The effect of 1,25-dihydroxyvitamin d on gene and protein expression of enzymes protecting from glucolipotoxicity in vitro
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| Rok publikování | 2015 |
| Druh | Konferenční abstrakty |
| Citace | |
| Popis | Main Objectives: Beside its classical function as orchestrator of calcium and phosphorus homeostasis, vitamin D also affects insulin secretion and tissue efficiency. Number of studies consistently reports inverse relationship between vitamin D deficiency and type 2 diabetes. Activation of certain metabolic pathways and down-stream transcription factors may protect from glucolipotoxicity and their targeted activation – e.g. by vitamin D – might explain the detrimental role of vitamin D deficiency in diabetes. The aim of the study was to quantify gene and protein expression of selected enzymes involved in protection from glucolipotoxicity, specifically glyoxalase 1 (GLO1), and other enzymes with antioxidant activity – hemoxygenase (HMOX), thiamin pyrophosphokinase (TPK1) and transketolase (TKT), in normo- and hyperglycaemic conditions and upon addition of vitamin D in peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVEC). Strategy and Methods: Peripheral blood samples were provided by healthy donors (n = 6). PBMCs separated using Histopaque were pooled and cultured 24 hours in RPMI medium containing 5 or 25mM glucose with or without 100 nM 1,25-dihydroxycholecalciferol or the same amount of solvent (ethanol). HUVEC were cultured in the same conditions. RNA was isolated and reverse transcribed using commercial kits. Total protein was isolated using RIPA buffer. Gene expression of genes of interest was determined using quantitative PCR with predesigned probes (TaqMan™ Assay) with ß-actin as a reference gene. Protein expression was determined using imunoblot analysis with specific antibodies and ß-actin as a control. Main Results: In PBMC vitamin D significantly increased gene expression of TKT (by 90 – 100 %), GLO1 (by 110 – 130 %) and TPK (90 – 100 %) in both normo- and hyperglycaemia compared to normo- and hyperglycaemia without vitamin D. Gene expression in HUVEC did not change. Vitamin D did not affect protein expression in both cell lines. Conclusions: The results of our study indicate that the active form of vitamin D regulates gene expression of enzymes opposing harmful effect of glucolipotoxicity whose activities appear suppressed by hyperglycaemia. However, we were unable to confirm this effect on protein expression. Enzyme activities will be further studied. While we cannot speculate on the effect of vitamin D on diabetes itself our results support its role in the protection against existing glucolipotoxicity therefore possibly translating into the prevention of development of diabetic complications. |
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