Interaction between Butyrate and Tumor Necrosis Factor alpha in Primary Rat Colonocytes

Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNF alpha) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNF alpha are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNF alpha in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNF alpha, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism (Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Ppargc1a, Cs, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Sod1, Sod2, and Cat) were measured, and untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNF alpha. We found that both butyrate and TNF alpha lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNF alpha-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNF alpha-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNF alpha-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 (Pdk4) were increased via co-treatment of butyrate and TNF alpha, suggesting the synergistic inhibition of glycolysis. TNF alpha alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNF alpha on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNF alpha-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNF alpha and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNF alpha and butyrate in colonocytes that may be important in understanding conditions of the colon.