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Mutations have been identified in the insulin receptor (IR) gene in patients who are insensitive to insulin action. We studied an extremely insulin resistant patient whose insulin binding to Epstein-Barr virus (EBV) transformed lymphocytes was severely reduced. Transmembrane signalling, evaluated as insulin receptor autophosphorylation, was normal. The patient's IR was immunoprecipitated normally by AbP6, a polyclonal antibody directed to the beta subunit. However, there was an approximately 50% decrease in the affinity of IR immunoprecipitation by a monoclonal antibody (MA-10) directed against the alpha subunit. These observations suggested that there were likely to be a mutation in the patient's insulin receptor that caused misfolding of the IR alpha subunit. Analysis of gene structure by Southern blotting experiments did not reveal any major deletion in the IR gene of the proband. Northern blot analysis showed a normal level of expression of IR gene. We applied denaturing gradient gel electrophoresis (DGGE) as well as direct sequence analysis to study the 22 exons of IR gene amplified by polymerase chain reaction (PCR) using the proband's genomic DNA as a template. We identified a new missense mutation substituting leucine (CTG) for proline (CCG) in homozygous state at codon 193 in exon 3. Both parents are heterozygous for the Leu193 mutation. The Leu193 mutation was not detected in any of 75 normal subjects (150 chromosomes), indicating that it is not a common sequence variant of the insulin receptor. In addition, during the course of screening the patient's DNA with perpendicular DGGE, we identified two previously unreported silent substitutions in exon 9.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism (both at the whole body and cellular level) by which metformin improves insulin sensitivity has yet to be defined. In the present study, we examined in vivo insulin-mediated whole-body glucose disposal, glycogen synthesis, hepatic glucose production, and insulin secretion, as well as in vitro muscle insulin receptor tyrosine kinase activity in eight control, eight neonatal streptozotocin diabetic rats, and eight diabetic rats before and after treatment with metformin. Ten weeks after birth diabetic rats had higher fasting (132 + 5 v 101 + 2 mg/dL) and postmeal (231 + 10 v 133 + 3) plasma glucose levels compared with controls (P less than .001). Metformin treatment was followed by a significant decrease in the growth rate and normalized glucose tolerance without enhancing the deficient insulin response. Insulin-mediated glucose uptake in diabetic versus control rats was reduced (P less than .01) during the high-dose (15.4 + 0.6 v 18.3 + 1.0 mg/kg.min) insulin clamp study and was increased to values greater (P less than .05) than controls following metformin treatment. Muscle glycogen synthetic rate in vivo, measured by incorporation of 3H-3-glucose radioactivity, was diminished by 25% (P less than .01) in diabetic rats, restored to normal values with metformin, and correlated closely (r = .82, P less than .002) with total-body glucose uptake during the insulin clamp in all three groups. Insulin receptor tyrosine kinase activity, measured in partially purified insulin receptors, was reduced in diabetic rats and increased to supernormal levels after metformin. The decrease in muscle tyrosine kinase activity in diabetic versus control animals was entirely accounted for by a reduction in maximal velocity (Vmax) (32 v 45 pmol/mg.min, P less than .01) and increased to supernormal levels following metformin (91 pmol/mg.min, P less than .001) without any change in affinity (Km). Muscle tyrosine kinase activity was closely correlated with both the muscle glycogen synthetic rate (r = .82, P less than .002) and total-body insulin-mediated glucose disposal (r = .64, P less than .01) in vivo. The close correlation between in vivo insulin action, muscle glycogen synthesis, and muscle insulin receptor tyrosine kinase activity is consistent with an important role of the enzyme in the insulin resistance of diabetes and its improvement following metformin treatment.

Liver-purified insulin receptor tyrosine kinase (IRTK) activity was examined in partially pancreatectomized rats following normalization of blood glucose concentration by either phlorizin or vanadate treatment. Chronic moderate hyperglycemia did not modify the IRTK activity, despite the presence of in vivo and in vitro insulin resistance. Oral vanadate administration for 3 weeks normalized glucose tolerance and caused a 2.5-fold increase in basal IRTK activity. In contrast, correction of hyperglycemia with phlorizin, an inhibitor of renal glucose reabsorption, did not change the IRTK activity, although glucose tolerance was returned to normal. The vanadate-induced effect on basal IRTK was due to an increase in Vmax of the enzyme; the Km remained unchanged. The insulin-stimulated IRTK activity was not affected by either vanadate or phlorizin treatment. These results suggest that: 1) partial (90%) pancreatectomy in rats causes insulin resistance in the absence of in vitro alterations in IRTK and 2) correction of chronic hyperglycemia with vanadate, but not with phlorizin, is associated with an increased basal activation of the protein tyrosine kinase in liver insulin receptors.

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.

Insulin receptor beta-subunit autophosphorylation, the first event occurring after insulin binding, plays a crucial role in modulation of receptor-associated kinase activity towards exogenous substrates and possibly in the transmission of biological signals of insulin. Receptor autophosphorylation strongly depends on insulin receptor occupancy. Till now the effects of receptor phosphorylation on insulin binding itself have not been clarified. In the present report we demonstrate the absence of any feedback mechanism by which insulin receptor activation by phosphorylation affects binding affinity of insulin receptor itself.

The effect of monoclonal anti-insulin receptor antibody MA 10 on [125I]insulin binding and on insulin receptor protein tyrosine kinase activity was investigated in human and rat tissues. It was observed that MA 10 inhibits insulin binding to human, but not rat, tissues while inhibiting insulin-stimulated receptor autophosphorylation and protein tyrosine kinase activity in both human and rat tissues. These data suggest that MA 10 is directed against a region of the insulin receptor that is in between the insulin-binding domain and the beta-subunit and that in human, but not rat, tissues, this region is involved in insulin binding.

Insulin receptor associated kinase activity and its relationships with the insulin resistance of streptozotocin-induced diabetes were investigated in rats, using solubilized, partially purified insulin receptors from liver membranes. Insulin receptor kinase activity was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyr1. Diabetes was associated with a 45% reduction in kinase activity, in the same number of insulin receptors, with no change in insulin binding affinity. To investigate the independent roles of hyperglycemia and hypoinsulinemia on the observed impairment of receptor kinase activity, diabetic rats were fasted for 24 h in order to normalize blood glucose levels only. After this short fast, no change in kinase activity, from the values measured in fed diabetic animals, was observed. Our findings suggest that streptozotocin diabetes is associated with a reduction of insulin receptor kinase activity, which a short fast is not able to reverse.

IGF I receptor is a tyrosine kinase capable of phosphorylating the receptor itself and other substrates. A high degree of homology does exist in tyrosine kinase domain among receptors for several polypeptide growth factor receptors and this enzymic activity has been indicated as a possible mediator of biological action. Nevertheless growth factor receptors possess peculiar specificities both in their functions and tissue distribution. A human polyclonal IgG (pIgG), previously characterized as anti insulin receptor antibody, able to inhibit insulin receptor kinase activity, was used to further investigate subunit homologies and differences in antigenicity and functional regulation between IGF I and insulin receptors, IGF I receptor tyrosine kinase was stimulated by a IGF I analog (aIGF I), produced by DNA recombinant technology, pIgG was able to inhibit IGF I receptor kinase activity, thus revealing antigenic homologies between the kinase domains of insulin and IGF I receptors. However the more pronounced inhibition of IGF I receptor-compared with insulin receptor kinase activity by pIgG suggests the existence of different regulatory mechanisms.

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.

The immunoglobulin G of a polyclonal antiserum (pIgG) from a patient with insulin resistance and hypoglycemia was tested for its ability to inhibit insulin binding and to affect the autophosphorylation of partially-purified insulin receptors extracted from rat liver membranes. pIgG, when added 4 hr prior to insulin, inhibited subsequent insulin binding by 50% at 30 micrograms added protein; however, insulin previously bound to the receptor could not be displaced by a 4 hr subsequent exposure of up to 70 micrograms pIgG. pIgG, independent of its effect on insulin binding, inhibited both basal and insulin-stimulated autophosphorylation of the insulin receptor in a dose-dependent manner with a half maximal effect at 3.3 to 7 micrograms protein. Furthermore, pIgG also reduced basal autophosphorylation of the EGF receptor. The effect of pIgG to inhibit basal autophosphorylation of insulin and EGF receptors, together with its ability to reduce autophosphorylation of insulin receptors fully occupied by insulin, imply that the effect of pIgG on receptor autophosphorylation is largely independent of its effect on ligand binding. Moreover, these findings suggest that pIgG may inhibit autophosphorylation by acting on domains which are similar in the insulin and EGF receptors.

The effect of a polyclonal anti-insulin receptor antibody (pIgG) on the insulin receptor tyrosine kinase (IRTK) activity toward poly-(Glu-Tyr) was examined using wheat germ agglutinin agarose-purified insulin receptors from rat liver membranes. The main effect of pIgG was a reduction of Vmax (from 60.8 to 31.8 pmol/min/mg), without changes of Km, when IRTK was activated by insulin. In contrast, when IRTK was activated by ATP preincubation, pIgG was unable to affect the reaction, suggesting that IRTK possesses at least two regulatory mechanisms, one of which can be affected by pIgG.

The metabolic parameters of 40 insulin-dependent patients (22 females and 18 males) have been studied to evaluate their intercorrelations. We obtained positive correlation between glycosylated haemoglobin and triglycerides (r = +0,460; p less than 0,01) and between HbA1 and mean diurnal plasma glucose (MDPG) (r = +0,652; p less than 0,001). There was a negative correlation between HbA1 and high density lipoproteins (HDL) cholesterol (r = -0,406; p less than 0,05). No significant correlation was found between HbA1 and either uric acid or total cholesterol. Correlations were also found between MDPG and triglycerides, MDPG and HDL-cholesterol, triglycides and HDL-cholesterol, uric acid and HDL-cholesterol. These correlations are discussed.

Insulin binding on circulating red cells has been studied in hypertriglyceridemic (HTG) patients before and after normalization of plasma TG levels by a low fat and low CHO diet, followed for 2 months. Under basal condition HTG patients showed lower insulin binding on red cells (B/T) than control subjects. The reduction in binding was due to a lower receptor number (binding capacity). After the diet and normalization of TG levels, insulin binding was identical in HTG patients and always lower than in controls. The fasting values of blood glucose, IRI, FFA, were also unchanged after TG reduction, suggesting, together with the low insulin binding, a state of insulin resistance. We conclude that our lean patients, affected by HTG, present a state of insulin resistance. Despite normalization of plasma TG, obtained with diet alone, insulin receptor binding on red cells in unchanged.