Proinflammatory cytokines increase the rate of glycolysis and adenosine-5′-triphosphate turnover in cultured rat enterocytes

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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. Design In vitro “reductionist” model of sepsis. Setting University laboratory. Subjects Cultured rat enterocyte-like cells. Interventions 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-α (10 ng/mL), interleukin-1β (1 ng/mL), and interferon-γ (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 μM). 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. Measurements and Main 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 μg/mL cycloheximide and 5 μg/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.