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İnsülin direnci gelişiminde pankreatik β-hücre farklılaşmasının moleküler mekanizmasının in vitro araştırılması Skip to content Tez Künye Durumu İnsülin direnci gelişiminde pankreatik β-hücre farklılaşmasının moleküler mekanizmasının in vitro araştırılması / The in vitro investigation of molecular mechanism of pancreatic β-cell differentiation in the development of insulin resistance Yazar:MERVE ERÇİN Danışman: DOÇ. DR. SELDA OKTAYOĞLU Yer Bilgisi: İstanbul Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı / Moleküler Biyoloji Bilim Dalı Konu:Biyoloji = Biology Dizin: Onaylandı Yüksek Lisans Türkçe 2017 115 s. Bu çalışmanın amacı, tip2 diyabetik bireylerin açlık kan örneklerinde glukoz ve insülin seviyelerinin yükselmesi ile belirlenen insülin direnci ve özellikle obeziteye bağlı olarak gelişen açlık kan örneklerinde leptin seviyelerinin yükselmesi ile belirlenen leptin direncinin in vitro şartlarda taklit edilerek, bu uyaranların pankreatik adacık kaynaklı mezenkimal kök hücre (PAK-MKH)’leri beta hücrelerine farklılaştırma olasılığının araştırılması ve ilgili moleküler mekanizmanın ortaya konmasıdır. Bu amaçla PAK-MKH’lere 24 saat süreyle 20 mM glukoz, 1 nM insülin ve 0,5 ng/mL leptin uygulandı. Beta hücre farklılaşmasının tespit edilebilmesi için insülin üretiminde rol alan transkripsiyon faktörleri veya beta hücrelerine özgün proteinler olan Pdx-1, Pax4, Nkx2.2, Nkx6.1 ve ins2 genlerinin ekspresyon seviyeleri gerçek zamanlı geri çevrim polimeraz zincir reaksiyonu (qRT-PCR) yöntemiyle gösterildi. Gen ekspresyonu düzeyinde insülin üretimi ile ilgili elde edilen bulgular, insülin+ hücrelerin oranının akım sitometrik analizi ile desteklendi. Reaktif oksijen türleri (ROS)’nin üretiminde meydana gelen artış bir farklılaşma belirteci olarak kullanıldı ve ROS üreten hücrelerin oranı DCFDA+ hücrelerin akım sitometrik analizi ile gösterildi. Ayrıca, yeni farklılaşan beta hücrelerinin fonksiyon edinimi, yükselen glukoz uyarısına yanıt olarak salınan insülin seviyelerinin enzim bağlı immünosorbent tekniği ile belirlenmesi yoluyla gösterildi. p-IRS1 (Ser307), PTEN ve p-AKT (Ser473) seviyelerinde meydana gelen artışa karşın p-IRS1 (Try) seviyesinin azalması insülin direnci belirteci olarak kabul edilirken, SOCS3 seviyesindeki artış leptin direnci göstergesi olarak kullanıldı. Bu belirteçlerin seviyelerindeki değişimler western emdirimi tekniği ile gösterildi. PI3K/AKT sinyal yolağının negatif bir düzenleyicisi olan PTEN’in bu mekanizmadaki olası rolü bu molekülün farmakolojik bir inhibitörü olan bpV(HOpic) uygulanması yolu ile araştırıldı. Aktivitesi AKT sinyali ile düzenlenen p-GSK-3β (Ser9 ve Try216) ve β-katenin seviyelerindeki değişimler western emdirimi yöntemiyle gösterildi. β-katenin’in N-kaderin ile olan etkileşimi immunopresipitasyon yöntemini takiben western emdirimi tekniği ile gösterildi. İlaveten insülin ve leptin sinyalinde önemli bir molekül olan Tub’un membran, sitoplazmik ve nuklear fraksiyonlardaki seviyelerinin western emdirimi tekniği ile gösterilmesi sonucunda bu proteinin aktivasyonu ile ilgili fikir edinildi. β-katenin’in Tub ile olası etkileşimi immunopresipitasyon yöntemini takiben western emdirimi tekniği ile araştırıldı. β-katenin ve Tub’un bu mekanizmadaki rolleri özgün susturucu RNA’lar kullanılarak teyit edildi. β-katenin ve Tub genlerinin ekspresyon seviyelerindeki değişimler qRT-PCR tekniği ile gösterildi. Tub’un insülin sentezinde önemli rol oynayan transkripsiyon faktörleri olan ins1, ins2, pdx1 ve MafA üretimindeki olası rolleri kromozom immünopresipitasyon tekniğiyle araştırıldı. Sonuç olarak, uyaran uygulanan tüm gruplarda beta hücre farklılaşmasının başlaması söz konusu olmakla birlikte, insülin ve leptinin birlikte uygulandığı grupta yeni oluşan beta hücrelerinin çok daha yüksek oranda olduğu, hücrelerin farklılaşmanın daha ileri aşamasında ve daha olgun olduğu tespit edildi. Ancak, yeni oluşan beta hücreleri fonksiyonel değildi. Bu gruptaki hücrelerde yüksek oranda SOCS3, p-IRS1 (Ser307) ve PTEN, düşük oranda p-IRS1 (Try) varlığı ile birlikte p-AKT (Ser473) seviyesinin değişmediği tespit edildi. Bu bulgular PAK-MKH’lere insülin ve leptinin birlikte uygulanması sonucu insülin ve leptin direncinin geliştiğini göstermektedir. Yüksek oranda olgun beta hücrelerinin oluşmasını uyaran bu sinyal yolağında esasen AKT/GSK-3β/β-katenin ve Tub’un rol aldığı gösterildi. β-katenin ve Tub’un nukleusta birbirine bağlandıkları tespit edildi. Ayrıca, Tub’un insülin sentezinde anahtar rol oynayan ins1, ins2, pdx1 ve MafA genlerinin promotör bölgelerine bağlandığı tespit edildi. Bu bulgular, tip2 diyabet gelişiminin ilk basamağı olan kompenzasyon aşamasında PAK-MKH’lerde gelişen insülin ve leptin direncinin beta hücre farklılaşmasını belirgin olarak artırdığını açıkça ortaya koymaktadır. Bu farklılaşmanın engellenebilmesi amacıyla AKT/GSK-3β/β-katenin ve özellikle Tub’u inhibe eden yeni teröpatik ajanların geliştirilebilmesi tip2 diyabet gelişiminin önlenebilmesi açısından önemli bir adım olabilme potansiyeli taşımaktadır. Diğer taraftan, pankreatik beta hücrelerinin sayıca azaldığı tip2 diyabetin ileri seviyeleri veya beta hücrelerinin hemen hiç bulunmadığı tip1 diyabetin tedavisi için ise tersine AKT/GSK-3β/β-katenin ve özellikle Tub’u aktive eden yeni teröpatik ajanların geliştirilebilmesi yoluyla endojen PAK-MKH’lerden yeni beta hücrelerinin oluşturulması yoluyla bu hastalıkların tedavisine yeni bir bakış açısı kazandırabilinir. The purpose of this study is to investigate the molecular mechanism of pancreatic islet-derived mesenchymal stem cell (PID-MSC) differentiation into beta cells in presence of insulin and leptin resistance stimulators by mimicing insulin resistance which is determined by the elevation of glucose and insulin levels in fasting blood samples, and leptin resistance which is determined by the elevation of leptin levels in fasting blood samples especially due to obesity, in type 2 diabetic patients. For this aim, 20 mM glucose, 1 nM insulin and 0.5 ng/mL leptin were administered to PID-MSCs for 24 hours. Expression levels of the pdx-1, pax4, nkx2.2, nkx6.1 and ins2 genes, which are specific transcription factors involved in insulin production or beta cell-specific proteins, have been demonstrated by the real time reverse transcription-polimerase chain reaction (qRT-PCR) method for detecting beta cell differentiation. Findings related to insulin production at the level of gene expression were supported by flow cytometric analysis of the insulin+ cell ratio. The increase in the production of reactive oxygen species (ROS) was used as a differentiation indicator and the ratio of ROS producing cells was shown by flow cytometric analysis of DCFDA+ cells. Function acquisition of newly differentiating beta cells was also demonstrated by determining the level of insulin release in response to elevated glucose stimulation by enzyme-linked immunosorbent assay (ELISA). The increase in SOCS3 level was used as a leptin resistance indicator, while the decrease in p-IRS1 (Try) level versus the increase in p-IRS1 (Ser307), PTEN and p-AKT (Ser473) levels were used as insulin resistance markers. The changes in the levels of these markers were indicated by western blotting. The possible role of PTEN, a negative regulator of the PI3K/AKT signaling, was investigated by the application of bpV (HOpic), a pharmacological inhibitor of this molecule. Changes in p-GSK-3β (Ser9 and Try216) and β-catenin levels were demonstrated by western blotting. The interaction of β-catenin with N-cadherin was demonstrated by immunoprecipitation followed by western blotting. In addition, the level of Tub, which is an important molecule in insulin and leptin pathway, in plasma membrane, cytoplasm, and nuclear fractions demonstrated by western blotting. The possible interaction of β-catenin with Tub was investigated by immunoprecipitation followed by western blotting. The roles of β-catenin and Tub in this mechanism were confirmed using specific silencer RNAs. Changes in expression levels of β-catenin and Tub genes were shown by qRT-PCR technique. Possible roles of Tub in the production of ins1, ins2, pdx1 and MafA, which are important transcription factors in insulin synthesis, were investigated by chromosome immunoprecipitation technique. As the result, it was determined that beta cell differentiation was stimulated in all groups, while in the group in which insulin and leptin were applied together, it was found that the newly formed beta cells were much higher, the differentiation was more advanced and they were more mature. However, newly formed beta cells were not functional. It was found that in the cells of this group the level of p-AKT (Ser473) did not change while SOCS3, p-IRS1 (Ser307) and PTEN levels were increased p-IRS1 (Try) level was decreased. These findings suggest that combined administration of insulin and leptin to PID-MSCs results in the development of the insulin and leptin resistance. It was shown that this signaling pathway, which stimulates the formation of mature beta cells at a high rate, is mainly mediated by AKT/GSK-3β/β-catenin and Tub. β-catenin and Tub were linked to each other in the nucleus under this condition. It was also found that Tub binded to promoter regions of ins1, ins2, pdx1 and MafA genes, which play key roles in insulin synthesis. These findings clearly demonstrate that insulin and leptin resistance developed in PID-MSCs in the first stage of the type 2 diabetes development, markedly increases beta cell differentiation. The ability to develop new therapeutic agents that inhibit AKT/GSK-3β/β-catenin and in particular Tub with the aim of beta cell differentiation inhibition has the potential to be an important step in the prevention of the development of type 2 diabetes. Inversely, endogenous PID-MSCs can be differentiated into beta cells through the development of new therapeutic agents that activate AKT/GSK-3β/β-catenin and in particular Tub, for the treatment of advanced type 2 diabetes, in which pancreatic beta cells are reduced, or type 1 diabetes, in which beta cells are almost never found.
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