賴德仁林志立李欣樺Hsin-Hua Li醫學研究所2026-10-299999-12-312016https://handle.ncl.edu.tw/11296/bp36muhttps://scholars.csmu.edu.tw/handle/123456789/36247已知胰島素訊息缺損和糖尿病與老化相關疾病之間有緊密關聯性，如血管疾病、阿茲海默症和非酒精性脂肪肝等，本研究證實老化相關疾病和神經退化性疾病的致病機轉確實與胰島素訊息有相關聯。第一部分研究證實在SK-N-MC神經細胞中如腐植酸血管疾病因子的存在會加重乙型類澱粉(β-Amyloid, Aβ)所誘發的神經毒性，其機制主要透過刺激PERK和Eukaryotic Initiation Factor 2 α (eIF2α) 的磷酸化活化內質網壓力。研究結果也發現Aβ誘發的氧化壓力(oxidative stress)和粒線體功能失調主要因為減少Silent information regulator-mammalian ortholog 1 (Sirt1)/peroxisome proliferators-activated receptor γ co-activator 1α (PGC-1α)的表現，導致神經細胞加速凋亡。然而，在腐植酸和Aβ共同處理情況下，若外加內質網壓力抑制劑salubrinal (eIF2α inhibitor)或Sirt1過量表現則顯著地恢復細胞存活率。研究推論血管性疾病如腐植酸可以透過活化內質網壓力和氧化傷害而加重Aβ誘導的神經細胞毒性，此結果提供一個新的解釋在血管疾病誘發阿茲海默症的致病機轉。第二部分證實一個特別的microRNA miR-302沉默phosphatase and tensin homolog (PTEN)基因而活化Akt的表現，隨後刺激Nanog的表現，進而抑制Aβ誘發的pSer307 insulin receptor substrate 1 (IRS-1) 表現，並增加IRS在 tyrosine 的磷酸化和活化pSer 473-Akt/pSer 9-glycogen synthase kinase 3β (GSK3β) 。此外也發現miR-302可經由調控Akt路徑進而刺激nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)的表現，最後抑制Aβ誘發的氧化傷害。更進一步還在人體實驗研究阿茲海默症病人的peripheral blood mononuclear cell (PBMC)中Nanog和La ribonucleoprotein domain family member 7 (LARP7)的mRNA表現，阿茲海默症病人皆顯著地減少Nanog和LARP7的mRNA的表現，本研究認為以上結果在未來可有助於開發新的診斷方法。第三部分則說明在非酒精性脂肪肝疾病中，miR-302如何減少肝臟脂肪酸的合成和減緩肝臟胰島素阻抗的一個可能機制。結果發現miR-302透過沉默超長鏈脂肪酸延伸酶(elongation of very long chain fatty acids protein 6, Elovl6)，其功能為延伸C16到C18的酵素，減少高脂肪酸誘導的脂肪堆積、氧化傷害和肝臟胰島素阻抗等現象。綜合以上的研究結果證實氧化壓力似乎與老化相關疾病，如血管性疾病、阿茲海默症和非酒精性脂肪肝等疾病中扮演著重要的致病危險因子。而本研究發現透過減少氧化傷害或保護胰島素訊息路徑可以做為保護老化相關的致病機制，希望經由發現這些機制在未來對於這些疾病的預防與治療上能有所貢獻。Impaired insulin signaling was known to be linked to diabetes and aging-related diseases such as vascular disease, Alzheimer’s disease (AD) and non-alcoholic fatty liver disease (NAFLD). In the present study, we demonstrated the possible pathogenic mechanism of aging-associated disease and neurodegeneration via affecting insulin-related signaling. In the part I, the results showed that humic acid (HA) exhibits a synergistic effect enhancing Aβ-induced neurotoxicity in SK-N-MC neuronal cells. Furthermore, this deterioration was mediated through activation of endoplasmic reticulum (ER) stress by stimulating phosphorylation of PERK and Eukaryotic Initiation Factor 2 α (eIF2α). Our results also observed HA and Aβ-induced oxidative stress was associated with mitochondrial dysfunction caused by down-regulation of Silent information regulator-mammalian ortholog 1 (Sirt1)/peroxisome proliferators-activated receptor γ co-activator 1α (PGC-1α) pathway. However, treating with the ER stress inhibitor salubrinal, or overexpression of Sirt1 significantly restored cell viability affected by HA and Aβ. These findings suggested a new mechanism by which HA can deteriorate Aβ-induced neurotoxicity through modulation of ER stress, which may provide significant insights into the pathogenesis of AD co-occurring with vascular injury. Part II demonstrated a specific microRNA, miR-302, silenced phosphatase and tensin homolog (PTEN) activated Akt signaling, which subsequently stimulated Nanog expression to suppress Aβ-induced p-Ser307 insulin receptor substrate 1 (IRS-1) expression and thus enhanced tyrosine phosphorylation and p-Ser 473-Akt/p-Ser 9-GSK3β formation. In addition, we also found that miR-302-mediated Akt signaling further stimulated nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) elevation and hence inhibited Aβ-induced oxidative stress. Furthermore, our in vivo studies revealed that the expression of Nanog and La ribonucleoprotein domain family member 7 (LARP7) mRNA levels were significantly reduced in AD patients'' blood cells, providing a novel potential diagnosis marker for AD. Part III demonstrated a possible mechanism of miR-302 regulated fatty acid formation and hepatic insulin resistance in NAFLD. Our results demonstrated that miR-302 targeted elongation of very long chain fatty acids protein 6 (Elovl6), mainly elongated C16-18 aliphatic acid, which attenuated high fatty acid-induced hepatic lipid accumulation, oxidative stress and insulin resistance. The above-listed observations reveal that oxidative stress indeed exhibits aging-related diseases such as vascular disease, Alzheimer’s disease (AD) and Non-alcoholic fatty liver disease (NAFLD) and display an important risk factor. Our results suggest that reducing oxidative stress or preventing insulin signaling show protection effects in aging-related diseases, and may help to provide novel therapeutic strategies or drug targets in future.老化血管疾病阿茲海默症非酒精性脂肪肝胰島素訊息miR-302agingvascular diseaseAlzheimer’s disease (AD)non-alcoholic fatty liver disease (NAFLD)insulin signalingthesis