Wrede C E, Dickson L M, Lingohr M K, Briaud I, Rhodes C J
Pacific Northwest Research Institute and Department of Pharmacology, University of Washington, Seattle, WA 98122, USA.
J Mol Endocrinol. 2003 Jun;30(3):271-86. doi: 10.1677/jme.0.0300271.
It is possible that activation of protein kinase C (PKC) isoforms by free fatty acids (FFA) plays a role in the failure of pancreatic beta-cell mass expansion to compensate for peripheral insulin resistance in the pathogenesis of type-2 diabetes. The effect of lipid moieties on activation of conventional (PKC-alpha and -beta1), novel (PKC-delta) and atypical (PKC-zeta) PKC isoforms was evaluated in an in vitro assay, using biotinylated neurogranin as a substrate. Oleoyl-Coenzyme A (CoA) and palmitoyl-CoA, but not unesterified FFA, significantly increased the activity of all PKC isoforms (P< or =0.05), particularly that for PKC-delta. It was found that FFA (0.4 mM oleate/complexed to 0.5% bovine serum albumin) inhibited IGF-I-induced activation of protein kinase B (PKB) in the pancreatic beta-cell line (INS-1), but this was alleviated in the presence of the general PKC inhibitor (Gö6850; 1 microM). To further investigate whether conventional or novel PKC isoforms adversely affect beta-cell proliferation, the effect of phorbol ester (phorbol 12-myristate 13-acetate; PMA)-mediated activation of these PKC isoforms on glucose/IGF-I-induced INS-1 cell mitogenesis, and insulin receptor substrate (IRS)-mediated signal transduction was investigated. PMA-mediated activation of PKC (100 nM; 4 h) reduced glucose/IGF-I mediated beta-cell mitogenesis (>50%; P< or =0.05), which was reversible by the general PKC inhibitor Gö6850 (1 microM), indicating an effect of PKC and not due to a non-specific PMA toxicity. PMA inhibited IGF-I-induced activation of PKB, correlating with inhibition of IGF-I-induced association of IRS-2 with the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, in contrast, PMA activated the mitogen-activated protein kinases, Erk1/2. Titration inhibition analysis using PKC isoform inhibitors indicated that these PMA-induced effects were via novel PKC isoforms. Thus, FFA/PMA-induced activation of novel PKC isoforms can inhibit glucose/IGF-I-mediated beta-cell mitogenesis, in part by decreasing PKB activation, despite an upregulation of Erk1/2. Thus, activation of novel PKC isoforms by long-chain acyl-CoA may well contribute to decreasing beta-cell mass in the pathogenesis of type-2 diabetes, similar to their inhibition of insulin signal transduction which causes insulin resistance.
在2型糖尿病发病机制中，游离脂肪酸（FFA）激活蛋白激酶C（PKC）亚型可能在胰腺β细胞质量扩张失败以补偿外周胰岛素抵抗方面发挥作用。在体外试验中，以生物素化的神经颗粒蛋白为底物，评估了脂质部分对传统（PKC-α和-β1）、新型（PKC-δ）和非典型（PKC-ζ）PKC亚型激活的影响。油酰辅酶A（CoA）和棕榈酰辅酶A，而非未酯化的FFA，显著增加了所有PKC亚型的活性（P≤0.05），尤其是PKC-δ的活性。研究发现，FFA（0.4 mM油酸/与0.5%牛血清白蛋白复合）抑制了胰腺β细胞系（INS-1）中胰岛素样生长因子-I（IGF-I）诱导的蛋白激酶B（PKB）激活，但在存在通用PKC抑制剂（Gö6850；1 μM）时这种抑制作用得到缓解。为了进一步研究传统或新型PKC亚型是否对β细胞增殖产生不利影响，研究了佛波酯（佛波醇12-肉豆蔻酸酯13-乙酸酯；PMA）介导的这些PKC亚型激活对葡萄糖/IGF-I诱导的INS-1细胞有丝分裂以及胰岛素受体底物（IRS）介导的信号转导的影响。PMA介导的PKC激活（100 nM；4小时）降低了葡萄糖/IGF-I介导的β细胞有丝分裂（>50%；P≤0.05），这可被通用PKC抑制剂Gö6850（1 μM）逆转，表明是PKC的作用而非非特异性的PMA毒性。PMA抑制了IGF-I诱导的PKB激活，这与抑制IGF-I诱导的IRS-2与磷脂酰肌醇-3激酶的p85调节亚基的结合相关。然而，相反的是，PMA激活了丝裂原活化蛋白激酶Erk1/2。使用PKC亚型抑制剂的滴定抑制分析表明，这些PMA诱导的效应是通过新型PKC亚型介导的。因此，FFA/PMA诱导的新型PKC亚型激活可部分通过降低PKB激活来抑制葡萄糖/IGF-I介导的β细胞有丝分裂，尽管Erk1/2上调。因此，长链酰基辅酶A激活新型PKC亚型很可能在2型糖尿病发病机制中导致β细胞质量减少，类似于它们对胰岛素信号转导的抑制从而导致胰岛素抵抗。
