Summary
We have studied autophosphorylation and tyrosine kinase activity of the insulin receptor purified from liver and muscle of fasted rats before and after infusion of insulin (100 mU/h) during a 2.5 h glucose clamp. Recovery of insulin receptors and insulin binding to the solubilised receptors was unaffected by the glucose clamp. Autophosphorylation of the insulin receptor β subunit was increased in liver receptors prepared from rats at the end of the glucose clamp compared to rats in the basal state both in the absence of insulin in vitro (109% increase, p<0.001) and after in vitro stimulation with 10−7 mol/l insulin (clamped vs fasted; 96% increase, p<0.001). Insulin (10−7 mol/l) stimulated autophosphorylation was also increased in muscle receptor preparations from clamped rats compared with rats in the basal state (58% increase, p<0.05). In both liver and muscle receptors, the clamp increased the amount of [32P]-phosphate incorporated into the β subunit without changing the sensitivity of the insulin stimulation. HPLC analysis of the tryptic phosphopeptides derived from the β subunit after insulin stimulated autophosphorylation of liver receptors revealed an increase of 32P in all phosphorylation sites without any change in the overall pattern. Tyrosine kinase activity of liver and muscle insulin receptors from clamped rats was also increased approximately twofold (p<0.05) when analysed using a synthetic substrate (poly Glu4 Tyr1). Our results support the notion that the insulin receptor exists in an active and inactive form, and that elevated plasma insulin concentrations increases the proportion of active receptors.
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Kasuga M, Karlsson FA, Kahn CR (1982) Insulin stimulates the phosphorylation of the 95 000-Dalton subunit of its own receptor. Science 215: 185–187
Nemenoff RA, Kwok YC, Shulman GI, Blackshear PJ, Osathanondh R, Avruch J (1984) Insulin-stimulated tyrosine protein kinase. Characterisation and relation to the insulin receptor. J Biol Chem 259: 5058–5065
White MF, Stegmann EW, Dull TJ, Ullrich A, Kahn CR (1987) Characterisation of an endogenous substrate of the insulin receptor in cultured cells. J Biol Chem 262: 9769–9777
White MF, Takayama S, Kahn CR (1985) Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro. J Biol Chem 260: 9470–9478
Ellis L, Clauser E, Morgan DO, Edery M, Roth RA, Rutter WJ (1986) Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell 45: 721–732
Ebina Y, Araki E, Taira M, Shimada F, Mori M, Craik C, Siddle K, Pierce S, Roth RA, Rutter WJ (1987) Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin — and antibody-stimulated glucose uptake and receptor kinase activity. Proc Natl Acad Sci USA 84: 704–708
Morgan DO, Roth RA (1987) Acute insulin action requires insulin receptor kinase activity. Introduction of an inhibitory monoclonal antibody into mammalian cells blocks the rapid effects of insulin. Proc Natl Acad Sci 84: 41–45
Goldfine ID (1987) The insulin receptor: molecular biology and transmembrane signalling. Endocrinol Rev 8: 235–255
Kahn CR, Goldstein BJ (1989) Molecular defects in insulin action. Science 245: 13
Friedenberg GR, Henry RR, Klein HH, Reichard DR, Olefsky JM (1987) Decreased kinase activity of insulin receptors from adipocytes of non-insulin dependent diabetic subjects. J Clin Invest 79: 240–250
Caro J, Ittopp O, Poris WJ, Meelhem D, Flickinger EG, Thomas F, Jenquin M, Silverman JF, Khanzanie PG, Sinha MK (1986) Studies of the mechanisms of insulin resistance in the liver from humans with non-insulin dependent diabetes. J Clin Invest 78: 249–258
Arner P, Pollare T, Lithell H, Livingston JN (1987) Defective insulin receptor tyrosine kinase in human skeletal muscle in obesity and Type 2 (non-insulin-dependent) diabetes mellitus. Diabetologia 30: 437–440
Obermaier-Kusser B, White MF, Pongratz DE, Su Z, Ermel B, Muhlbacher C, Haring HU (1989) A defective intramolecular autoactivation cascade may cause the reduced kinase activity of the skeletal muscle insulin receptor from patients with non-insulin-dependent diabetes mellitus. J Biol Chem 264: 9497–9504
Burant CF, Treutelaar MK, Buse MG (1986) Diabetes induced functional and structural changes in insulin receptors from rat skeletal muscle. J Clin Invest 78: 260–270
Okamoto M, White MF, Maron R, Kahn CR (1986) Autophosphorylation and kinase activity of insulin receptor in diabetic rats. Am J Physiol 251: E542-E550
Freidenberg GR, Klein HH, Cordera R, Olefsky JM (1985) Insulin receptor kinase activity in rat liver: regulation by fasting and high carbohydrate feeding. J Biol Chem 260: 12444–12453
Takata Y, Sasaoka T, Iwasaki M, Shigeta Y (1988) Alteration of the insulin-receptor kinase activity by high fat feeding. Diabetes 37: 1397–1404
White MF, Shoelson SE, Keutmann H, Kahn CR (1988) A cascade of tyrosine autophosphorylation in the β subunit activates the phosphotransferase of the insulin receptor. J Biol Chem 263: 2969–2980
Tavare JM, Denton RM (1988) Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of sites phosphorylated by two-dimensional peptide mapping. Biochem J 252: 607–615
Tornquist HE, Avruch J (1988) Relationship of site-specific β subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine protein kinase activity). J Biol Chem 263: 4593–4601
Shoelson SE, White MF, Kahn CR (1988) Tryptic activation of the insulin receptor. Proteolytic truncation of the α-subunit releases the β-subunit from inhibitory control. J Biol Chem 263: 4852–4960
Takayama S, White MF, Kahn CR (1988) Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity. J Biol Chem 263: 3440–3447
Tornquist HE, Gunsalus JR, Nemenoff RA, Frackelton AR, Pierce MW, Avruch J (1988) Identification of the insulin receptor tyrosine residues undergoing insulin-stimulated phosphorylation in intact rat hepatoma cells. J Biol Chem 263: 350–359
Kraegen EW, James DE, Bennett SP, Chisholm DJ (1983) In vivo insulin sensitivity in the rat determined by euglycaemic clamp. Am J Physiol 245: E1-E7
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72: 246–254
Desbuquois B, Aurbach GD (1971) Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays. J Clin Endocrinol 33: 732–738
Pang DT, Sharma BR, Shafer JR (1985) Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies. Arch Biochem Biophys 242: 176–186
Goren HJ, White MF, Kahn CR (1987) Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity. Biochemistry 26: 2374–2382
Heding LG (1972) Determination of total serum insulin (IRI) in insulin-treated diabetic patients. Diabetologia 8: 260–266
Reddy SSK, Kahn CR (1988) Insulin resistance: a look at the role of insulin receptor kinase. Diabetic Med 5: 621–629
Merion M, Sly WS (1983) The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J Cell Biol 96: 644–650
Posner BI, Khan MN, Bergeron JJM (1987) Internalisation of insulin and its receptor: role in signaling. In: Raizada MK, Phillips MR, Le Roith DD (eds). Insulin, insulin-like growth factors and their receptors in the central nervous system. Plenum Press, New York London, pp 1–10
Ballotti R, Kowalski A, White MF, Le Marchand-Brustel Y, Van Obberghen E (1987) Insulin stimulated tyrosine phosphorylation of its receptor β-subunit in intact rat hepatocytes. Biochem J 241: 99–104
Pang DT, Sharma B, Shafer JA, White MF, Kahn CR (1985) Predominance of tyrosine phosphorylation of insulin receptors during the insulin response of intact cells to insulin. J Biol Chem 260: 7131–7136
Takayama S, White MF, Lauris V, Kahn CR (1984) Phorbol esters modulate insulin receptor phosphorylation and insulin action in cultured hepatoma cells. Proc Natl Acad Sci USA 81: 7797–7801
Kruszynska YT, Home PD, Alberti KGMM (1985) Comparison of portal and peripheral insulin delivery on carbohydrate metabolism in streptozotocin diabetic rats. Diabetologia 28: 167–171
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Kruszynska, Y.T., Halban, P.A., Kahn, C.R. et al. Activation of liver and muscle insulin receptor tyrosine kinase activity during in vivo insulin administration in rats. Diabetologia 33, 77–83 (1990). https://doi.org/10.1007/BF00401044
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DOI: https://doi.org/10.1007/BF00401044