• BACKGROUND
    • The pyruvate dehydrogenase complex (PDC) catalyzes the conversion of pyruvate to acetyl CoA, effectively controlling the entrance of glycolysis products into aerobic metabolism. Because hyperlactatemia is one of the hallmarks of sepsis, we hyphothesized that gram-positive and negative bacterial toxin treatment will interfere with mRNA levels of regulatory enzymes of the PDC and overall enzyme activity in hepatocytes.
  • METHODS
    • HEP G2 hepatocarcinoma cells were incubated for 24 hours in the presence of lipopolysaccaride (LPS) or lipoteichoic acid. Total RNA was then isolated and message RNA levels for both pyruvate dehydrogense kinase 4 and phosphatase 2 were determined by RTPCR. Amplified DNA fragments were visualized by ethidium bromide in agarose gels and densitometry of the bands was performed. Data were then normalized to the housekeeping gene, GAPDH. Enzyme activity was then determined by capturing intact PDC on nitrocellulose membranes then determining PDC-dependent production of NADH.
  • RESULTS
    • LPS treatment led to a time dependent increase in PDK4 message while decreasing PDP2 levels. Enzyme activity, in these cells, also significantly decreased 24 hours after exposure to LPS. Cells cultured in the presence of lipoteichoic acid and insulin exhibited differing message ratios and activity levels when evaluated at 4 hours, but at 24 hours shifted to mimic those observed in LPS treated cells.
  • CONCLUSION
    • This data may indicate that exposure to bacterial cell wall components and insulin could create cellular environments that result in a build-up of lactate.