The effect of fasting on the activation in vivo of the insulin receptor kinase.

Fasting causes insulin resistance in liver and fat, and increases insulin sensitivity in muscle. We studied the response in vitro and in vivo to insulin of the insulin receptor tyrosine kinase in muscle and liver from 72 h fasted and control rats. Insulin was injected intraperitoneally together with glucose, and blood and tissue samples were obtained 0, 5, 15 and 30 min later. Basal serum glucose and insulin levels were significantly higher in control than in fasting rats. Serum glucose rose to approximately 300 mg/dl at 5 min and then progressively declined without hypoglycaemia. Receptors were prepared from whole tissue by wheat germ lectin affinity chromatography. 125I-insulin binding to purified receptors was increased by fasting in both muscle (18%) and liver (50%). In untreated fasting and control animals, muscle and liver insulin receptor tyrosine kinase activity was stimulated to similar levels by insulin added in vitro. With only insulin treatment in vivo, muscle receptor tyrosine kinase behaved similarly in fasting and control animals with maximal activation at 15 min post injection. In liver, insulin in vivo stimulated receptor tyrosine kinase activity maximally at 5 min post injection in both fasting and control, but in fasting animals the treatment in vivo caused a significantly larger and more prolonged activation of the enzymic activity, possibly due to a decrease in the rate of dephosphorylation and deactivation of the beta subunits.     

Tyrosine kinase activity of brain insulin and IGF-1 receptors

Lectin-purified rat brain preparations demonstrate specific [125I]insulin and [125I]-IGF-1 binding. Insulin-stimulable tyrosine kinase activity as measured by exogenous substrate phosphorylation was present in brain and liver lectin purified preparations with the delta kinase activity/B/F of brain approximately 2.5 fold greater than that of liver. Insulin-stimulable tyrosine kinase activity was abolished in liver but decreased by only approximately 50 percent in brain after immuno-depletion with antiserum which recognizes insulin but not IGF-1 receptors. Insulin and IGF-1 dose responses for phosphorylation of the immunodepleted brain preparations suggested that the remaining tyrosine kinase activity was IGF-1 receptor mediated. Thus, functional IGF-1 receptors are present in rat brain, and the doses of insulin typically used to evaluate insulin receptor tyrosine kinase activity will stimulate IGF-1 receptor tyrosine kinase activity as well.     

Insulin binding and insulin receptor tyrosine kinase activity are not altered in the liver of rats with non-insulin-dependent diabetes

Using the insulin-glucose clamp technique, we have previously shown that an increased sensitivity to insulin in vivo is a characteristic of the liver in rats with non-insulin-dependent diabetes induced by neonatal streptozotocin administration. We have thus studied the properties of liver insulin receptor in that model. 125I-porcine insulin binding was found normal both in isolated plasma membranes and in solubilized, wheat germ agglutinin purified receptors prepared from livers of rats with non-insulin-dependent diabetes, when compared to controls. Basal and insulin-stimulated insulin receptor kinase activities were also found normal for both the autophosphorylation of the beta subunit of the insulin receptor and the phosphorylation of the artificial substrate poly (Glu-Tyr) 4:1. Thus, in that model of chronic insulin deficiency and mild hyperglycemia: 1) liver insulin receptors are not up-regulated; 2) tyrosine kinase activity remains unaffected. This last observation supports the hypothesis that the increased insulin effect in the liver of rats with non-insulin-dependent diabetes is probably distal to the insulin receptor kinase.     

Tumor necrosis factor alpha in various tissues of insulin-resistant obese Koletsky rats: relations to insulin receptor characteristics.

Tumor necrosis factor alpha (TNFalpha) was found to be significantly increased in skeletal muscles and retroperitoneal fat of obese insulin-resistant Koletsky rats as compared to control Wistar rats. This increase was accompanied by a depression of insulin receptor protein tyrosine kinase (PTK) activity. Neither the insulin-binding capacity nor insulin receptor affinity were related to this TNFalpha increase in these tissues. In the liver, no significant changes of TNFalpha content and only a lowering of insulin-binding capacity were found. It is concluded that an increased TNFalpha content in muscles and fat (but not in the liver) contributes to insulin resistance by lowering insulin receptor protein tyrosine kinase activity, while other insulin receptor characteristics (insulin-binding capacity and affinity of insulin receptors to the hormone) do not seem to be influenced by this factor.     

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies

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.     

Dephosphorylation increases insulin-stimulated receptor kinase activity in skeletal muscle of obese Zucker rats

Serine/threonine phosphorylation of insulin receptor has been implicated in the development of insulin resistance. To investigate whether dephosphorylation of serine/threonine residues of the insulin receptor may restore the decreased insulin-stimulated receptor tyrosine kinase activity in skeletal muscle of obese Zucker rats, insulin receptor tyrosine kinase activity was measured before and after alkaline phosphatase treatment. Compared to lean controls, insulin-stimulated glucose transport was depressed by 61% (p < 0.05) in obese Zucker rats. The insulin receptor and insulin receptor substrate-1 contents were decreased by 14% (p < 0.05) and 16% (p < 0.05), respectively, in skeletal muscle of obese Zucker rats. In vivo insulin-induced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 was depressed by 82% (p < 0.05) and 86% (p < 0.05), respectively. In the meantime, in vitro insulin-stimulated receptor tyrosine kinase activity in obese rats was decreased by 39% (p < 0.05). Dephosphorylation of the insulin receptor by prior alkaline phosphatase treatment increased insulin-stimulated receptor tyrosine kinase activity in both lean and obese Zucker rats, but the increase was three times greater in obese Zucker rats (p < 0.05). These findings suggest that excessive serine/threonine phosphorylation of the insulin receptor in obese Zucker rats may be a cause for insulin resistance in skeletal muscle.     

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.     

Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) regulation of insulin receptor function: phosphorylation of insulin receptor with A kinase decreases the insulin binding activity.

The effect of phosphorylation of insulin receptor with adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) on its insulin binding activity was investigated by using insulin receptors prepared from rat liver in vitro. A 95 KDa protein was phosphorylated by stimulation of insulin receptor kinase. This protein was also phosphorylated by A kinase. Analysis of phosphoamino acid showed that tyrosine residue(s) was phosphorylated by activation of insulin receptor kinase, whereas phosphoserine and phosphothreonine were dominantly generated by activation of A kinase. [125I] Iodoinsulin binding activity was decreased by prior phosphorylation of the receptor with A kinase. Scatchard analysis showed that the affinity for insulin was decreased by the phosphorylation with A kinase. Although the maximal activity of insulin receptor kinase was not affected by phosphorylation with A kinase, the insulin concentration which induced half maximal activity (ED50) of the receptor kinase was increased by the phosphorylation with A kinase. These results suggested that counter regulatory hormones whose actions are mediated by the generation of adenosine 3',5'-cyclic monophosphate regulate the insulin binding to the alpha subunit through phosphorylation of the beta subunit of insulin receptor.     

The Male Obese Wistar Diabetic Fatty Rat Is a New Model of Extreme Insulin Resistance

The male obese Wistar Diabetic Fatty (WDF) rat is a genetic model of obesity and non-insulin dependent diabetes (NIDDM). The obese Zucker rat shares the same gene for obesity on a different genetic background but is not diabetic. This study evaluated the degree of insulin resistance in both obese strains by examining the binding and post binding effects of muscle insulin receptors in obese, rats exhibiting hyperinsulinemia and/or hyperglycemia. Insulin receptor binding and affinity and tyrosine kinase activity were measured in skeletal muscle from male WDF fa/fa (obese) and Fa/? (lean) and Zucker fa/fa (obese) and Fa/Fa (homozygous lean) rats. Rats were fed a high sucrose (68% of total Kcal) or Purina stock diet for 14 weeks. At 27 weeks of age, adipose depots were removed for adipose cellularity analysis and the biceps femoris muscle was removed for measurement of insulin binding and insulin-stimulated receptor kinase activity. Plasma glucose (13.9 vs. 8.4 mM) and insulin levels (14,754 vs. 7440 pmoI/L) were significantly higher in WDF obese than in Zucker obese rats. Insulin receptor number and affinity and TK activity were unaffected by diet. Insulin receptor number was significantly reduced in obese WDF rats (2.778 ± 0.617 pmol/mg protein), compared to obese Zucker rats (4.441 ± 0.913 pmol/mg potein). Both obese strains exhibited down regulation of the insulin receptor compared to their lean controls. Maximal tyrosine kinase (TK) activity was significantly reduced in obese WDF rats (505 ± 82 fmol/min/mg protein) compared to obese Zucker rats (1907 ± 610 fmol/min/mg protein). Only obese WDF rats displayed a decrease in TK activity per receptor. These observations establish the obese WDF rat as an excellent model for exploring mechanisms of extreme insulin resistance, particularly post-receptor tyrosine kinase-associated defects, in non-insulin dependent diabetes.     

Fasting and refeeding alter the insulin receptor tyrosine kinase in chicken liver but fail to affect brain insulin receptors

Insulin receptors from chicken liver and brain were studied following alterations in the nutritional state. Chickens were either fasted for 48 h, fasted for 48 h and then refed for 24 h, or fed a regular diet ad libitum. 125I-Porcine insulin binding was significantly elevated in liver membranes from the fasted animals and lowered in refed chickens when compared to preparations from ad libitum fed chickens. These changes in 125I-insulin binding were inversely related to the levels of plasma insulin and since receptor affinities for insulin were similar in each group, they probably represent alterations in receptor number. Apparent Mr of alpha subunits of the insulin receptors was unaffected by alterations in the nutritional states. The presence of ATPase-like activities that co-eluted with liver insulin receptors from wheat germ agglutinin lectin columns but not from pea lectin columns necessitated the use of both pea and wheat germ agglutinin for liver insulin receptor purification. The insulin receptors purified from both lectin columns were recognized by anti-insulin receptor antiserum and had similar affinities for insulin which were unaltered by the nutritional state. Insulin-stimulatable autophosphorylation of the beta subunit of the insulin receptor was lower in livers from fasted chickens and intermediate in refed chickens. Furthermore, basal and insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr) 4:1 was significantly less in the fasting state and intermediate in the refed state compared to the ad libitum fed state. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in all three states suggesting that the differences in insulin-induced phosphorylation are due to a change in maximal stimulation and not a change in insulin sensitivity. In contrast to the alterations seen with liver receptors, brain insulin receptors were unaffected by these alterations in nutritional state. These findings suggest that: liver insulin receptors are affected by altering the nutritional state; insulin binding to liver membranes is inversely related to plasma insulin levels; and tyrosine kinase is decreased both in fasted and refed animals suggesting an uncoupling of the normal interaction between alpha subunit and beta subunit in liver insulin receptors.     

Effect of aging on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipocytes.

The effect of aging on the insulin binding parameters and on the kinetic characteristics of the insulin receptor autophosphorylation in rat adipose tissue has been investigated. Using solubilized receptors from adipocyte plasma membranes, no significant differences were identified in either affinity or receptor number in adult vs old rats. Time courses for in vitro receptor phosphorylation revealed that both the initial rate of autophosphorylation and the maximal 32P incorporation were decreased by 40% in old (24-month) animals as compared to adult (3-month) control rats. The tyrosine phosphatase activity associated with the adipocyte plasma membranes does not account for the decreased kinase activity found in old rats. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in both groups of rats. However, the kinase activity showed a decreased responsiveness to the hormone in the old rats. Double reciprocal plot analysis of receptor phosphorylation revealed that the Km for ATP was not modified. In contrast, the insulin-stimulated Vmax value was decreased by two-fold in 24-month-old rats. The decrease in Vmax does not appear to be related to an increased basal phosphorylation level on Ser/Thr residues of the C terminus of the receptor beta-subunit. Thus, we conclude that the reduced insulin receptor kinase activity in adipose tissue from old rats is due, at least in part, to a defect of the intrinsic kinase activity of the insulin receptor.     

Does the insulin-mimetic action of vanadate involve insulin receptor kinase?

Effects of vanadate administration on the insulin receptor status in liver were examined in streptozotocin-induced diabetic rats. Diabetic rats were characterized by hyperglycemia (4-fold increase), hypoinsulinemia (81% decrease) and a significant (P<0.01) increase in hepatic insulin receptor numbers. Autophosphorylation of the subunit of insulin receptor and its tyrosine kinase activity towards the synthetic peptide (poly glut₄tyr₁) decreased by approximately 60% as a result of diabetes. After chronic treatment of these rats with sodium orthovanadate, the plasma glucose levels were normalized to near control values with the hypoinsulinemia remaining unaltered. The insulin-stimulated phosphorylation of the subunit increased significantly (P<0.001) in diabetic rats after treatment with vanadate. However, the improvement in the tyrosine kinase activity was marginal.In vitro, vanadate prevented the dephosphorylation of the phosphorylated insulin receptor and increased its tyrosine kinase activity in the absence as well as presence of insulin. The findings of this study further support the view that insulin receptor is one of the sites involved in the insulin-mimetic actions of vanadate.     

Insulin receptor kinase activity in rat adipocytes is decreased during aging

The tyrosine kinase activity of the insulin receptor derived from rat adipocyte plasma membranes was examined during aging. In the absence of insulin, autophosphorylation and histone H2B phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among 3- and 24-month-old rats. In contrast, insulin-stimulated kinase activity was significantly reduced in the old animals. We have also found that the insulin dependent phosphorylation of a putative endogenous substrate of 60 kDa was drastically reduced in old animals. These results suggest that the decrease in kinase activity in old rats could be related with the insulin resistance of aging.     

Insulin receptor kinase activity in rat liver. Regulation by fasting and high carbohydrate feeding

Insulin receptor kinase activity was measured in partially purified receptor preparations from livers of rats fed a standard diet or subjected to either prolonged fasting or a high carbohydrate (CHO) diet, conditions known to decrease (fasting) and increase (CHO) insulin action. Basal and insulin-stimulated phosphorylation of the beta subunit of the insulin receptor was comparable in all groups with a half-maximal effect at approximately 2.0 ng/ml free insulin and a 10-12-fold maximal effect. The kinase activity of insulin receptors from the three groups was further examined using the synthetic polypeptide Glu 4:Tyr 1. Basal and insulin-stimulated rates of Glu 4:Tyr 1 phosphorylation were highest in the CHO-fed and lowest in the fasted group. The magnitude of these differences was the same in the absence or presence of insulin; thus, the alterations in receptor kinase activity in fasting and CHO feeding were entirely expressed in the basal rate of peptide phosphorylation. Antireceptor antibody immunoprecipitated 70-80% of the basal Glu 4:Tyr 1 kinase activity in each group; the remaining 20-30% showed minor group differences when normalized for the amount of protein present in the receptor preparations. These results indicate that the group differences in basal kinase were intrinsic to the insulin receptor. Insulin increased the Vmax of Glu 4:Tyr 1 phosphorylation by approximately 30 fmol of phosphorus/fmol of binding activity/30 min in all three groups; however, the absolute Vmax was highest in the CHO-fed and lowest in the fasted group. The Km of Glu 4:Tyr 1 phosphorylation was unaffected by insulin and was comparable (approximately 0.25 mg/ml) in the three groups. These findings indicate that fasting and CHO feeding produce changes in receptor kinase activity which are regulated by mechanisms independent of insulin and that the alterations show substrate specificity so that differences are detected with one substrate (Glu 4:Tyr 1) but not another (the beta subunit).     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

The insulin receptor-related receptor. Tissue expression, ligand binding specificity, and signaling capabilities.

In 1989, Shier and Watt identified a gene which was predicted to encode a new member of the insulin receptor (IR) family, and they called it the insulin receptor-related receptor (IRR) (Shier, P., and Watt, V. M. (1989) J. Biol. Chem. 264, 14605-14608). However, the tissues expressing this receptor, its ligand binding specificity and its signaling capability have remained unknown. In the present studies we report Northern blot analyses and polymerase chain reaction data, which indicate that the IRR mRNA is expressed in a variety of tissues, including the human kidney, heart, skeletal muscle, liver, and pancreas. In order to examine the ligand(s) recognized by IRR, we constructed a chimeric receptor with the extracellular domain of the IR replaced with that of IRR. This chimera was found not to bind radioactively labeled insulin, insulin-like growth factor I (IGF-I), or IGF-II. These ligands and relaxin, the only other known member of the mammalian insulin family, also failed to stimulate the tyrosine kinase activity of this chimeric receptor. A second chimeric receptor with the extracellular domain of IR and the kinase domain of IRR was also constructed and utilized to study the signaling capabilities of the kinase domain of IRR. This chimera exhibited high affinity insulin binding and insulin-stimulated tyrosine kinase activity. The kinase domains of the IR and IRR were found capable of phosphorylating the same spectrum of exogenous and endogenous substrates. However, Chinese hamster ovary (CHO) cells stably overexpressing the kinase domain of IRR exhibited elevated basal thymidine incorporation and 2-deoxyglucose uptake compared with CHO cells and CHO cells overexpressing wild-type IR. We conclude that: 1) IRR is expressed in the human kidney, heart, skeletal muscle, liver, and pancreas, 2) IRR does not appear to be the receptor of any known member of the insulin family, and 3) the tyrosine kinase of IRR appears to be similar to that of IR in both the spectrum of substrates phosphorylated and the biological responses stimulated.     

Increased protein kinase C activity is linked to reduced insulin receptor autophosphorylation in liver of starved rats

Phosphorylation of the insulin receptor beta-subunit on serine/threonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor beta-subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serine/threonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor beta-subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and cAMP-dependent protein kinase A in liver were studied. A 2-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.     

Intrinsic differences of insulin receptor kinase activity in red and white muscle

The sensitivity and responsiveness of glucose uptake and glycogen synthesis to insulin are 3-4-fold greater in red than in white skeletal muscle (James, D. E., Jenkins, A. B., and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574). In the present study, the insulin receptor tyrosine kinase activity has been examined in red and white muscle of rats. Partially purified insulin receptors were obtained from muscle following solubilization in detergent, ultracentrifugation, and lectin affinity chromatography. Total insulin receptor number per gram of tissue was slightly higher in red (30%) than in white muscle. In contrast, basal and insulin-stimulated autophosphorylation, normalized for receptor number, were 2.3-fold higher in red muscle. A similar difference was observed in the ability of partially purified receptors to phosphorylate the exogenous substrate polyglutamate/tyrosine. The integrity of the insulin receptor preparation in the two fiber types was identical as determined by affinity cross-linking of [125I-TyrB26]insulin to the receptor. Mixing partially purified receptors from red and white muscle resulted in an additive response for exogenous substrate phosphorylation, suggesting that the difference in tyrosine kinase activity was not due to the presence of an inhibitor or activator. The results suggest that there are differences in the insulin receptors of red and white muscles that lead to discordance in their basal and insulin-stimulated intrinsic tyrosine kinase activity. The correlation between these differences and insulin action in red and white muscle supports the concept that the insulin receptor tyrosine kinase activity is involved in the initiation of insulin action.     

A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis

The cDNAs encoding the normal human insulin receptor (HIRc) and a receptor that had lysine residue 1018 replaced by alanine (A/K1018) were used to transfect Rat 1 fibroblasts. Lysine 1018 is a critical residue in the ATP binding site of the tyrosine kinase domain in the receptor beta-subunit. Untransfected Rat 1 cells express 1700 endogenous insulin receptors. Expressed HIRc receptors had levels of insulin-stimulable autophosphorylation in vitro comparable to normal receptors, whereas A/K1018 receptors had less than 1% of that activity. Stimulation by insulin of HIRc receptors in situ in intact cells led to phosphorylation of beta-subunit tyrosine residues and activation of tyrosine kinase activity that could be preserved and assayed in vitro after receptor purification. In contrast, A/K1018 receptors showed no such activation, either of autophosphorylation or of kinase activity toward histone. Cells expressing HIRc receptors display enhanced sensitivity to insulin of 2-deoxyglucose transport and glycogen synthase activity. This increased sensitivity was proportional to insulin receptor number at low but not at high levels of receptor expression. A/K1018 receptors were unable to mediate these biologic effects and actually inhibited insulin's ability to stimulate glucose transport and glycogen synthase through the endogenous Rat 1 receptors. Expressed HIRc receptors mediated insulin internalization and degradation, whereas A/K1018 receptors mediated little, if any. Endocytotic uptake of the expressed A/K1018 insulin receptors was also markedly depressed compared to normal receptors. Unlike HIRc receptors, A/K1018 receptors also fail to undergo down-regulation after long (24 h) exposures to high (170 nM) concentrations of insulin. We conclude the following. 1) Normal human insulin receptors expressed in Rat 1 fibroblasts display active tyrosine-specific kinase, normal intracellular itinerary after endocytosis, and normal coupling to insulin's biologic effects. 2) A receptor mutated to alter the ATP binding site in the tyrosine kinase domain had little if any tyrosine kinase activity. 3) This loss of kinase activity was accompanied by a nearly complete lack of both endocytosis and biologic activity.     

The role of antireceptor antibodies in stimulating phosphorylation of the insulin receptor

Four polyclonal antisera directed against the insulin receptor were tested for their capability to activate the tyrosine-specific protein kinase associated with the receptor. All four antisera were shown to inhibit insulin binding to the receptor in cultured human lymphoblastoid cells and to stimulate lipogenesis in isolated rat adipocytes. Although two antisera (B-d, B-8) stimulated the activity of the tyrosine kinase of partially purified receptor preparations from rat liver, two other antisera (B-2 and B-10) failed to do so. This failure could not be explained by lack of antibody binding to receptor, by interference with the receptor as a substrate for the kinase, or by blocking of the enzyme's active site. We conclude that these two antireceptor antibodies bind to the receptor but fail to activate the kinase. The simplest interpretation of these observations is that activation of the tyrosine-specific protein kinase might not be an obligatory step in coupling insulin binding to insulin action. However, it is also possible that the mechanism by which polyclonal antireceptor antisera mimic insulin's bioactivity may differ from the mechanism of action of insulin itself.