Synthesis of an insulin-like compound consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I

An insulin-like hybrid molecule consisting of the A chain of insulin and a B chain corresponding to the B domain of human insulin-like growth factor I (growth factor I sequence 1-30) has been synthesized essentially by the procedures developed in this laboratory for the synthesis of insulin and analogues. The hybrid competed with 125I-insulin for insulin receptors in rat liver plasma membranes and was a full agonist in stimulating incorporation of [3(-3)H]glucose into lipids in rat adipocytes. In both assays, the compound displayed ca. 2% of the potency of insulin. The compound was recognized by anti-insulin antibodies but was only ca. 0.25% as potent as insulin in this activity. The hybrid exhibited growth-promoting activity in fibroblasts, displaying 3-8% of the activity of insulin. In contrast, the compound was recognized by insulin-like growth factor carrier proteins, a property not associated with insulin. Two points of nonhomology between the B chain of insulin and the B domain of insulin-like growth factor I are considered in connection with these observations.     

T cell-independent responses to an Ir gene-controlled antigen. I. Characteristics of the immune response to insulin complexed to Brucella abortus

Immune responses by mice to heterologous insulins are controlled by H-2-linked Ir genes. Antibody responses to insulin are T cell dependent (TD), and nonresponder mice fail to make detectable insulin-specific antibodies. To further analyze the role of T cells in regulation of immune responses to insulin, we have developed a method for induction of insulin-specific B cells in the relative absence of T cells. Insulin has been chemically coupled to the T cell-independent (TI) organism Brucella abortus (insulin-BA). Studies reported here demonstrate that in terms of kinetics of responses, isotype expression, and induction of responses in X-linked immunodeficient mice, insulin-BA behaves as a typical type-1 TI antigen. Despite these characteristic features, T cells appear to augment the response to insulin-BA. More importantly, insulin-BA stimulates IgM and IgG anti-insulin antibodies in all strains tested regardless of whether the mice were responders or nonresponders to the particular insulin tested. Thus insulin-BA should be a useful antigen for dissecting the cell interactions required for development of insulin-specific immunity.     

Mouse antibodies to the insulin receptor developing spontaneously as anti-idiotypes. I. Characterization of the antibodies

Mice immunized to ungulate insulins were found to develop antibodies of two specificities: insulin antibodies that were mostly IgG1 and IgG2 antibodies that acted both as anti-idiotypes to specific mouse insulin antibodies and as antibodies to the insulin receptor. There was a negative association between the presence of anti-idiotypic receptor antibodies and insulin antibodies bearing the specific idiotype; the specific idiotypic antibodies were confined to the early phase of the primary response while the anti-idiotypic receptor antibodies were detected only after the idiotypic antibodies had disappeared. To map the insulin epitope that triggered the specific idiotypic response, we chemically altered the insulin molecule so as to inhibit its interaction with the insulin receptor. The altered insulins triggered high titers of antibodies binding to antigenic determinants on native insulin, but no anti-idiotypic receptor antibodies. Thus, the epitope responsible for the specific idiotypic-anti-idiotypic network was probably the part of the insulin molecule whose conformation is recognized by the insulin receptor.     

Antibodies to deduced sequences of the insulin receptor distinguish conserved and nonconserved regions in the IGF-I receptor

Antipeptide antibodies directed to two amino acid sequences predicted from the cDNA encoding the insulin proreceptor have been used to study the relationship between the human receptors for insulin and insulin-like growth factor I (IGF-I). An antibody directed to a cytoplasmic domain near the membrane spanning region of the proreceptor inhibited the protein tyrosine kinase activity of both receptors whereas an antibody directed to the C terminus of the insulin receptor showed no cross-reactivity with the IGF-I receptor. The results establish that the cloned cDNA from the human placenta encodes the insulin receptor and not the closely related IGF-I receptor, that the IGF-I and insulin receptors share a specific amino acid sequence necessary for the expression of enzymatic activity, and that the C terminus of the insulin receptor is not conserved in the IGF-I receptor.     

Receptor aggregation is necessary for activation of the soluble insulin receptor kinase

Purified polyclonal human antibodies (B-8) against the receptor for insulin (anti-R IgG), and their F(ab')2 and Fab' fragments, were used to study a possible role of receptor aggregation in the process that couples insulin binding with the activation of the insulin receptor kinase. Anti-R IgG, F(ab')2, and Fab' fragments were shown to inhibit insulin binding to solubilized partially purified receptor preparations from rat liver. This suggests that the antibodies and fragments bind near or at the insulin-binding site. Only anti-R IgG and its bivalent F(ab')2 fragments were capable of stimulating the receptor kinase activity. Monovalent Fab' fragments were completely devoid of such activity. Cross-linking of anti-R Fab' with goat anti-human Fab' restored the capability of the Fab' fragments to activate the receptor kinase. These data strongly suggest that receptor cross-linking or aggregation constitutes a sufficient trigger to activate the insulin-receptor kinase and could, therefore, be an important step in the transmembrane signaling process. This step presumably precedes the activation of the receptor kinase and the resulting phosphorylation of its protein substrates.     

Mapping surface structures of the human insulin receptor with monoclonal antibodies: localization of main immunogenic regions to the receptor kinase domain

A panel of 37 monoclonal antibodies to the human insulin receptor has been used to characterize the receptor's major antigenic regions and their relationship to receptor functions. Three antibodies recognized extracellular surface structures, including the insulin binding site and a region not associated with insulin binding. The remaining 34 monoclonal antibodies were directed against the cytoplasmic domain of the receptor beta subunit. Competitive binding studies demonstrated that four antigenic regions (beta 1, beta 2, beta 3, and beta 4) are found on this domain. Sixteen of the antibodies were found to be directed against beta 1, nine against beta 2, seven against beta 3, and two against beta 4. Antibodies to all four regions inhibited the receptor-associated protein kinase activity to some extent, although antibodies directed against the beta 2 region completely inhibited the kinase activity of the receptor both in the autophosphorylation reaction and in the phosphorylation of an exogenous substrate, histone. Antibodies to the beta 2 region also did not recognize autophosphorylated receptor. In addition, antibodies to this same region recognized the receptor for insulin-like growth factor I (IGF-I) as well as the insulin receptor. In contrast, antibodies to other cytoplasmic regions did not recognize the IGF-I receptor as well as the insulin receptor. These results indicate that the major immunogenic regions of the insulin receptor are located on the cytoplasmic domain of the receptor beta subunit and are associated with the tyrosine-specific kinase activity of the receptor. In addition, these results suggest that a portion of the insulin receptor is highly homologous to that of the IGF-I receptor.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

In vitro insulin-like actions of human growth hormone: a study with an insulin antibody

When polyclonal insulin antibodies were preincubated with either adipose tissue from hypophysectomized rats or adipocytes from normal rats, human growth hormone failed to stimulate glucose oxidation. Removal of insulin from adipocytes through incubation with pyruvate at pH 7.0, followed by washing three times, also abolished subsequent in vitro insulin-like action of hGH. Administration of the same insulin antibody to hypophysectomized rats 30 minutes prior to injection of hGH did not inhibit the insulin-like activity of the hGH as measured by its ability to decrease serum glucose and non-esterified fatty acid levels. It is concluded that the in vitro promotion of glucose oxidation by hGH requires insulin. Because of the uncertainty of complete removal of insulin in intact animals, such a conclusion cannot be made regarding in vivo insulin-like action of hGH.     

Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules.

The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.     

B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies

Chronic inflammation characterized by T cell and macrophage infiltration of visceral adipose tissue (VAT) is a hallmark of obesity-associated insulin resistance and glucose intolerance. Here we show a fundamental pathogenic role for B cells in the development of these metabolic abnormalities. B cells accumulate in VAT in diet-induced obese (DIO) mice, and DIO mice lacking B cells are protected from disease despite weight gain. B cell effects on glucose metabolism are mechanistically linked to the activation of proinflammatory macrophages and T cells and to the production of pathogenic IgG antibodies. Treatment with a B cell-depleting CD20 antibody attenuates disease, whereas transfer of IgG from DIO mice rapidly induces insulin resistance and glucose intolerance. Moreover, insulin resistance in obese humans is associated with a unique profile of IgG autoantibodies. These results establish the importance of B cells and adaptive immunity in insulin resistance and suggest new diagnostic and therapeutic modalities for managing the disease.     

Structural features involved in the biological activity of insulin and the insulin-like growth factors: A27 insulin/BIGF-I

A synthetic insulin-like compound consisting of the A-chain of insulin extended at its carboxyl terminus with the hexapeptide "D-domain" of insulin-like Growth Factor II, linked via disulfide bonds to a B-chain corresponding to the "B-domain" of insulin-like Growth Factor I, has been examined for insulin-like metabolic activity and for mitogenic activity. The synthetic material (A27 insulin/BIGF-I) is less potent than insulin in metabolic assays, and less potent than both insulin and IGF-I in mitogenic assays. It is proposed that neither the "D-domain" nor the "B-domain" of the IGFs is a major contributor to mitogenic activity. Their presence in the same molecule does not result in significant growth-promoting activity.     

Activation of insulin signal transduction pathway and anti-diabetic activity of small molecule insulin receptor activators

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T. , Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974-977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.     

Increased P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85alpha, p85beta, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85alpha is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85alpha-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85alpha, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85alpha in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85alpha or p85beta, respectively (p85alpha (+/-) and p85beta(-/-)). Wild type and p85beta(-/-) mice developed in vivo insulin resistance and demonstrated overexpression of p85alpha and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85alpha(+/-)mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85alpha expression. These findings demonstrated the importance of increased p85alpha in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85alpha as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.     

High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor

Large quantities of high affinity antibodies directed against the human insulin receptor and the human insulin-like growth factor-I (IGF-I) receptor were obtained from hen's eggs. Hens were immunized with human placental membranes and human liver membranes by intramuscular injections. Specific antibodies to the receptors were demonstrated in serum and egg yolks at 5 weeks and these antibodies presisted for at least 6 months. Antibodies from egg yolks were purified by the polyethylene glycol precipitation technique of Polson et al. The eggs provided the equivalent of about 450 ml of immunized serum per month per bird. The purified antibodies were approximately equally reactive with receptors for insulin or IGF-I. Antibodies immunoprecipitated affinity-labeled receptors, inhibited binding of each ligand, and were capable of stimulating 2-deoxyglucose uptake in rat adipocytes and thymidine incorporation in cultured fibroblasts. The presence of antibodies directed against the IGF-I receptor in those hens immunized with human liver membranes was unexpected, since liver membranes possess little or no IGF-I binding activity. We conclude that antibodies against human antigens may be relatively easily obtained by immunization of hens and purification of those antibodies from eggs.     

Insulin-like and insulin-inhibitory effects of monoclonal antibodies for different epitopes on the human insulin receptor.

Monoclonal antibodies previously shown to react with five distinct epitopes on the human insulin receptor were tested for their metabolic effects on isolated human adipocytes. Two antibodies which reacted with receptor alpha-subunit and completely inhibited 125I-insulin binding mimicked the actions of insulin to stimulate lipogenesis from [14C]glucose and to inhibit catecholamine-induced lipolysis. On a molar basis, these antibodies were comparable in potency with insulin itself. Two other antibodies which decreased insulin binding only slightly or not at all also mimicked these metabolic effects of insulin. One of these antibodies reacted with receptor beta-subunit. In contrast, a further antibody which reacted with alpha-subunit and inhibited insulin binding did not affect basal lipogenesis or catecholamine-induced lipolysis, but was able to antagonize the effects of insulin on these processes. The same antibody antagonized the insulin-like effect of another antibody with which it competed in binding to insulin receptor, but not the effect of an antibody which bound independently to the receptor. It is concluded that binding of ligand at or close to the insulin-binding site is neither necessary nor sufficient to trigger insulin-like metabolic effects, which may rather depend on some general property of antibodies, such as their ability to cross-link and aggregate receptor molecules.     

Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors

Madin-Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites (Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen, nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface-labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipitates were analyzed on SDS-gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells wee incubated first with serum containing antibodies to the insulin receptor and then with 125I-protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin-like activity were tested on MDCK cells. The insulinomimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [3H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin-insensitive. However, since insulin-associated responses could be elicited by some insulin mimickers, the post-receptor limb of response in MDCK cells was apparently intact.     

From insulin and insulin-like activity to the insulin superfamily of growth-promoting peptides: a 20th-century odyssey

In 1941, Gellhorn reported that administration of human blood to hypophysectomized/adrenodemedullated rats caused a fall in blood sugar. This was among the early demonstrations that human blood possesses glucose-lowering or insulin-like activity (ILA). Gellhorn assumed he had detected only insulin. During the 1960s, however, it became evident that plasma ILA contained at least two components: one, suppressible ILA (SILA), was inactivated by anti-insulin antibody and was therefore considered to be indistinguishable from pancreatic insulin; the other, nonsuppressible ILA (NSILA), was unaffected by anti-insulin antibody. Subsequent work resolved NSILA into insulin-like growth factors I and II (IGF-I and IGF-II), two 7.5 kilodalton peptides with potent mitogenic properties; established their identity with the somatomedins; and investigated both their therapeutic potential and role in the pathogenesis of neoplastic and other human diseases. Insulin and the IGFs exhibit striking homologies in amino acid composition and some degree of overlap in their signaling pathways and actions. Moreover, insulin-like proteins have been identified not only in all vertebrate classes but also in molluscs, insects, and worms. These observations are the basis for the hypothesis that the genes encoding vertebrate insulins and IGFs and invertebrate insulin-like molecules evolved from a common ancestral gene, and for the concept of an insulin superfamily of growth-promoting peptides.     

Isolation and partial characterization of insulin of the honeybee (Apis mellifica)

In the honeybee (Apis mellifica), insulin-like material was partially purified with acid ethanol extractions by a classic method for recovering insulin and following gel filtration on a Sephadex G-50 column. The preparations were characterized by their ability to cross-react with porcine insulin antibodies. Insulin-like biological activity was demonstrated using the insulin bioassay. Stimulation of glucose oxidation or lipogenesis was measured by isolated rat adipocytes. Insulin seems to be more widespread in invertebrates than was previously assumed.     

Characterization and application of monoclonal antibodies directed to separate epitopes of glutathione-insulin transhydrogenase

Five monoclonal antibodies specific for glutathione-insulin transhydrogenase were characterized. None of the monoclonal antibodies cross-reacted with another insulin-degrading enzyme, neutral thiopeptidase. The isotype of four antibodies was IgG1 and of the fifth IgG2b. Affinity studies, competitive binding studies and immunoblot analysis of CNBr and trypsin cleavage products of glutathione-insulin transhydrogenase demonstrated that the four IgG1 antibodies were directed to an epitope of the enzyme which was distinct from the epitope recognized by the IgG2b antibody. Inhibition studies indicated that each monoclonal antibody, when added singly to glutathione-insulin transhydrogenase, was unable to inhibit the insulin-degrading activity of the enzyme. However, when monoclonal antibodies directed against separate epitopes of glutathione-insulin transhydrogenase were presented together (i.e., the IgG2b with any one of the four IgG1 antibodies), a loss in enzymatic activity was noted. Immunoblot analysis of rat organ extracts with the IgG1 antibodies demonstrated one immunoreactive protein band of Mr 56,000 in all tissues examined (liver, fat, pancreas and kidney) except the spleen, which demonstrated two immunoreactive protein bands of Mr 56,000 and 51,000. The same immunoblots, when probed with the IgG2b antibody, demonstrated the same immunoreactive protein banding pattern as above plus an additional immunoreactive protein band of Mr 67,000 in all tissues. Studies with spleen extracts from steptozotocin-induced diabetic rats demonstrated that there was a loss of the 51,000 immunoreactive band in diabetes. This 51,000 protein was restored upon insulin treatment of the diabetic rats and nullified upon concomitant administration of cycloheximide or actinomycin D with insulin. Immunoblots of human liver, adipose and skeletal muscle extracts indicated that each monoclonal antibody cross-reacted with the human form of the enzyme which had a molecular weight of Mr 63,000; a second minor immunoreactive band of 67,000 was detected with the IgG2b antibody. The physiological significance of additional molecular forms of the enzyme (i.e., 67,000 and 51,000) remains to be determined.     

Effects of zinc supplementation on the plasma glucose level and insulin activity in genetically obese (ob/ob) mice

The effects of zinc supplementation (20 mM ZnCl₂ from the drinking water for eight weeks) on plasma glucose and insulin levels, as well as its in vitro effect on lipogenesis and lipolysis in adipocytes were studied in genetically obese (ob/ob) mice and their lean controls (+/?). Zinc supplementation reduced the fasting plasma glucose levels in both obese and lean mice by 21 and 25%, respectively (p < 0.05). Fasting plasma insulin levels were significantly decreased by 42% in obese mice after zinc treatment. In obese mice, zinc supplementation also attenuated the glycemic response by 34% after the glucose load. The insulin-like effect of zinc on lipogenesis in adipocytes was significantly increased by 80% in lean mice. However, the increment of 74% on lipogenesis in obese mice was observed only when the zinc plus insulin treatment was given. This study reveals that zinc supplementation alleviated the hyperglycemia of ob/ob mice, which may be related to its effect on the enhancement of insulin activity.