Butyrate Protects against Diet-Induced NASH and Liver Fibrosis and Suppresses Specific Non-Canonical TGF-β Signaling Pathways in Human Hepatic Stellate Cells.

Eveline Gart,Jaap Keijer,Kanita Salic,Aswin Menke,Martien P M Caspers,Mette Juul Nielsen,Karin Toet,Everton Souto Lima,Roeland Hanemaaijer,Diana Julie Leeming,Martine C Morrison,Lars Verschuren,Wim Van Duyvenvoorde,Robert Kleemann

doi:10.3390/biomedicines9121954

Abstract

In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, which is the main determinant of NASH-associated mortality. The short-chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remain unclear. Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared with a HFD-control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). HFD-fed mice developed obesity, insulin resistance, increased plasma leptin levels, adipose tissue inflammation, gut permeability, dysbiosis, and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels, and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 levels and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-β-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-β signaling pathways Rho-like GTPases and PI3K/AKT and other important pro-fibrotic regulators (e.g., YAP/TAZ, MYC) by butyrate, providing a potential rationale for its antifibrotic effects. In conclusion, butyrate protects against obesity development, insulin resistance-associated NASH, and liver fibrosis. These antifibrotic effects are at least partly attributable to a direct effect of butyrate on collagen production in hepatic stellate cells, involving inhibition of non-canonical TGF-β signaling pathways.

Highlights

Liver fibrosis is a result of chronic liver damage for which non-alcoholic steatohepatitis (NASH) is one of the main causal factors [1,2]
Long-term high-fat diet (HFD) feeding over 38 weeks significantly increased body weight relative to chow, while energy intake was comparable in the HFD and chow group
Mice fed BU for 38 weeks were protected against obesity-associated white adipose tissue (WAT) inflammation, hyperinsulinemia, and NASH, and BU strongly suppressed development of liver fibrosis

Summary

Introduction

Liver fibrosis is a result of chronic liver damage for which non-alcoholic steatohepatitis (NASH) is one of the main causal factors [1,2]. Biomedicines 2021, 9, 1954 diets can induce hepatic lipids deposition (steatosis) which can progress to NASH (steatosis and inflammation) and fibrosis [3]. Hepatic stellate cells (HSCs) are the main producers of these ECM proteins and play a central role in liver fibrosis. In response to TGF-β, the canonical signaling pathway (involving SMADs) and non-canonical signaling pathways (i.e., ERK, JNK/p38, Rho-like GTPases, PI3K/AKT) promote HSC activation [8]. The activation of these signaling pathways by TGF-β stimulates trans-differentiation of quiescent HSC to undergo a transcriptional and morphological program into proliferative HSCs that produce excessive amounts of ECM proteins

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Conclusion