Research Unit of Diabetes and Endocrine Diseases, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy.
PLoS One. 2011 May 5;6(5):e19462. doi: 10.1371/journal.pone.0019462.
The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.
本研究旨在深入探讨 ENPP1（胰岛素受体（IR）激活的负调节剂）在胰岛素信号转导、胰岛素分泌以及最终葡萄糖代谢中的作用机制。将携带 K121 或 Q121 变异体的 ENPP1 cDNA 转染入 HepG2 肝、L6 骨骼肌和 INS1E 胰岛β细胞。研究胰岛素诱导的 IR 自身磷酸化（HepG2、L6、INS1E）、Akt-Ser(473)、ERK1/2-Thr(202)/Tyr(204)和 GSK3-β Ser(9)磷酸化（HepG2、L6）、PEPCK mRNA 水平（HepG2）和 2-脱氧-D-葡萄糖摄取（L6）。还研究了 GLUT4mRNA（L6）、胰岛素分泌和 caspase-3 激活（INS1E）。与未转染细胞相比，HepG2-K、L6-K 和 INS1E-K 中的胰岛素诱导的 IR 自身磷酸化分别降低了 20%、52%和 11%（HepG2-K、L6-K 和 INS1E-K），而 HepG2-Q、L6-Q 和 INS1E-Q 中的胰岛素诱导的 IR 自身磷酸化则降低了 44%、92%和 30%（HepG2-Q、L6-Q 和 INS1E-Q）。在 HepG2 和 L6 中也得到了类似的 Akt-Ser(473)、ERK1/2-Thr(202)/Tyr(204)和 GSK3-β Ser(9)的数据。未转染、HepG2-K 和 HepG2-Q 细胞中胰岛素诱导的 PEPCK mRNA 降低幅度逐渐降低（65%、54%、23%）。未转染的 L6 中胰岛素诱导的葡萄糖摄取增加了 60%（基础水平以上），而 L6-K 和 L6-Q 细胞中的葡萄糖摄取则完全被消除。L6-K 和 L6-Q 中的 GLUT4mRNA 略有减少（与未转染细胞相比分别减少 13%和 25%）。与未转染细胞相比，INS1E-K 中的葡萄糖诱导的胰岛素分泌减少了 60%，而 INS1E-Q 中的胰岛素分泌几乎被消除。在未转染的 INS1E、INS1E-K 和 INS1E-Q 中，血清缺乏使 caspase-3 激活了两倍、三倍和四倍。与 KK 和 KQ 个体相比，来自纯合子 QQ 供体的分离人胰岛中，glyburide 诱导的胰岛素分泌减少了 50%。我们的数据清楚地表明，ENPP1，尤其是当 Q121 变异体起作用时，会影响骨骼肌和肝细胞中的胰岛素信号转导和葡萄糖代谢，以及胰岛素分泌β细胞的功能和存活，因此它是一种导致胰岛素抵抗、胰岛素分泌缺陷和葡萄糖代谢异常的强致病因素。
