Insulin and insulin mixtures; nph insulin

There are several distinct types of commercial insulins available, and with combinations of these many curves of timing of insulin action may be obtained, but none can parallel the action of a normal pancreas.NPH insulin, the newest addition, has a wide range of usefulness and may supplant many of the other types and combinations.     

Engineering of insulin receptor isoform-selective insulin analogues

Background

The insulin receptor (IR) exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues.

Methodology/Principal Findings

Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference) even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity.

Conclusions/Significance

We have discovered a new class of IR isoform-selective insulin analogues with 2–4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.     

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.     

The dissociation of insulin from human insulin antibodies

The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k₁ = 2·1^?3 min^?1 and a fast (k₂ = 4·10^?2 min^?1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k₁ and k₂ values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6–850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achivement of maximal binding there is a time-dependent rise in binding to the slow dissociating component, with a concomitant fall in k₁. The traditional concept that equilibrium is established at maximum binding requires further examination.     

Human insulin receptor juxtamembrane domain independent insulin signaling

The exon 16-encoded juxtamembrane (JM) domain of human insulin receptor (hIR) harbors the NPEY motif which couples the insulin-activated hIR kinase to downstream signal transduction molecules. We sought to determine if signal transduction requires the entire exon 16-encoded 22-amino acid JM domain. Transfected CHO cells were generated stably expressing either the wild-type hIR (hIR-WT) or two mutant hIRs (hIRDeltaEx16 in which the JM domain was deleted, and hIRrosJM in which the deleted segment was replaced by the corresponding domain of v-ros protein). The mutant hIRDeltaEx16 and hIRrosJM exhibited similar insulin-binding as the hIRWT. Insulin internalization and insulin dose-response experiments toward activation of downstream signal transduction molecules demonstrated that: i) the presence of intact hIR-JM domain which harbors the NPEY motif is essential for Shc phosphorylation but not for IRS-1 phosphorylation; ii) insulin signal transduction can occur independent of the JM domain of hIR and without participation of the NPEY motif; iii) engagement of this putative alternative downstream signal transduction is Shc independent and is dependent on insulin concentration; and iv) insulin internalization does not necessarily require the hIR specific aa sequence of the JM domain which can be partially substituted by the JM domain of the v-ros tyrosine kinase.     

Human insulin

The two human insulins of clinical importance are (a) semisynthetic human insulin prepared from pork pancreas by enzymatically substituting threonine for alanine-the last amino acid in the beta chain-thereby transforming pork insulin in vitro to human insulin; and (b) biosynthetic human insulin synthesized biotechnologically in Escherichia coli-K12. Using this latter technique, it is possible to produce mass quantities of highly purified insulin for the treatment of insulin-dependent diabetics, avoiding the problems inherent in supplies of insulin produced from animal pancreas. It has been suggested that to avoid confusion the two human insulins should be called semisynthetic human insulin of pork origin and biosynthetic human insulin of E. coli origin, respectively. These insulins have four advantages over highly purified animal insulins: (a) they induce lower titers of circulating insulin antibodies; (b) their subcutaneous injection is associated with fewer skin reactions; (c) they are absorbed more rapidly from the injection site; and (d) less degradation occurs at the site of injection. These data indicate that newly diagnosed insulin-dependent diabetes, particularly in children, should be treated with either of the two human insulins. The warranty against inadequate supplies of insulin offered by biosynthetic human insulin makes the use of pork insulins unnecessary and beef insulins totally useless.     

Relation of insulin receptors to insulin resistance.

The status of insulin-receptor interactions in a variety of insulin-resistant states is reviewed. Utilizing large adipocytes from adult rats and small fat cells from young rats, we have conducted a series of in vitro experiments in an attempt to determine the cellular alteration(s) responsible for the insulin resistance associated with obesity. Stimulation of glucose oxidation by insulin is reduced in large cells. Studies using a mimicker of insulin action, spermine, as well as measurements of 125I-insulin binding to large and small cells indicate that receptor number and affinity are not responsible for hormone resistance. Furthermore, when rapid and direct measurements of sugar uptake were made, insulin stimulation was virtually identical in both cell types. These findings indicate that large adipocytes have an efficient insulin-responsive D-glucose transport system and suggest that the apparent hormone resistance may be due to alterations in intracellular glucose metabolism. It has been proposed that altered insulin-receptor interaction underlies the insulin resistance of human obesity. We have investigated this particular aspect of insulin action by 125I-insulin binding studies. Similar numbers of insulin receptors per cell and affinity for insulin were observed in adipocytes obtained from normal weight subjects and morbidly obese patients. Thus, the initial step in insulin action is unaltered in human obesity.     

Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. Sequencing of the B chain fragments demonstrated that the Drosophila enzyme cleaves the B chain of insulin at four sites between residues B10 and B11, B14 and B15, B16 and B17, and B25 and B26. These cleavage sites correspond to four of the seven cleavage sites generated by the mammalian insulin protease. These results demonstrate that all the insulin cleavage sites generated by the Drosophila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function.     

Glucagon increases insulin levels by stimulating insulin secretion without effect on insulin clearance in mice

Circulating insulin is dependent on a balance between insulin appearance through secretion and insulin clearance. However, to what extent changes in insulin clearance contribute to the increased insulin levels after glucagon administration is not known. This study therefore assessed and quantified any potential effect of glucagon on insulin kinetics in mice. Prehepatic insulin secretion in mice was first estimated following glucose (0.35 g/kg i.v.) and following glucose plus glucagon (10 μg/kg i.v.) using deconvolution of plasma C-peptide concentrations. Plasma concentrations of glucose, insulin, and glucagon were then measured simultaneously in individual mice following glucose alone or glucose plus glucagon (pre dose and at 1, 5, 10, 20 min post). Using the previously determined insulin secretion profiles and the insulin concentration-time measurements, a population modeling analysis was applied to estimate the one-compartment kinetics of insulin disposition with and without glucagon. Glucagon with glucose significantly enhanced prehepatic insulin secretion (Cmax and AUC_0-20) compared to that with glucose alone (p < 0.0001). From the modeling analysis, the population mean and between-animal SD of insulin clearance was 6.4 ± 0.34 mL/min for glucose alone and 5.8 ± 1.5 mL/min for glucagon plus glucose, with no significant effect of glucagon on mean insulin clearance. Therefore, we conclude that the enhancement of circulating insulin after glucagon administration is solely due to stimulated insulin secretion.     

Down-regulation of insulin receptors is related to insulin internalization

In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of 125I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.     

Measuring the balance between insulin synthesis and insulin release

The absolute rates of hormone synthesis and release were determined in purified pancreatic B cells. Newly synthesized proteins were labeled with L-[3,5-3H]tyrosine or L-[2,5-3H]histidine. When medium glucose was less than or equal to 10 mM, the production of insulin exceeded or equaled its release. Raising the glucose levels above 10 mM did not further increase the rate of insulin synthesis (67 +/- 10 fmol/10(3) cells/2 hour) but elevated that of insulin release up to 3-fold the production rates (181 +/- 10 fmol/10(3) cells/2 hour). In the presence of glucagon or of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate the cells also released 3-fold more hormone that they synthesized; release was however reduced to 25% of the rate of production in the presence of epinephrine. It is concluded that glucose as well as hormonal regulators of islet B cells can influence, bi-directionally, the balance between the rates of insulin synthesis and release.     

Enhanced endogenous insulin secretion after treatment with monocomponent insulin

Eight maturity-onset diabetic patients who had no infections, liver diseases or other endocrine diseases which might affect carbohydrate metabolism were treated with monocomponent lente insulin, which is known to have little antigenicity, until blood sugar was controlled. The diurnal values of the blood sugar (BS) and immunoreactive insulin (IRI) were obtained before treatment and on the third day after the withdrawal of insulin treatment. One patient was dropped from the study because of insulin antibody formation. The total integrated IRI area in the diurnal profile was increased from 98.9 +/- 23.7 to 186.1 +/- 37.7 muU.h/ml (p less than 0.05), while the total blood sugar area was decreased from 7,500 +/- 1,200 to 5,510 +/- 1,214 mg-h% (p less than 0.05). Oral Glucose Tolerance Test on the fourth day after the withdrawal of insulin treatment showed increased IRI response compared to pretreatment. It is concluded that endogenous insulin secretion could be increased in the diabetic patient by the metabolic control with exogenous insulin injections.