Original articleKetogenesis alleviates TNFα-induced apoptosis and inflammatory responses in intestinal cells
Graphical abstract
Introduction
The mammalian intestinal epithelium undergoes a highly regimented and tightly controlled process of continuous self-renewal, which includes balanced proliferation, apoptosis and differentiation [1]. An imbalance of this orderly process is associated with various intestinal pathologies such as colorectal cancer and inflammatory bowel diseases (IBDs) [2,3].
Patients with IBD, a chronic inflammatory disorder characterized by periods of remission and relapse [4], suffer from a number of debilitating symptoms such as abdominal pain, diarrhea, weight loss and rectal bleeding [5]. These diseases include primarily Crohn's disease and ulcerative colitis, which differ in several features including the location and the nature of the inflammation [5,6]. Recent studies have shown that IBDs are a result of a combination of multi-factorial etiologies such as genetic, microbial, and environmental factors [4,7]. Although the exact pathogenesis for IBDs is still not entirely known, IBDs are characterized by a deregulated production of pro-inflammatory mediators including tumor necrosis factor α (TNFα) [7,8].
TNFα is associated with cellular processes such as cell proliferation, survival and death and is a key regulator of the inflammatory response in a variety of human diseases, including psoriasis, rheumatoid arthritis, and IBDs [[8], [9], [10]]. TNFα has an important role in maintaining the intestinal integrity and in the pathogenesis of intestinal inflammation [8,10]. Increased systemic and intestinal tissue levels of TNFα in IBD patients and IBD risk alleles, associated with TNF signaling components that have been identified by genome wide association studies, suggest an essential function for TNFα in the pathogenesis of IBD [[11], [12], [13]]. In addition, anti-TNFα therapy is considered as a beneficial therapeutic option for some IBD patients [8,10,11,14].
Ketogenesis represents a series of metabolic reactions that produce ketone bodies, which provide a substitute source of energy in the body during fasting and starvation [15,16]. This process primarily occurs in the mitochondria of liver cells but is also carried out in the kidney, brain and colon to a lesser extent [16,17]. Ketogenesis is regulated by transcriptional and posttranslational modifications of some key ketogenic enzymes [15,17]. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) is the rate-limiting enzyme of ketogenesis, which generates ketone bodies such as β-hydroxybutyrate (βHB) [17,18]. We and others have shown that the expression of HMGCS2 is regulated by PPARs, c-Myc or the Wnt/β-catenin pathway [[19], [20], [21]]. Cytokines (e.g., TNFα) have been reported to inhibit ketone body production in animal models and isolated rat hepatocytes [[22], [23], [24]]. However, detailed mechanisms and the potential role of ketogenic enzymes, especially HMGCS2, in this process are not known.
In our current study, we investigated the regulation of HMGCS2 expression and βHB concentration by TNFα in intestinal epithelial cells and mouse intestinal organoids and the protein level of HMGCS2 in IBD patient tissues. We also determined the effect of HMGCS2 and βHB on TNFα-induced responses in the intestinal epithelial cells. Furthermore, we showed the effect of HMGCS2 on TNFα-induced reactive oxygen species (ROS) generation and the influence of ROS on ketogenesis regulation in intestinal cells. Our findings suggest a novel protective role for ketogenesis in TNFα-induced apoptosis and inflammatory responses in intestinal epithelial cells.
Section snippets
Materials and methods
Cell culture, transfection and treatment. Human colorectal cancer epithelial cell lines, HT29, Caco2 and DLD1, were obtained from American Type Culture Collection (ATCC, Manassas, VA). HT29 and Caco2 cells were cultured in McCoy's 5 A medium supplemented with 10% FBS and MEM with 15% FBS, 1% sodium pyruvate and 1% nonessential amino acids, respectively. DLD1 cells were grown in RPMI1640 with 10% FBS. The cells were maintained at 37 °C in a humidified 5% CO2 incubator. Transfection with
Results
TNFα inhibits ketogenesis through suppression of HMGCS2 expression in intestinal cells. Previous studies have shown that cytokines (e.g., TNFα) inhibit ketone body production in several animal models and isolated rat hepatocytes [[22], [23], [24]]. To determine how ketogenesis is regulated by TNFα, the expression of HMGCS2, a rate-limiting ketogenic enzyme, was determined in human colon epithelial cancer cell lines, HT29 and Caco2 cells. As shown in Fig. 1A, treatment of HT29 (left) and Caco2
Discussion
The intestinal epithelium closely interacts with the microbiota and immune system to maintain a tight equilibrium and balance between proliferation and programmed cell death. Disruption of this highly-regimented process can lead to a number of intestinal diseases [2,3]. Cytokines and chemokines play a critical role in the integrity of the intestinal epithelium and can initiate and drive intestinal damage [31,32]. Previously, the inhibitory effect of cytokines, especially TNFα, on ketone body
Declaration of competing interest
The authors have no conflicts of interest to disclose.
Acknowledgments
We thank the Markey Cancer Center Research Communications Office for editorial support and manuscript preparation. We also thank the Biostatistics and Bioinformatics Shared Resource Facility, the Biospecimen Procurement and Translational Pathology Shared Resource Facility, and the Flow Cytometry and Immune Monitoring Shared Resource Facility of the University of Kentucky Markey Cancer Center. This work was supported by the National Cancer Institute (R01 DK048498 and P30 CA177558).
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