Glucosylceramide synthase inhibitors D-PDMP and D-EtDO-P4 decrease the GM3 ganglioside level, differ in their effects on insulin receptor autophosphorylation but increase Akt1 kinase phosphorylation in human hepatoma HepG2 cells
Glucosylceramide synthase inhibitors D-PDMP and D-EtDO-P4 decrease the GM3 ganglioside level, differ in their effects on insulin receptor autophosphorylation but increase Akt1 kinase phosphorylation in human hepatoma HepG2 cells
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DOI：
10.18388/abp.2014_930
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发表时间：
2016-01-01
影响因子：
1.7
通讯作者：
Pacuszka, Tadeusz
中科院分区：
文献类型：
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作者：
Fedoryszak-Kuska, Natalia;Panasiewicz, Miroslawa;Pacuszka, Tadeusz
神经节苷脂是几种细胞生长相关受体的调节剂。研究表明，对于富含小窝蛋白的脂肪细胞，GM3神经节苷脂与胰岛素受体(IR)结合，使其复合体与小窝蛋白解离，从而降低胰岛素治疗后IR的自磷酸化。我们将这些研究扩展到人类肝细胞来源的HepG2细胞，其特征是高水平的IR但低的小窝蛋白。为了降低神经鞘糖脂的含量，用葡萄糖神经酰胺合成酶抑制剂d-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol(D-PDMP)和D-苏氨酸-1-(3，4-乙二氧基)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol(D-EtDO-P4)。40 mU M的D-PDMP和1 mU M的D-EtDO-P4使GM3含量分别下降到对照的22.3%(17.8-26.1%)和18.1%(13.7-24.4%)。用D-PDMP获得的GM3减少的同时，细胞用100nM胰岛素刺激后IR自动磷酸化水平显著增加185.1%(153.5-423.8%)。D-EtDO-P4对IR自磷酸化的影响较小，达对照水平的134.8%(111.3-167.8%)，差异无统计学意义。D-PDMP和D-EtDO-P4的作用也可以在Akt1激酶的水平上检测到。在D-PDMP存在下生长的细胞中，Akt1的磷酸化水平是对照组的286.0%(151.4%-621.1%)。在这种情况下，D-EtDO-P4的作用类似：223.0%(181.4-315.4%)显着增加磷酸化Akt1。我们推测，HepG2细胞中鞘糖脂的耗竭不仅可能影响IR的自磷酸化，还可能通过改变Akt1的膜微环境而独立地影响Akt1的磷酸化。
Gangliosides function as modulators of several cell growth related receptors. It was shown for caveolin-rich adipocytes, that GM3 ganglioside binds to insulin receptor (IR), dissociates its complex with caveolin, and thus lowers IR autophosphorylation following insulin treatment. We extended those studies into human hepatocyte-derived HepG2 cells, characterized by a high level of IR but low of caveolin. To lower the glycosphingolipid content, estimated by GM3 concentration, two glucosylceramide synthase inhibitors d-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and D-threo-1-(3,4,-ethylenedioxy) phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (D-EtDO- P4) were used. D-PDMP at 40 mu M or D-EtDO-P4 at 1 mu M concentrations in culture medium decreased the GM3 content to 22.3% (17.8-26.1%) and 18.1% (13.7-24.4%), respectively, of the control value. The reduction of GM3 obtained with D-PDMP was accompanied by a 185.1% (153.5-423.8%) significant increase in the level of IR autophosphorylation following cell stimulation with 100 nM insulin. The effect of D-EtDO-P4 on IR autophosphorylation was smaller amounting to an increase by 134.8% (111.3-167.8%) of the control level and statistically non-significant. The effects of D-PDMP and D-EtDO-P4 could also be detected at the level of Akt1 kinase. In cells grown in the presence of D-PDMP the level of phosphorylated Akt1 was 286.0% (151.4%-621.1%) of that in the control. In this case the effect of D-EtDO-P4 was similar: 223.0% (181.4-315.4%) significant increase in phosphorylated Akt1. We assume that glycosphingolipid depletion in HepG2 cells may affect not only IR autophosphorylation but also, independently, the phosphorylation of Akt1, by modifying the membrane microenvironment of this kinase.