肥胖是引發胰島素不敏感以及第二型糖尿病的高度危險因子， 而目前已經被證實，當血液中脂肪酸的濃度提高時會造成胰島素所誘導的葡萄糖吸收能力受到阻斷。神經醯胺 (ceramide)，為一脂肪酸的衍生物，會透過降低AKT/protein kinase B (PKB)和 glycogen synthase kinase-3 (GSK3)活性而影響胰島素的訊息傳遞，然而神經醯胺對於上游分子，例如IRS1以及PI3K則沒有影響。本研究目的主要是進一步探討神經醯胺在胰島素訊息傳遞的詳細作用機轉。胰島素刺激的C2C12肌肉細胞中，100 μM C2-神經醯胺會造成AKT在絲胺酸473 (Serine 473)位點磷酸化程度降低，這一點同文獻所報導。另一方面，在訊息傳遞的上游分子中，例如IRS1在酪胺酸 (Tyrosine)位點磷酸化程度以及IRS1總蛋白表現量上則沒有太大的影響。而在同樣的處理條件中，我們發現S6K的磷酸化程度卻有顯著的增加。我們利用mTORC1的抑制劑Rapamycin處理，進一步證實C2-神經醯胺所造成AKT在絲胺酸473 (serine 473)位點磷酸化程度降低和增加的S6K的磷酸化可能有因果關係。所以根據目前已建立的胰島素訊息傳遞模式中，S6K上游分子如TSC1/TSC2，Rheb，mTORC1均有可能為C2-神經醯胺的直接作用標的。我們利用shRNA減少這些蛋白質在細胞中的表現以進一步檢測C2-神經醯胺對於胰島素訊息傳遞破壞作用是否因此受到影響，發現在C2C12肌肉細胞中減弱了Rheb分子表現量時確實會對C2-神經醯胺所造成Akt473磷酸化程度下降的作用產生干擾，因此我們找出C2-神經醯胺的作用應該位於PI3K的下游，但Rheb分子/或上游。此一發現對於C2-神經醯胺在肌肉細胞所造成之胰島素不敏感的現象，可謂訂出了C2-神經醯胺的標的分子，而詳細的作用層級及其作用機轉將有助於藥物標的的開發。

Obesity is a high risk factor to develop insulin resistance and type II diabetes. Chronic elevation of free fatty acid levels in plasma has been found to be closely associated with impaired insulin-mediated glucose uptake. Ceramide, a fatty acid derived lipid, was confirmed as a negative regulator of insulin signaling pathway by reducing Akt/protein kinase B (PKB) and glycogen synthase kinase-3 (GSK3) activity. However, there are no effects on the upstream signaling molecules, such as IRS1 and PI3K. In this study, we intend to investigate the detail molecular mechanism of C2-ceramide on insulin signaling. We showed that 100 μM C2-ceramide caused a decrease of Akt Ser473 phosphorylation in insulin-stimulated C2C12 myotubes, which is in consistent with the results on the literatures. While, the phosphorylation of the upstream signaling molecules, IRS1 tyrosine residue, and IRS1 total abundance were not changed. Interestingly, an increased phosphorylation of S6K was observed in the same condition. Utilizing rapamycin, an mTORC1 inhibitor, we further demonstrated that the decreased Akt Ser473 phosphorylation might be mediated through the activated S6K. According to current model of insulin signaling pathway, the upstream molecules of S6K such as TSC1/2, Rheb and mTORC1 could have been the targets of C2-ceramide. Utilizing shRNA to individually knockdown these molecules in order to examine the effect of loss-of-function on C2-ceramide impaired insulin signaling pathway, we found that Rheb knockdown in C2C12 myoblasts can block the decreased Akt Ser473 phosphorylation induced by C2-ceramide. Taken together, we explored a possible novel pathway of C2-ceramide impaired insulin signaling through activated S6K to interfere with Akt Ser473 phosphorylation. The affected signaling molecules had also been pinpointed to upstream of Rheb but downstream of PI3K. Further investigation on mapping and detail mechanism will provide great therapeutically strategy on treatment of type II diabetes.

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