Madlala 2016 Abstract MitoFit Science Camp 2016
|Fructose-induced defects in selected mitochondrial enzymes is detected by respirometry using glutamate&malate but not pyruvate&malate substrates.|
There are increasing health concerns about the excess consumption of fructose which has become a major constituent of modern diet . Research suggests that excessive intake of fructose (>50 g.day-1) is associated with mitochondrial dysfunction via increased production of reactive oxygen species and reduced mitochondrial DNA content [2,3]. However, the effects of fructose on hepatic mitochondrial respiration has not been characterized and the viability of using respirometry to detect impairment in liver mitochondrial enzymes remains unknown. Therefore, the aims of this study were to determine the effects of fructose exposure on activities of selected mitochondrial enzymes and characterize mitochondrial respiration using complex I-linked substrates in HepG2 cells.
HepG2 cells were exposed to 15 mM fructose for 72 h, and after permealization with digitonin, respiration with pyruvate & malate (PM) and glutamate & malate (GM) were measured at different respiratory states using high resolution respirometry. The activities of aconitase and glutamate-oxaloacetate transaminase (SGOT) were measured enzymatically and the levels of citrate and 2-oxoglutarate in the respiratory medium were determined by Mass spectrometry.
Fructose decreased the activities of aconitase and SGOT by 30% and 40%, respectively, compared to controls. Respiration at LEAK, OXPHOS and ET-pathway states, as well as oxygen flux attributable to ATP synthesis (GM_P – GM_Lomy), were significantly reduced with GM but were unchanged with PM. Citrate but not 2-oxogluterate accumulated in respiratory medium when cells were allowed to respite at OXPHOS state for 30 minutes.
These data suggest that respirometry does not detect the defects in aconitase and SGOT caused by fructose exposure when PM is used as substrates whereas use of GM does. Accumulation of citrate in the respiratory medium with little change in the oxygen flux for ATP synthesis when PM is used as substrate indicates incomplete and inefficient metabolism of PM with no defect in the ATPase machinery in fructose-treated cells.
Labels: MiParea: Respiration, Exercise physiology;nutrition;life style
Organism: Human Tissue;cell: Liver Preparation: Permeabilized cells
Regulation: Substrate Coupling state: LEAK, OXPHOS, ET Pathway: N HRR: Oxygraph-2k Event: B2 MitoFit Science Camp 2016
Affiliations and Support
1- Exercise Sc Sports Med Unit, Dept Human Biol, Univ Cape Town, South Africa. - [email protected]
Funding agent: National Research Foundation of South Africa.
Figure 1. Effects of 15 mM fructose on activities of aconitase and glutamate-oxaloacetate transaminase in HepG2 cells.
Figure 2. Effects of 15 mM fructose on oxygen consumption using PM and GM in digitonin-permeabilised HepG2 cells.
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