Hyperacetylation of Toll‐like receptor 4 (TLR4): a potential factor contributing to non‐alcoholic liver disease (NAFLD), a silent killer in obesity

Abstract

BackgroundObesity frequently leads to non‐alcoholic fatty liver disease (NAFLD), the most common liver problem affecting the American population. In fact, the prevalence of NAFLD in adult Americans ranges from 25‐46%. To date, there is no specific treatment for NAFLD. In severe cases, NAFLD can lead to fibrosis and cirrhosis which evolves into nonalcoholic steatohepatitis (NASH). Most patients with NAFLD experience no symptoms, making it a silent killer. Evidence has suggested that Toll‐like receptor 4 (TLR4) signaling, a key component of innate immunity, is implicated in the pathogenesis of NAFLD; however, the mechanisms of TLR4 activation remain unclear. We hypothesized that hyperacetylation of TLR4 leads to its overactivation in obesity, contributing to NAFLD development.MethodsEight‐week‐old male C57BL/6 mice were randomized into two experimental groups: the control group (n=4) received a regular chow diet (5% fat, 48.7% carbohydrates [3.2% sucrose], 24.1% protein) and the Western diet (WD) group (n=4) received a WD (21% fat, 50% carbohydrates [34% sucrose], and 20% protein) for 36 weeks. Body weight was obtained and intraperitoneal glucose tolerance test (IPGTT) was performed over the course of the dietary protocol at weekly and monthly intervals, respectively. During terminal experiments, liver tissue was isolated and processed for cross‐section histology. Staining with H&E and Oil‐Red‐O were used for grading of liver injury with the NAFLD Score, which involves summation of steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2) scores. A final score of 0–2 indicates no NASH, 3–4 indicates borderline NASH, and 5–8 indicates NASH. TLR4 and its downstream protein expression was detected by western blot and immunoprecipitation was utilized to determine the acetylation levels of TLR4.ResultsThe WD group exhibited increased body weight (41.22 ± 7.71 g vs 29.94 ± 1.16 g controls, p<0.05) and liver weight (1.27 ± 0.0.07 g vs 1.27 ± 0.07 g controls, p<0.05). Results from IPGTT demonstrated that male mice develop early intolerance to glucose, within 8 weeks on WD. Hepatic histology showed ballooned hepatocytes, lobular inflammation, and steatosis, confirming presence of NAFLD with borderline NASH. Livers from the WD group exhibited increased TLR4 expression (1.4‐fold increase, p<0.05). Of note, this was accompanied by an increase in TLR4 signaling in WD livers as confirmed by elevated expression of the downstream signaling protein TNF receptor associated factor 6 (TRAF6) (0.8‐fold increase, p<0.05). Strikingly, immunoprecipitation assay revealed that TLR4 is hyperacetylated in the livers from the WD group.ConclusionsOur results demonstrate for the first time that hepatic TLR4 is hyperacetylated in obesity‐related NAFLD. This hyperacetylation may serve as a trigger for the TLR4/TRAF6 signaling activation and may prove to be crucial to the development of NAFLD and NASH in obesity.