title: Proinflammatory cytokines increase the rate of glycolysis and adenosine-5'-triphosphate turnover in cultured rat enterocytes.
abstract: OBJECTIVE
  Measurements of steady-state adenosine-5'-triphosphate (ATP) levels in tissue samples from patients or experimental animals with sepsis or endotoxemia provide little information about the rate of ATP production and consumption in these conditions. Accordingly, we sought to use an in vitro "reductionist" model of sepsis to test the hypothesis that proinflammatory cytokines modulate ATP turnover rate.
  
  
  METHODS
  In vitro "reductionist" model of sepsis.
  
  
  METHODS
  University laboratory.
  
  
  METHODS
  Cultured rat enterocyte-like cells.
  
  
  METHODS
  IEC-6 nontransformed rat enterocytes were studied under control conditions or following incubation for 24 or 48 hrs with cytomix, a mixture of tumor necrosis factor-alpha (10 ng/mL), interleukin-1beta (1 ng/mL), and interferon-gamma (1000 units/mL). To measure ATP turnover rate, ATP synthesis was acutely blocked by adding to the cells a mixture of 2-deoxyglucose (10 mM), potassium cyanide (8 mM), and antimycin A (1 microM). ATP content was measured at baseline (before metabolic inhibition) and 0.5, 1, 2, 5, and 10 mins later. Log-linear ATP decay curves were generated and the kinetics of ATP utilization thereby calculated.
  
  
  RESULTS
  ATP consumption rate was higher in cytomix-stimulated compared with control cells (3.11 +/- 1.39 vs. 1.25 +/- 0.66 nmol/min, respectively; p <.01). Similarly, the half-time for ATP disappearance was shorter in cytomix-stimulated compared with control cells (2.63 +/- 1.00 vs. 6.21 +/- 3.49; p <.05). In contrast to these findings, the rate of ATP disappearance was similar in cytokine-naïve and immunostimulated IEC-6 cells when protein and nucleic acid synthesis were inhibited by adding 50 microg/mL cycloheximide and 5 microg/mL actinomycin D to cultures for 4 hrs. The rates of glucose consumption and lactate production were significantly greater in cytomix-stimulated compared with controls cells.
  
  
  CONCLUSIONS
  Incubation of IEC-6 cells with cytomix significantly increased ATP turnover. Increased ATP turnover rate was supported by increases in the rate of anaerobic glycolysis. These findings support the view that proinflammatory mediators impose a metabolic demand on visceral cells. In sepsis, cells may be more susceptible to dysfunction on the basis of diminished oxygen delivery and/or mitochondrial dysfunction.
authors.affiliation: Department of Cardiothoracic Anesthesia (SB), Linköping University, Linköping, Sweden.
authors.forename: Sören
authors.initials: S
authors.lastname: Berg
authors.forename: Penny L
authors.initials: PL
authors.lastname: Sappington
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Guzik
authors.forename: Russell L
authors.initials: RL
authors.lastname: Delude
authors.forename: Mitchell P
authors.initials: MP
authors.lastname: Fink
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D020895:Enterocytes / Q000378:metabolism* / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects*; D018836:Inflammation Mediators / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D019344:Lactic Acid / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D051381:Rats; D018805:Sepsis / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D016207:Cytokines; D018836:Inflammation Mediators; D007375:Interleukin-1; D011506:Proteins; D014409:Tumor Necrosis Factor-alpha; D019344:Lactic Acid; D012313:RNA; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D005947:Glucose