Eggimann 2014 Abstract MiP2014
|Adaptation to nutrient availability in human fibroblasts with mitochondrial dysfunction: the role of sirtuins.|
Defects in the mitochondrial oxidative phosphorylation system (OXPHOS) lead to an extremely heterogeneous group of disorders with an incidence of ≈1 in 5,000 live births . Diagnosis of mitochondrial disorders is still challenging and requires extensive clinical and laboratory evaluation. One screening test for detection of defects in the OXPHOS is the incubation of primary patient fibroblasts cell lines (FBs) in galactose based medium, where some FBs with OXPHOS defects fail to survive [2,3]. The adaptation to galactose medium in FBs is not well understood. It is hypothesized that metabolic sensors may play a role in this process. The sirtuins seem to fulfil the role of metabolic sensors during conditions of caloric restriction or change from glucose to galactose based medium [4,5]. Sirtuins are a family of NAD+-dependent protein deacetylases (SIRT1 - SIRT7). SIRT1 is located in the nucleus/cytoplasm, whereas SIRT 3 - 5 are mainly located in the mitochondria. SIRT1 and SIRT3 are deacetylases, in contrast to SIRT4 (ADP-ribosyltransferase activity) and SIRT5 (demalonylase, desuccinylase and weak deacetylase activity) . For this study six FBs were selected (one control and five with a mitochondrial defect). The basis for the experiments was a 20 h incubation time in galactose based medium and, as control, an incubation in glucose medium. After 20 h the change in the expression level of acetylated proteins, as well as the expression level of SIRT1 and SIRT3 - 5, in the mitochondrial and cytosolic fraction was analyzed by Western Blot. The process of adaptation to galactose and the role of glutamine was further investigated with an XF24 extracellular flux analyzer (Seahorse Bioscience) measuring cellular oxygen consumption rates (OCR) and extracellular acidification rates (ECAR; an indirect estimate of glycolysis). We observed a general decrease of acetylated proteins in the mitochondrial fraction in all cell lines after incubation in galactose. The extent of this decrease could not be correlated with respiratory defects. Because SIRT3 is the main-deacetylase in mitochondria, we suspected this deacetylation process to be the result of a higher SIRT3 activity in galactose [4,7]. This could be either because of an altered SIRT3 expression at the protein level, which was not the case in these experiments, or an altered SIRT3 activity due to a changed mitochondrial NAD+ concentration, which would require further testing.
• O2k-Network Lab: CH Bern Nuoffer JM
Labels: MiParea: Respiration, mt-Medicine, Patients
Stress:Mitochondrial disease Organism: Human Tissue;cell: Fibroblast Preparation: Intact cells
Regulation: Aerobic glycolysis Coupling state: ROUTINE
Event: C1, Oral MiP2014
1-Univ Inst Clinical Chem; 2-Interdisciplinary Metabolic Team; Bern Univ Hospital; Switzerland. - [email protected]
We observed a general decrease of acetylated proteins in the mitochondrial fraction in all cell lines after incubation in galactose. The extent of this decrease could not be correlated with respiratory defects. Because SIRT3 is the main-deacetylase in mitochondria, we suspected this deacetylation process to be the result of a higher SIRT3 activity in galactose [4,7]. This could be either because of an altered SIRT3 expression at the protein level, which was not the case in these experiments, or an altered SIRT3 activity due to a changed mitochondrial NAD+ concentration, which would require further testing. Mitochondrial SIRT4 was decreased at the protein level in all FB after galactose incubation. This uniform down-regulation suggests that SIRT4 is involved in the adaption to galactose, unaffected by respiratory function. Under standard conditions, the FBs of all patients with an OXPHOS defect had a decreased cytosolic expression of SIRT1, SIRT3 and SIRT5 compared to the control. In glucose (+Gln), OCR differed in the FBs, whereas the control FBs and a Complex I deficient FB had the highest values. ECAR values were generally decreased in galactose medium in all FBs (± Gln). Two FBs with Complex I deficiency showed higher OCR in galactose than in glucose. This may reflect a compensatory increase in OXPHOS, induced by galactose. The OCR with glucose was dependent on glutamine in several FBs, whereas glutamine had no effect on OCR in galactose-based medium.
In conclusion: (1) all FBs show lower levels of acetylated proteins in mitochondria in galactose-based media. This deacetylation cannot be attributed to changing sirtuin expression; (2) the lower cytosolic SIRT1, 4 and 5 protein expression under normal conditions in fibroblasts with a mitochondrial defect needs to be further investigated as a potential marker; (3) in contrast to galactose-based medium, OCR was dependent on glutamine in glucose-based medium.
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