Biosynthetic Human Proinsulin Research Articles

The Insulin Receptor Isoform A: A Mitogenic Proinsulin Receptor?

The existence of a precursor (“proinsuline”) for the putative hormone “insuline” was already postulated in 1916 (before the isolation of insulin) by Sir Edward Sharpey Schafer (1). It took another half-century for proinsulin to be isolated and characterized by Don Steiner (for recent review, see Ref. 2). Historically and currently, proinsulin has been regarded as a weak-affinity ligand for the insulin receptor (IR) with a low metabolic potency (3). The availability of biosynthetic human proinsulin in the 1980s allowed the exploration of possible clinical usefulness of human proinsulin as a soluble, intermediate acting insulin agonist by the Eli Lilly Co. (Indianapolis, IN) in 1984 (3). Low receptor affinity (and hence low receptor-mediated endocytosis) translated into a longer half-life and slower metabolic clearance rate. It also showed a marginal hepatoselectivity. Lack of demonstrable clinical advantage and an increase in the occurrence of acute myocardial infarctions led to a close of the clinical trials in early 1988. Proinsulin as an active biological agent went into oblivion since, with the notable exception of the work by the lab of Flora de Pablo in Madrid, suggesting that proinsulin is an active agent of its own in early embryonic development even before the appearance of the pancreatic primordium (4). Renewed interest in the actions of proinsulin is likely to arise from the publication in this issue of an article by Malaguarnera et al. (5) in the group that has pioneered a series of elegant studies on the biochemical properties (ligand binding selectivity and signal transduction) of the two splice variants of the IR, isoforms A and B (IR-A and IR-B), and their homodimers and heterodimers with IGF-I receptors (for review, see Ref. 6). IR-A and IR-B differ by the absence or presence of a 12-amino acid peptide sequence encoded by IR exon 11. The IR-A is predominantly expressed during prenatal life, and in adult life is expressed preferentially in noninsulin target tissues. It has a greater affinity for IGFs than IR-B, especially for IGF-II. IR-A appears to be especially potent in mitogenic signaling and may play a role in cancer progression (6). In this new study, using fibroblasts devoid of IGF-IR (R ) stably transfected with similar amounts of IR-A or IR-B, the authors report that proinsulin binds to IR-A with a 7-fold higher affinity than IR-B [dissociation constant (Kd) for IR-A 4.6 nM vs. Kd for IR-B 28.3 nM] and activates IR-A with a greater potency than IR-B, behaving essentially like IGF-II except for lack of binding to the IGF-I receptor, as previously shown (3). This is the first direct comparison of the affinities of the two receptor isoforms for proinsulin. It is interesting to compare the data with historical figures using cells that were later shown to express predominantly one or the other isoform or both. The authors define binding of proinsulin to IR-A as “high affinity.” One may question this statement given a Kd of 4.6 nM. In my laboratory, the Kd for high-affinity insulin binding to IR-A has been consistently around 0.2 nM (7, 8), in agreement with Ref. 9, which is 23 times lower than the proinsulin Kd reported here. The authors do not report on the relative values of proinsulin’s Kd to insulin’s Kd in their cells. Inspection of figure 1 in Ref. 5 suggests about 10% for IR-A and 2% for IR-B. Relative binding of porcine proinsulin relative to porcine insulin in IM-9 lymphocytes, which express only IR-A, was initially reported to be 10% (10). In liver plasma membranes, which predominantly express IR-B, it was 3–5% (due to a 20-fold lower asso-

Hypoglycemic Potency and Metabolic Clearance Rate of Intravenously Administered Human Proinsulin and Metabolites*

Since circulating proinsulin has been suggested to be important in the pathogenesis of noninsulin-dependent diabetes, and biosynthetic human proinsulin (HPI) may have a therapeutic role in patients with diabetes mellitus, the biological activity of proinsulin metabolites is of potential significance. Moreover, recent studies have suggested that the majority of circulating proinsulin immunoreactivity consists of metabolites. We, therefore, compared the blood glucose-lowering ability and MCR of the two proinsulin metabolites des-(31,32)HPI and des-(64,65)HPI with intact HPI in seven anesthetized dogs after an overnight fast. Intravenous bolus injections of 12.5 micrograms HPI/kg BW and equimolar amounts of des-(31,32)HPI and des-(64,65)HPI were given on three separate occasions. In addition to blood glucose, des-(31,33)HPI, des-(64,65)HPI, and HPI were measured using an insulin RIA and peptide-specific standard curves. Kinetic parameters were derived by fitting two exponentials to the respective decay curves. The MCR of HPI (3.3 +/- 0.1 ml/kg.min) was significantly lower (P less than 0.05) than that of des-(64,65)HPI (6.4 +/- 0.6 ml/kg.min), but was not significantly different from that of des-(31,32)HPI (3.8 +/- 0.4 ml/kg.min). The MCR of biosynthetic insulin (17.2 +/- 1.8 ml/kg.min), as measured in three of the dogs, was higher than that of HPI or the two metabolites. The blood glucose-lowering ability (defined as nadir glucose/fasting glucose, expressed as a percentage) of des-(64,65)HPI (49.3 +/- 5.0%) was significantly greater (P less than 0.05) than that of intact HPI (87 +/- 2.2%), and the glucose-lowering ability of des-(31,32)HPI (75.2 +/- 3.8%) was intermediate. In conclusion, HPI metabolites are more biologically active than intact HPI. The extent of in vivo conversion of proinsulin to metabolites may enhance the biological activity of proinsulin and, thus, have physiological, pathophysiological, and therapeutic significance.

Comparisons of Insulin and Biosynthetic Human Proinsulin Actions in Cultured Hepatocytes

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.

Metabolic effects of biosynthetic human proinsulin in type 2 diabetes mellitus

Due to a longer plasma half-life and half-time of action on glucose metabolism biosynthetic human proinsulin was thought to be an alternative to long-acting insulin preparations. To test this hypothesis we studied 23 type 2 diabetic patients who could no longer be treated sufficiently with oral hypoglycaemic agents. After an initial 1 week phase during which all patients received protamine bound insulin twice daily, the patients either continued on NPH insulin (Group A, n = 11) or were randomly switched to human proinsulin (Group B, n = 12). Glucose profiles and peripheral and hepatic insulin sensitivity (euglycaemic clamp: 120 mU m-2 min-1) were measured at the end of the initial period (Time 1) and 1 week later (Time 2). The insulin-mediated glucose disposal (RD) was not changed after either treatment (group A: 176 +/- 18 vs. 192 +/- 19 mg m-2 min-1; group B: 175 +/- 15 vs. 174 +/- 12 mg m-2 min-1 for times 1 and 2, respectively, NS). Suppression of hepatic glucose output (HGO) was complete in both groups at both times. Fasting blood glucose levels (FBG) and basal HGO were equally low at times 1 and 2 (group A: FBG 118 vs. 123 mg dl-1, BHGO 81 vs. 79 mg m-2 min-1; group B: FBG 118 vs. 106 mg dl-1, BHGO 87 vs. 84 mg m-2 min-1; NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Scintigraphic distribution of 123 I labelled proinsulin, split conversion intermediates and insulin in rats.

Insulin, biosynthetic human proinsulin and 2 human proinsulin conversion intermediates, des (64, 65) human proinsulin and des (31, 32) human proinsulin, were labelled with 123 I and the derivatives monosubstituted on Tyr A14 were purified by reverse phase high performance liquid chromatography. The four tracers were injected into anaesthetized rats via a jugular or a portal vein and time activity curves were generated for the liver and kidneys using a gamma camera and an online computer. Liver extraction coefficients varied in the order insulin (38%), des (64, 65) human proinsulin (11.7%), des (31, 32) human proinsulin (3.2%), human proinsulin (1.6%); whereas half-life of hepatic activity varied in the reverse order, from 6 min for insulin, to 45 min for human proinsulin. As expected for a non-receptor mediated process, kidney extraction varied conversely to liver extraction, being highest for human proinsulin and lowest for insulin. It is concluded that the kinetics of human proinsulin conversion intermediates depends upon the site of cleavage and deletion and is intermediate between those of insulin and intact human proinsulin.

The effect of biosynthetic human proinsulin on intermediary metabolism

The effect of biosynthetic human proinsulin on intermediary metabolism

Semisynthetic human [[3H2]Phe1]proinsulin.

Biosynthetic human proinsulin (obtained by recombinant DNA techniques) was used as the starting material for the preparation, by semisynthetic methods, of [3H]proinsulin with the label at the N-terminal phenylalanine residue. The labelled proinsulin was characterized by its retention time on reversed-phase h.p.l.c., by polyacrylamide-gel electrophoresis, by the time course of its enzymic conversion into insulin and by chromatographic analysis after extensive proteolytic degradation. The specific radioactivity of the product was 5 Ci/mmol. Experimental details of the preparation of human [[3H]Phe1]proinsulin, the isolation of this product by isocratic h.p.l.c. and gel filtration, and further characterization of protein intermediates have been deposited as supplement SUP 50138 (12 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on prepayment [see Biochem. J. (1987) 241, 5].

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)

Regulation of ketogenesis, gluconeogenesis, and glycogen synthesis by insulin and proinsulin in rat hepatocyte monolayer cultures.

The metabolic actions of porcine insulin and biosynthetic human proinsulin on fatty acid and glucose metabolism were studied in rat hepatocytes cultured in monolayer for 24 h. Our aim was to establish whether proinsulin action in the liver is similar to insulin action and whether the relative potencies of the two hormones are the same for different metabolic processes. Proinsulin and insulin exerted a similar maximal inhibitory effect on ketone body formation from palmitate and on gluconeogenesis from pyruvate. The half-maximal effective concentration of proinsulin was 11-13 times that of insulin. The antiketogenic effects of insulin and proinsulin were associated with an increased glycerol 3-phosphate content and a decreased affinity of carnitine palmitoyltransferase for its substrate palmitoyl-CoA. When the basal rate of ketogenesis was increased with isobutyl methylxanthine, the half-maximal effective concentrations of both proinsulin and insulin were decreased, but the relative potency of the two hormones was unchanged. Proinsulin and insulin exerted similar maximal stimulatory effects on glycogen synthesis and on the activities of pyruvate kinase, glucose 6-phosphate dehydrogenase, phosphogluconate dehydrogenase, and malic enzyme. The half-maximal effective concentration of proinsulin was 10-30 times that of insulin. These findings are consistent with receptor binding studies on liver membranes that suggest that proinsulin interacts with insulin-specific and not proinsulin-specific receptors. Our findings also suggest that proinsulin action does not differ from insulin action at a postreceptor site.

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.

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.

Kinetics of biosynthetic human proinsulin action in isolated rat adipocytes

Kinetics of biosynthetic human proinsulin action in isolated rat adipocytes

Kinetics of biosynthetic human proinsulin action in isolated rat adipocytes.

The binding and biologic potencies of human biosynthetic proinsulin (HPro) were determined in isolated rat adipocytes. At both 16 degrees C and 37 degrees C, proinsulin was found to have 3% (on a molar basis) the potency of porcine insulin for displacing bound [(125I)TyrA14]-insulin from insulin receptors. Human biosynthetic proinsulin also had 3% of the molar potency of insulin for stimulation of deoxyglucose transport (EC50 = 8.8 +/- 0.05 X 10(-11) M for insulin and 2.9 +/- 0.55 nM for HPro). However, both hormones produced the same maximal effect on glucose transport. In order to determine if the delay in onset and persistence of proinsulin action seen in vivo was due to any differences at the cellular level, the time course of HPro action on glucose transport was determined. Biologically equivalent submaximal concentrations of insulin (0.166 nM) and HPro (4.44 nM) gave identical time courses for stimulation of deoxyglucose transport at 37 degrees C with half-maximal effects at 4 min and full effects by 30 min. Maximally stimulating concentrations of insulin (1.66 nM) and HPro (22.2 nM) also had superimposable time courses. Deactivation of stimulated glucose transport was determined by incubating equivalent concentrations of insulin (0.166 and 1.66 nM) and HPro (4.44 and 22.2 nM) until full stimulation was achieved, washing cells free of unbound hormone, and initiating dissociation and deactivation by resuspension in hormone-free buffer. Both the absolute activities of transport and rates of deactivation were the same for insulin and HPro. At the submaximal concentrations, 50% of the hormones' effects were lost by 20 min, while 50 min was required after maximal stimulation for 50% deactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of circulating human proinsulin concentrations using a proinsulin-specific antiserum.

Antibodies have been raised against biosynthetic human proinsulin that show less than 1% cross-reactivity with human insulin and C-peptide. A sensitive (IC50 0.16 pmol/ml; minimum detectable concentration 0.004 pmol/ml) radioimmunoassay has been developed using this antiserum and 125I-proinsulin that will measure proinsulin-like immunoreactivity in human serum without the need for prior separation of insulin or C-peptide. In healthy, fasted subjects (N = 23), the serum proinsulin concentration was 0.015 +/- 0.001 pmol/ml (mean +/- SEM). In six healthy subjects, serum proinsulin rose to 250% of basal after 120 min in response to 100 g oral carbohydrate, but to only 130% after 60 min following 25 g oral carbohydrate. The proinsulin/total immunoreactive insulin ratio and the proinsulin/C-peptide ratio fell sharply after both high and low carbohydrate loads. Endogenous human serum proinsulin-like immunoreactivity released into the circulation after 100 g carbohydrate was eluted from a Mono Q high-performance, ion-exchange column with the same retention time as biosynthetic human proinsulin. Treatment of biosynthetic proinsulin with trypsin under mild conditions led to a decrease in proinsulin-like immunoreactivity concomitant with an increase in C-peptide and insulin-like immunoreactivity, indicating that the proinsulin-specific antiserum did not preferentially recognize intermediates of proinsulin cleavage.

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes.

The receptor binding characteristics and biologic protency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15 degrees C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 +/- 0.8 X 10(-7) M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 +/- 0.3 X 10(-9) M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37 degrees C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 +/- 0.4 X 10(-8) M, whereas the same response was observed at an insulin concentration of 5.2 +/- 1 X 10(-11) M. Proinsulin also demonstrated an antilipolytic potency that was less than 1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes

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.

The effects of biosynthetic human proinsulin on carbohydrate metabolism.

Large quantities of biosynthetic human proinsulin have recently become available through recombinant DNA technology. Since the in vivo effects of human proinsulin have not been studied in man, we compared the dose-response relationship for stimulation of glucose disposal and suppression of hepatic glucose output by proinsulin and insulin. Ten normal subjects were studied using the euglycemic glucose clamp technique. The human proinsulin and insulin infusion rates were chosen to achieve steady-state proinsulin levels 10-fold higher than insulin levels on a molar basis, based on previous observations that porcine proinsulin has approximately 10% the potency of insulin. Proinsulin infusion rates of 2.75, 7.5, 22.5, and 45 micrograms/m2/min were compared with insulin infusion rates of 0.63, 1.67, 5, and 10 micrograms/m2/min. Primed, continuous infusions of insulin yielded steady-state levels within 25 min, whereas proinsulin levels did not reach a steady state for 120-180 min. The metabolic clearance rate of insulin was 11-12 ml/kg/min at the lower infusion rates but fell to 8.4 ml/kg/min at the highest infusion rate. The metabolic clearance rate of proinsulin was 3.0-3.5 ml/kg/min at all infusion rates. Dose-response analysis demonstrated that proinsulin-mediated glucose disposal was approximately 8% that of insulin. In contrast, proinsulin-mediated suppression of hepatic glucose output was approximately 12% that seen with insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of biosynthetic human proinsulin on carbohydrate metabolism

The effects of biosynthetic human proinsulin on carbohydrate metabolism

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.