers by adipocytes incubated at 37 C occurred at a rate proportional to their receptor binding affinity. The biological activity of iodoinsulins was also evaluated, based on the ability either to stimulate glucose oxidation or to exert an antilipolytic effect. The 127I-labeled (B26)iodoisomer exhibited the greatest potency in both assays compared to either (A14)- or (B16)iodoinsulins, consistent with its higher apparent affinity for receptor binding. The receptor-binding activity and biological potency of unmodified native insulin and 127I-labeled (A14)-iodoinsulin were directly compared. Identical results were obtained for each in both types of assay, suggesting that (A14)iodoinsulin is a valid tracer for insulin with isolated adipocytes. We conclude that in isolated rat adipocytes, (B26)iodoinsulin has greater activity than either unlabeled insulin or the other iodinated derivatives. (A14)Iodoinsulin is indistinguishable from native insulin; (B16)iodoinsulin has slightly reduced activity, while that of (A19)iodoinsulin is considerably reduced.

Adipose Tissue↗