Gıda Tatlandırıcıları Glikasyon Ürünleri Uyarımlı Amiloid Oluşumu Üzerine Fosfodiesteraz 1B Geni Ekspresyon Seviyelerinin Araştırılması

Year 2019, Volume: 2 Issue: 2, 80 - 87, 29.05.2019

Altuğ Küçükgül , Aykut Kütükoğlu

Abstract

Araştırmada yüksek früktoz diyeti tüketimine bağlı hücrede ortaya çıkan metabolik son ürün metilgliyoksalin in vitro beta amiloid oluşumu, ikincil haberci molekül siklik adenozin monofosfat (cAMP) ve bu molekülün parçalanmasını sağlayan fosfodiesteraz 1 (PDE1) üzerine etkilerinin araştırılması amaçlandı. Çalışmada insan orijinli glioblastoma U-87 MG (ATCC HTB-14) hücre hattı kullanıldı. Hücreler, içeriğinde %10 oranında fötal sığır serumu (FBS) ve penisilin/ streptomisin/amfoterisin B karışımı bulunduran Eagle’s Minimum Essential Medium (E-MEM) besi ortamında, 37°C, %5 CO2 ve %95 hava bulunduran steril inkübatörde üretildi. Çalışmada 1 gün önceden 24 kuyucuklu besi kaplarına ekilen hücrelere farklı konsantrasyonlarda metilgliyoksal (100- 400 um) eklendi. Süre sonunda kuyucuklara MTT (Thiazolyl Blue Tetrazolium Bromide) ayıracı eklenerek metilgliyoksalin etkin konsantrasyonu tespit edildi. Sonraki denemelerde bu konsantrasyon hücrelere uygulanarak 24 saatin sonunda alınan RNA örneklerinden PDE 1B, PDE 1B1 ve Aβ1-40 gen ekspreyon düzeyleri real time PCR metoduyla araştırıldı. Yine alınan protein örneklerinden cAMP düzeyi ise ELİSA metoduyla analiz edildi. Araştırmada yapılan canlılık kontrollerinde metilgliyoksalin 200 uM konsantrasyonu kontrol grubuna göre hücreleri %28 oranında azalttığı gözlendi. Bu konsantrasyon etkin konsantrasyon olarak kabul edilerek sonraki çalışmalar için kullanıldı. Yine metilglyoksalin hücreiçi cAMP düzeyini %31 oranında arttırdığı tespit edildi. Elde edilen gen ekspresyon verilerine göre metilgliyoksal Aβ1-40 gen ekspresyonunu 3.7 kat arttırıken, sırasıyla PDE1B ve PDE1B1 gen ekspresyonlarını ise 1.6 ve 3.3 kat baskıladığı ettiği tespit edildi. Yapılan araştırmada fruktoz diyetinin etkilenen bireylerde Alzheimer hastalığının patogenezinde anlamlı derecede etkileyeceği kanısına varıldı. Ancak konunun tam aydınlatılması için başta in vivo denemeler olmak üzere ve daha ileri araştırmalara ihtiyaç duyulmaktadır.

Keywords

Alzheimer, cAMP, Amiloid protein, Fosfodiesteraz

Investigation of Phosphodiesterase 1B Gene Expression Levels on Amyloid Formation Induced by Food Sweeteners Glycation Products

Abstract

In this study, it was aimed to investigate the effects of methylglyoxal, metabolic end product of high-fructose diets, on in vitro beta amyloid formation, secondary messenger molecule cyclic adenosine monophosphate (cAMP), and phosphodiesterase 1 (PDE1), which is responsible for the breakdown of this molecule. Human Glioblastoma U-87 MG (ATCC HTB-14) cell line was used in the study. Cells were produced in a sterile incubator at 37°C, 5% CO2 and 95% air in Eagle’s Minimum Essential Medium (E-MEM) containing 10% of fetal bovine serum (FBS) and penicillin/streptomycin/ amphotericin B mixture. In the study, different concentrations of methylglyoxal (100-400 µM) were added to the cells sown in 24-wells one day in advance. At the end of the period, MTT (Thiazolyl Blue Tetrazolium Bromide) reagent was added to the wells to determine the effective concentration of methylglyoxal. Next, PDE1B, PDE1B1 and Aβ1-40 gene expression levels were investigated by real time PCR method. The cAMP protein level was analyzed by ELISA method. In the viability tests, 200 µM concentration of methylglyoxal decreased the cell viability by 28% compared to the control group. This concentration was used as the active concentration and was used for subsequent studies. Again, methylglyoxal was found to increase intracellular cAMP level by 31%. According to the obtained gene expression data, it was found that methylglyoxal was increased Aβ1-40 gene expression by 3.7-fold, whereas PDE1B and PDE1B1 gene expressions were regulated down by 1.6 and 3.3-fold respectively. It is concluded that the fructose diet will have a significant effect on the pathogenesis of Alzheimer’s disease in affected individuals. However, further investigations are needed, especially in vivo trials, to fully elucidate the subject.

Keywords

Alzheimer, Amyloid protein, cAMP, Phosphodiesterase

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