Research Article Integrative Food, Nutrition and Metabolism Integr Food Nutr Metab, 2018 doi: 10.15761/IFNM.1000228 Volume 5(5): 1-5 ISSN: 2056-8339 Fructose impairs mitochondrial respiration and substrate utilization in hepatocytes via the enzyme, glutamate oxaloacetate transaminase Hlengiwe P Madlala*, Gerald J Maarman and Edward Ojuka Division of Exercise Science and Sports Medicine, Department of Human Biology, University of Cape Town, Western Cape, South Africa Introduction ere are increasing health concerns about the excess consumption of fructose in the form of high fructose corn syrup in modern diets [1]. Upon consumption, approximately 75% of fructose is metabolised by hepatocytes via fructolysis for glycogenesisA, lactate production and triglyceride synthesis [2]. e first step in this pathway is the phosphorylation of fructose, to fructose 1-phosphate by the high affinity ketohexokinase (fructokinase) enzyme. Because this reaction has a low Michaelis constant, and is not controlled allosterically or hormonally, plasma fructose is rapidly cleared and adenosine triphosphate (ATP) inside the hepatocyte is rapidly depleted [3]. Depletion of ATP stimulates adenosine monophosphate deaminase that activates the purine degradation pathway, leading to uric acid production [4,5]. Excess uric acid increases mitochondrial reactive oxygen species (ROS) production in hepatocytes [6,7] which induces defects in a number of ROS-sensitive mitochondrial enzymes including aconitase [8]. Excessive ROS production [9] and reduced aconitase activity [10] is known to affect mitochondrial metabolism. Yet there is a lack of studies providing extended investigation into the impact of excess fructose on mitochondrial metabolic pathways and substrate utilization. Such investigations is important, as the data generated could build upon previous research, and contribute to the body of knowledge on the adverse metabolic effects of excess fructose. We hypothesise that fructose impairs mitochondrial metabolic pathways and substrate utilization in hepatocytes. erefore, the aim of this study was to investigate the effects of excess fructose on mitochondrial metabolic pathways and substrate utilization. Materials and methods Cell culture e HepG2 cell line was obtained from the Council for Scientific and Industrial Research (CSIR) (Stellenbosch, South Africa). Cell *Correspondence to: Hlengiwe P Madlala, Division of Exercise Science & Sports Medicine (ESSM), Faculty of Health Sciences, Department of Human Biology, University of Cape Town, Boundary Road, Newlands Sports Complex, SISSA, 3rd floor, Newlands, South Africa, Tel: 27216504576; Fax: 27216501796; E-mail: hlengiwe.madlala@uct.ac.za Key words: fructose, mitochondrial enzymes, mitochondrial respiration, complex I-linked substrates Received: July 25, 2018; Accepted: August 17, 2018; Published: August 21, 2018 Abstract Excess fructose associates with increased production of reactive oxygen species (ROS) that inhibits enzymes such as aconitase, which should affect mitochondrial metabolism. Yet there is a lack of studies investigating the impact of excess fructose on mitochondrial metabolic pathways and substrate utilization. We evaluated the impact of excess fructose on hepatocyte mitochondrial enzymes; citrate synthase (CS), aconitase and glutamate-oxaloacetate transaminase (GOT). Also, high- resolution using complex I-linked substrates [pyruvate+malate (PM), glutamate+malate (GM) and PGM]. Fructose decreased the activities of aconitase and GOT by 35% and 47% respectively. Respiration at Leak, OXPHOS and ETS states were reduced with GM but not PM or PGM. us, excess fructose inhibits GOT activity, reduced mitochondrial leak respiration, OXPHOS and ETS capacity. ese changes were observed with complex-1 linked respiration (substrates GM). erefore, fructose impairs mitochondrial respiration and substrate utilization via the enzyme GOT. lines were initially propagated in 25 cm 2 flasks (Bibby Sterilin, Stone, Staffordshire, UK), at 37 °C in 5 mL DMEM (Gibco BRL, Inchinnan, Scotland) containing 10% (v/v) fetal bovine serum, 20 mM HEPES, 10 mM NaHCO 3 , 100 μg mL -1 penicillin G, and 100 μg mL -1 streptomycin sulfate (Whittaker Bioproducts, Walkersville, MD, USA) at pH 7.5. Cells were incubated for 24 h to permit attachment and grown to semi- confluence and passaged 1:3 every 4-5 days. One group of cells received normal growth medium (control group, DMEM for 72 hours), while the second group were exposed to growth medium supplemented with excess fructose (15 mM, 72 hours) as previously reported [6]. Enzyme activity measurements Sample preparation: HepG2 cells were homogenised in extraction buffer (0.1 M Tris-HCl; 15 mM Tricarballylic acid; pH 7.8) and incubated on ice for 20 min. e homogenate was centrifuged at 10 000 rpm for 20 min and the supernatant was used as a sample for further experimentation. Subsequent enzyme assays were performed according to modified protocols previously described by Wang et al. [11]. Citrate synthase: e following components were added in a cuvette: 473 µl of citrate synthase buffer (0.1 M Tris-HCl, 1.25 mM 5,5’-dithiobis-[2-nitrobenzoic acid] in deionized water, pH 8.0), 2 µl of sample and 25 µl of Oxaloacetate-Acetyl-CoA solution (50 mM