Dominici F P, Balbis A, Bartke A, Turyn D
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Junin 956, (1113) Buenos Aires, Argentina.
J Endocrinol. 1998 Oct;159(1):15-25. doi: 10.1677/joe.0.1590015.
Overexpression of bovine growth hormone (bGH) in transgenic (PEPCK-bGH) mice induces resistance to insulin, which is compensated by a major increase in insulin levels. In these animals, hepatic insulin receptors (InsRs) are downregulated while tyrosine kinase activity of wheat germ agglutinin (WGA)-purified InsRs towards exogenous substrates is unexpectedly increased. By normalizing insulinemia, we attempted to determine whether the alterations detected in the early steps of insulin signal transduction are due to exposure to chronically high GH levels or are secondary to hyperinsulinemia. Transgenic PEPCK-bGH animals were treated with a single intraperitoneal administration of streptozotocin (STZ) or were deprived of food for 48 h, to normalize insulin levels. Both fasting and STZ treatment were effective in reducing insulin blood levels to control values or below, while GH levels remained unchanged (STZ treatment) or increased (fasted animals). In the liver of untreated transgenic mice, the number of InsRs as determined by 125I-insulin binding was significantly diminished (65+/-5% and 60+/-6% of normal values in microsomes and solubilized membranes respectively;P<0.01 vs control mice). In treated transgenic mice, the number of InsRs increased to values similar to or slightly higher than those found in normal control mice (STZ-treated: 139+/-26% and 126+/-8%; fasted: 128+/-5% (P<0.05) and 102+/-1.5%, for microsomes and solubilized membranes respectively). Neither treatment altered InsR affinity. InsR concentration in liver as determined by immunoblotting using an antibody against the beta-subunit of the insulin receptor was found to be reduced in transgenic mice (69+/-3% of normal values,P<0.001) and was normalized after both STZ treatment (105+/-4%) and fasting (109+/-4%). Insulin-stimulated autophosphorylation activity of InsRs in transgenic mice was increased (154+/-13%,P<0.01 compared with the control group), essentially normalized by STZ treatment (96+/-14%), and reduced by fasting, to below the values measured in normal control mice (56+/-15%,P<0.05). The potential influence of basal serine/threonine (Ser/Thr) phosphorylation of the InsR beta-subunit on the regulation of the InsRs from transgenic mice was also investigated. The autophosphorylation activity of WGA-purified InsRs from all groups of mice studied was essentially unchanged after dephosphorylation with alkaline phosphatase or mild trypsinization. Consequently, our results suggest that the observed changes in InsR number and autophosphorylation activity in the liver of bGH transgenic mice are directly related to changes in insulin blood levels, and that Ser/Thr phosphorylation is apparently not involved in the regulation of the InsR autophosphorylation activity in this model of insulin resistance.
在转基因（PEPCK-bGH）小鼠中，牛生长激素（bGH）的过表达会诱导胰岛素抵抗，而胰岛素水平的大幅升高可对此进行代偿。在这些动物中，肝脏胰岛素受体（InsRs）下调，而经麦胚凝集素（WGA）纯化的InsRs对外源底物的酪氨酸激酶活性却意外增加。通过使胰岛素血症正常化，我们试图确定在胰岛素信号转导早期步骤中检测到的改变是由于长期暴露于高水平的生长激素，还是继发于高胰岛素血症。对转基因PEPCK-bGH动物进行单次腹腔注射链脲佐菌素（STZ）或禁食48小时，以使胰岛素水平正常化。禁食和STZ处理均能有效将胰岛素血水平降低至对照值或更低，而生长激素水平保持不变（STZ处理）或升高（禁食动物）。在未处理的转基因小鼠肝脏中，通过¹²⁵I-胰岛素结合测定的InsRs数量显著减少（微粒体和可溶性膜中分别为正常值的65±5%和60±6%；与对照小鼠相比，P<0.01）。在处理过的转基因小鼠中，InsRs数量增加至与正常对照小鼠相似或略高的值（STZ处理组：微粒体和可溶性膜中分别为139±26%和126±8%；禁食组：分别为128±5%（P<0.05）和102±1.5%）。两种处理均未改变InsR亲和力。使用抗胰岛素受体β亚基抗体通过免疫印迹法测定的肝脏中InsR浓度在转基因小鼠中降低（为正常值的69±3%，P<0.001），在STZ处理（105±4%）和禁食（109±4%）后均恢复正常。转基因小鼠中胰岛素刺激的InsRs自磷酸化活性增加（154±13%，与对照组相比，P<0.01），经STZ处理基本恢复正常（96±14%），禁食则使其降低至低于正常对照小鼠测得的值（56±15%，P<0.05）。还研究了InsRβ亚基的基础丝氨酸/苏氨酸（Ser/Thr）磷酸化对转基因小鼠InsRs调节的潜在影响。在用碱性磷酸酶或轻度胰蛋白酶处理去磷酸化后，所有研究小鼠组中WGA纯化的InsRs的自磷酸化活性基本未变。因此，我们的结果表明，bGH转基因小鼠肝脏中观察到的InsR数量和自磷酸化活性变化与胰岛素血水平变化直接相关，并且在这种胰岛素抵抗模型中，Ser/Thr磷酸化显然不参与InsR自磷酸化活性的调节。
