Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA.
Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, USA.
Biomolecules. 2023 Jan 30;13(2):258. doi: 10.3390/biom13020258.
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 (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα 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 TNFα 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 TNFα, 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 TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 () were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon.
丁酸盐是一种短链脂肪酸，可被肠道上皮用作能量来源，同时还可改善肠道上皮屏障。最近，人们发现它对免疫和心血管稳态有有益影响。相反，肿瘤坏死因子-α（TNFα）是一种促炎和致高血压的细胞因子。虽然丁酸盐和 TNFα 都与高血压有关，但目前的研究尚未涉及它们在结肠中的相互作用。在这里，我们研究了丁酸盐在体外对原代啮齿动物结肠细胞中 TNFα 多种作用的调节能力。我们测量了暴露于丁酸盐或 TNFα 或两者后，结肠细胞中的 ATP 水平、细胞活力、线粒体膜电位（MMP）、活性氧（ROS）、线粒体氧化磷酸化和糖酵解活性。为了研究潜在的机制，我们还测量了与氧化应激、细胞命运和细胞代谢相关的转录物（Pdk1、Pdk2、Pdk4、Spr、Slc16a1、Slc16a3、Ppargc1a、Cs、Lgr5、Casp3、Tnfr2、Bax、Bcl2、Sod1、Sod2 和 Cat），并采用非靶向液相色谱-串联质谱（LC-MS/MS）来分析结肠细胞暴露于丁酸盐和 TNFα 后的代谢反应。我们发现，丁酸盐和 TNFα 均使细胞 ATP 水平降低，向静息细胞能量表型发展，其特征是耗氧量和细胞外酸化减少。与丁酸盐共同处理可改善 TNFα 诱导的细胞毒性和细胞活力降低。丁酸盐还拮抗 TNFα 介导的 MMP 和线粒体与细胞内钙比的降低，表明丁酸盐可能通过降低线粒体钙流来保护结肠细胞免受 TNFα 诱导的细胞毒性。通过丁酸盐和 TNFα 的共同处理，丙酮酸脱氢酶激酶 4（）的相对表达水平增加，表明糖酵解的协同抑制。TNFα 单独降低单羧酸转运蛋白 slc16a1 和 slc16a3 的表达，表明 TNFα 对结肠细胞中丁酸盐摄取的影响。在通过 LC-MS 检测到的 185 种代谢物中，生物蝶呤的 TNFα 诱导增加产生了唯一显著变化，表明结肠细胞中线粒体生物发生的改变。考虑到在包括高血压在内的许多情况下，结肠中 TNFα 升高和丁酸盐代谢减少的报告，本工作阐明了 TNFα 和丁酸盐在结肠细胞中的细胞相互作用，这对于理解结肠疾病的发病机制可能很重要。
