Utjecaj različitih koncentracija glukoze na aktivnost piruvat kinaze u HepG2 stanicama

Šećerna bolest je metabolički poremećaj kojeg karakterizira stanje kronične hiperglikemije i poremećeni metabolizam ugljikohidrata, masti i proteina zbog oštećene sekrecije inzulina i/ili poremećaja u signalnim putevima. U održavanju odgovarajuće koncentracije glukoze u krvi ključnu ulogu ima jetra. Metabolizam glukoze u jetri reguliraju hormoni koji utječu na enzimsku aktivnost ili sintezu samog enzima. Ovisno o potrebama metabolizma izmijenjuju se dva procesa, glikoliza i glukoneogeneza. Kako bi se koncentracija glukoze održala unutar referentnog intervala bitna je regulacija ključnih enzima oba procesa. U šećernoj bolesti dolazi do povećane glukoneogeneze i glikogenolize u stanicama jetre. S druge strane smanjene su aktivnosti enzima glikolize i glikogeneze. Cilj ovog istraživanja je ispitati stupanj glikolize u tumorskim HepG2 stanicama u hiperglikemijskim uvjetima. HepG2 stanice tretirali smo s četiri otopine različitih koncentracija glukoze (5 mM, 20 mM, 30 mM i 50 mM) te su tako tretirane stanice predstavljale model hepatocita u šećernoj bolesti. Stupanj glikolize odredili smo na temelju mjerenja katalitičke aktivnosti glikolitičkog enzima piruvat kinaze. Piruvat kinaza ima ključnu ulogu u zadnjem koraku glikolize, prevođenju fosfoenolpiruvata u piruvat. Aktivnost jetrenog izoenzima regulirana je alosteričkim efektorima i fosforilacijom. Katalitičku aktivnost piruvat kinaze (PK) izračunali smo iz spektrofotometrijskog mjerenja apsorbancije reduciranog koenzima. Podaci su statistički obrađeni u komercijalnom statističkom programu GraphPad Prism 6.01 i Microsoft Office Excel 2016. Za obradu rezultata korišteni su one-way ANOVA i Sidakov test. Rezultati istraživanja pokazuju statistički značajan pad aktivnosti piruvat kinaze kako se povećavala koncentracija glukoze u stanicama. Katalitička aktivnost enzima bila je najniža u uvjetima 30 mM glukoze. Kod koncentracije 50 mM glukoze nije vidljiv statistički značajni pad u odnosu na kontrolu jer je došlo do zasićenja aktivnih mjesta na enzimu. Rezultati potvrđuju prethodne hipoteze da je stupanj glikolize manji kod osoba s uznapredovalim stadijem šećerne bolesti kod kojih je prisutna veća koncentracija glukoze u krvi (20 mM i 30 mM). Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia and disturbed metabolism of carbohydrates, fats and proteins that occurs due damaged insulin secretion or disorder in insulin signal pathways. Liver has the essential role in maintenance adequate glucose concentration in blood. Glucose metabolism in liver is regulated by hormones which effect enzyme activity or enzyme synthesis. Depending on the metabolic needs, glycolysis and gluconeogenesis are exchanged. Regulating the essential enzymes of these processes is important to maintain glucose concentration within reference range. In diabetes mellitus, gluconeogenesis and glycogenolysis in liver cells are increased. On the other hand, activity of glycolytic and glycogenesis enzymes is decreased. The aim of this study was to examine the rate of glycolysis in tumor HepG2 cells in hyperglycemic conditions. HepG2 cells were treated with four different glucose concentrations (5 mM, 20 mM, 30 mM and 50 mM) and such treated cells represented a model of hepatocytes in diabetes mellitus. The rate of glycolysis was determined by measurements of pyruvate kinase catalytic activity. Pyruvate kinase has the essential part in the last step in glycolysis, conversion phosphoenolpyruvate in pyruvate. Liver isoenzyme activity is regulated by allosteric effectors and phosphorylation. We calculated the pyruvate kinase activity (PK) from the spectrophotometric measurements of reduced coenzyme. The results were statistically analyzed in the statistical program GraphPad Prism 6.01 and Microsoft Office Excel 2016. For result analysis, we used one-way ANOVA and Sidak test. Research results show statistically significant drop of pyruvate kinase activity due to increased glucose concentration in cells. Catalytic enzyme activity was the lowest in 30 mM glucose conditions. In 50 mM glucose concentration, there was no statistically significant drop regards to control because saturation od active sites on the enzyme was occurred. Results confirm previous hypothesis that the rate of glycolysis is lower in people with advanced stage of diabetes mellitus with higher blood glucose concentrations (20 mM and 30 mM).