The metabolic phenotyping core has the capability to assess substrate metabolism in cell culture models, tissue homogenates, and/or isolated mitochondria utilizing radiolabeled substrates.

  • Complete fatty acid oxidation from measures of 14CO2 production from the oxidation of [1-14C]-palmitic or oleic acid.
  • Incomplete fatty acid oxidation from measures of 14C-acid soluble metabolite production from the oxidation of [1-14C]-palmitic or oleic acid.
  • Neutral lipid synthesis by measuring incorporation of [1-14C]-palmitic or oleic acid into monoacylglycerol, diacylglycerol, triacylglycerol, and phospholipid pools.
  • Pyruvate dehydrogenase activity from measures of 14CO2 production from the oxidation of [1-14C]-pyruvate.
  • Citric acid cycle flux from measures of 14CO2 production from the oxidation of [2-14C]-pyruvate.
  • Glucose oxidation from measures of 14CO2 production from the oxidation of [U-14C]-glucose.
  • Basal and insulin-stimulated [U-14C]-glucose uptake and glycogen synthesis.
  • Metabolic flexibility in isolated mitochondria and tissue homogenates from measures of 14CO2 production from [1-14C]-palmitic or oleic acid with pyruvate titration.
  • Enzyme kinetics for glycolytic, citric acid cycle, and electron transport enzymes:
    • Citrate Synthase: the enzyme that catalyzes the reaction of acetyl coenzyme A with oxaloacetate to form citrate and is the enzyme regulating entry into the tricarboxylic acid cycle. Located in the mitochondrial matrix, citrate synthase is commonly used as an index of mitochondrial content.
    • Beta-hydroxyacylcoA dehydrogenase (BHAD): a key enzyme in the beta oxidation of fatty acids to acetyl coenzyme A. BHAD is an oxidoreductase which converts a fatty acid hydroxyl group to a keto group in the third step of beta oxidation.
    • Phosphofructokinase: a glycolytic enzyme which phosphorylates fructose 6-phosphate to produce fructose 1,6-biphosphate. Phosphofructokinase is the key rate limiting enzyme of glycolysis.
    • Malate Dehydrogenase (MDH): a mitochondrial enzyme involved in the formation of oxaloacetate from malate in the tricarboxylic acid cycle. Also located in the cytosol, MDH converts malate back into oxaloacetate. Cytosolic vs. mitochondrial malate dehydrogenase has physiological implications as mitochondrial MDH participates in the citric acid cycle while cytosolic MDH is involved in the malate-aspartate shuttle.
    • Cytochrome C Oxidase: is located inner mitochondrial membrane and is a key enzyme involved in oxidative phosphorylation. Cytochrome C oxidase couples reactions of the electron transport chain and is commonly used as a marker for inner mitochondrial membrane content.