Ketogenesis alleviates TNFα-induced apoptosis and inflammatory responses in intestinal cells

Highlights

• TNFα suppresses intestinal cell ketogenesis by inhibition of HMGCS2 expression.

• Protein levels of HMGCS2 are decreased in human IBD tissues.

• HMGCS2 attenuates TNFα-induced apoptosis, CXCL1-3 expression and ROS generation.

• Treatment of β-hydroxybutyrate or rosiglitazone alleviates TNFα-induced apoptosis.

• ROS represses ketogenesis in intestinal cells.

Abstract

The disturbance of strictly regulated self-regeneration in mammalian intestinal epithelium is associated with various intestinal disorders, particularly inflammatory bowel diseases (IBDs). TNFα, which plays a critical role in the pathogenesis of IBDs, has been reported to inhibit production of ketone bodies such as β-hydroxybutyrate (βHB). However, the role of ketogenesis in the TNFα-mediated pathological process is not entirely known. Here, we showed the regulation and role of HMGCS2, the rate-limiting enzyme of ketogenesis, in TNFα-induced apoptotic and inflammatory responses in intestinal epithelial cells. Treatment with TNFα dose-dependently decreased protein and mRNA expression of HMGCS2 and its product, βHB production in human colon cancer cell lines HT29 and Caco2 cells and mouse small intestinal organoids. Moreover, the repressed level of HMGCS2 protein was found in intestinal epithelium of IBD patients with Crohn's disease and ulcerative colitis as compared with normal tissues. Furthermore, knockdown of HMGCS2 enhanced and in contrast, HMGCS2 overexpression attenuated, the TNFα-induced apoptosis and expression of pro-inflammatory chemokines (CXCL1-3) in HT29, Caco2 cells and DLD1 cells, respectively. Treatment with βHB or rosiglitazone, an agonist of PPARγ, which increases ketogenesis, attenuated TNFα-induced apoptosis in the intestinal epithelial cells. Finally, HMGCS2 knockdown enhanced TNFα-induced reactive oxygen species (ROS) generation. In addition, hydrogen peroxide, the major ROS contributing to intestine injury, decreased HMGCS2 expression and βHB production in the intestinal cells and mouse organoids. Our findings demonstrate that increased ketogenesis attenuates TNFα-induced apoptosis and inflammation in intestinal cells, suggesting a protective role for ketogenesis in TNFα-induced intestinal pathologies.

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.