scfas alleviated steatosis and inflammation in mice with

https://doi.org/10.1530/JOE-20-0018https://joe.bioscientifica.com © 2020 Society for Endocrinology

Printed in Great BritainPublished by Bioscientifica Ltd.

Journal of Endocrinology

245:3 425–437M Deng et al. SCFA alleviated NASH

-20-0018

RESEARCH

SCFAs alleviated steatosis and inflammation in mice with NASH induced by MCD

Mingjuan Deng1,2, Fang Qu2,3, Long Chen2,4, Chang Liu2,3, Ming Zhang5, Fazheng Ren1,2,3,4, Huiyuan Guo1,2,4, Hao Zhang1,2,3, Shaoyang Ge4,6, Chaodong Wu7 and Liang Zhao1,2,4

1Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China2Key Laboratory of Precision Nutrition and Food Quality, Ministry of Education, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China3Beijing Laboratory for Food Quality and Safety, China Agricultural University, Beijing, China4Research Center for Probiotics, China Agricultural University, Beijing, China5School of Food and Chemical Engineering, Beijing Technology and Business University, Beijing, China6Hebei Engineering Research Center of Animal Product, Sanhe, China7Department of Nutrition, Texas A&M University, College Station, Texas, USA

Correspondence should be addressed to C Wu or L Zhao: [email protected] or [email protected]

Abstract

This study aimed to assess the effects of three major SCFAs (acetate, propionate, and butyrate) on NASH phenotype in mice. C57BL/6 mice were fed a methionine- and choline-deficient (MCD) diet and treated with sodium acetate, sodium propionate, or sodium butyrate during the 6-week feeding period. SCFA treatment significantly reduced serum levels of alanine aminotransferase and aspartate transaminase, the numbers of lipid droplets, and the levels of triglycerides and cholesterols in livers of the mice compared with control treatment. SCFAs also reduced MCD-induced hepatic aggregation of macrophages and proinflammatory responses. Among the three SCFAs, sodium acetate (NaA) revealed the best efficacy at alleviating MCD-induced hepatic steatosis and inflammation. Additionally, NaA increased AMP-activated protein kinase activation in the liver and induced the expression of fatty acid oxidation gene in both the liver and cultured hepatocytes. In vitro, NaA decreased MCD-mimicking media-induced proinflammatory responses in macrophages to a greater extent than in hepatocytes. These results indicated that NaA alleviates steatosis in a manner involving AMPK activation. Also, NaA alleviation of hepatic inflammation appears to be due to, in large part, suppression of macrophage proinflammatory activation. SCFAs may represent as a novel and viable approach for alleviating NASH.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and progresses to nonalcoholic steatohepatitis (NASH) that is featured by overt hepatic inflammatory damage (Sanyal 2005, Chalasani et al. 2018). Recent epidemiologic data reveal that NASH affects 1.5–6.45% of the general population (Estes et al. 2018,

Younossi et al. 2018). Also, the incidence of NASH in all age groups has increased continuously due to the ongoing epidemics of obesity, which is the main cause of NAFLD/NASH (Estes et al. 2018). Of note, 10–20% of patients with NASH were lean or with normal body weight. (Younossi et al. 2018). To date, however, there is

3

Key Words

f short chain fatty acid

f non-alcoholic steatohepatitis

f steatosis

f inflammation

f macrophage

Journal of Endocrinology (2020) 245, 425–437

245

Downloaded from Bioscientifica.com at 03/17/2022 02:51:41PMvia free access

https://doi.org/10.1530/JOE-20-0018

https://joe.bioscientifica.com

mailto:[email protected]

mailto:[email protected]


Published by Bioscientifica Ltd.Printed in Great Britain

426SCFA alleviated NASHM Deng et al. 245:3Journal of Endocrinology

no effective medicine for treating NASH, especially for lean subjects with NAFLD/NASH.

Supplementation of dietary fibers has been shown to reduce body weight, improve insulin resistance, and alleviate NAFLD/NASH (Papathanasopoulos & Camilleri 2010, Chambers et al. 2019). As the main microbial metabolites of dietary fibers in the intestines, SCFAs function to mediate the metabolic regulation of fibers (Canfora et al. 2015). Several studies showed that oral supplementation of SCFAs alleviated HFD diet-induced NAFLD in mice, including reduced steatosis and inflammation (Yamashita et al. 2007, den Besten et al. 2015, Jin et al. 2015). Indeed, SCFAs, mainly acetate, propionate, and butyrate, are not only metabolic substrates, but also signaling molecules that regulate metabolism (Koh et al. 2016). For instance, acetate or propionate appears to act through G protein–coupled receptor 41 (GPR41) and GPR43 to promote gut-derived peptide YY (PYY) and glucagon-like peptide (GLP1) and alter satiety and energy intake (Samuel et al. 2008, Tolhurst et al. 2012). This process exemplifies how SCFAs indirectly improve hepatic lipid metabolism. After entering the systemic circulation, SCFAs can directly regulate metabolism or the function of adipose tissue, liver, muscle, and brain (Canfora et al. 2015, Sahuri-Arisoylu et al. 2016). As documented by an in vitro study, SCFA regulation of hepatic metabolism is related to increased hepatic AMP-activated protein kinase (AMPK) phosphorylation and altered expression of peroxisome proliferator-activated receptor alpha (PPARa) target genes which are involved in free fatty acid (FFA) oxidation, glycogen storage, thermogenesis, gluconeogenesis, and lipogenesis (den Besten et al. 2015). As such, SCFAs act to both directly and indirectly regulate hepatic metabolism via complicated processes. Also, SCFAs modulating NAFLD/NASH may be altered directly and indirectly by oral dosing. To date, there are few studies that have assessed the potential direct effects of SCFAs on NASH in vivo.

Previous studies indicated that hepatic infiltration of macrophages is the major cause of inflammatory responses in the pathology of NASH (Mridha et al. 2017). In terms of decreasing NASN-related inflammation, acetate, propionate, or butyrate inhibited macrophage production of proinflammatory cytokines and reduced the status of M1/M2 polarization (Liu et al. 2012, Li et al. 2018a). Moreover, Ye et al. showed that treatment with butyrate for 6 weeks prevented hepatic injury via reducing the inflammatory responses in mice with NASH as evidenced by alterations in tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Ilb), Il4, and Il10 mRNA levels (Ye et al. 2018). However, there is a critical need to not only further

validate the beneficial effects of SCFAs on NASH-related hepatic inflammation, but more importantly to elucidate the differential roles for different SCFAs in altering hepatic inflammation.

In this study, mice with MCD diet-induced NASH were treated with acetate, propionate, and/or butyrate and assessed for NASH phenotypes. Furthermore, the effects of sodium acetate on lipid metabolism and the proinflammatory responses in hepatocytes and macrophages were examined.

Materials and methods

Animal experiments

Male C57BL/6J mice (10 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were maintained on 12 h light:12 h darkness cycles (lights on at 06:00 h) and allowed access to diet and water ad libitum. After 1-week of acclimatization, the mice were randomly assigned to the following eight groups (n = 12 per group): control group in which mice were maintained on a standard chow diet; MCD model group and other treatment groups in which mice were fed a methionine- and choline-deficient (MCD) diet for 6 weeks. The mice in treatment groups were also intraperitoneally injected with acetate sodium, propionate sodium, or butyrate sodium solutions (Sigma), which were dissolved in sterile saline (0.9 % w/v) and filtered with 0.22 μm filter membrane. Group NaA-L and NaA-H were treated with sodium acetate solution daily (0.04 and 0.2 g/kg body, respectively) during the feeding period. Groups NaP-L, NaP-H, NaB-L, and NaB-H were treated with sodium propionate or sodium butyrate at the same dose as sodium acetate, respectively, while CON and MCD group were injected with sterile saline. During the experiment, the body weights of mice were monitored weekly and the food amount was recorded weekly to calculate food intake. After the 6-week feeding period, all mice were fasted for 12 h before killing for the collection of blood and tissue samples as previously described (Luo et al. 2018). All diets are products of Research Diets, Inc. (New Brunswick, NJ, USA). All the study protocols were reviewed and approved by the Animal Research Ethics Committee of China Agricultural University (number: KY180028).

Cell culture and treatment

AML-12 cells, a line of mouse hepatocytes, were kindly provided by Stem Cell Bank, Chinese Academy of Sciences,






427

Research

M Deng et al. SCFA alleviated NASH 245:3Journal of Endocrinology

and cultured in DMEM/F-12 (GibcoA) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin and streptomycin (Beyotime, Beijing, China), 1% ITS Liquid Media Supplement (Gibco), and 40 ng/mL dexamethasone (Sigma) in a 5% CO2 incubator at 37°C (Sahai et al. 2006, Kohli et al. 2007). AML-12 cells were grown to 70–80% confluence in six-well plates (Corning). After 24 h of serum starvation, the control group was incubated with fresh DMEM/F-12 and other groups were cultured with MCD DMEM/F-12 (Gibco) containing various concentrations of sodium acetate (NaA) (0, 0.05, 0.5, and 5 mmol/L, respectively) (Sigma) for 24 h. After treatment, the cells were stained with Oil Red O and/or harvested for total RNA isolation. To analyze proinflammatory signaling, some MCD medium- and/or NaA-treated hepatocytes were treated with or without lipopolysaccharide (LPS, 100 ng/mL, dissolved in 1× PBS) for the last 30 min to harvest cell lysates or LPS (20 ng/mL) for the last 6 h to harvest RNA samples (Luo et al. 2018). The cell lysates were used for Western blot analyses.

RAW264.7 cells, a line of mouse macrophages, were purchased from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and 100 U/mL penicillin and streptomycin (Beyotime) in a 5% CO2 incubator at 37°C. For RT-PCR and Western blot analyses, RAW264.7 cells were seeded into six-well plates (Corning) in 2 mL of serum-free DMEM medium at a density of 1 × 106 cells and cultured for 12 h (Wu et al. 2017). Thereafter, the cells in the control group were incubated with fresh DMEM and the cells in the other groups were incubated with MCD DMEM (Gibco) containing different concentrations of NaA (0, 0.5, and/or 5 mmol/L) for an additional 24 h. The cells were harvested for total RNA isolation. To analyze proinflammatory signaling, some MCD medium- and/or NaA-treated macrophages were treated LPS (100 ng/mL) for the last 30 min to harvest cell lysates or LPS (20 ng/mL) for the last 6 h to harvest RNA samples (Luo et al. 2018). The cell lysates were used for Western blot analyses.

Biochemical assays

The activities of serum alanine aminotransferase (ALT) and aspartate transaminase (AST) were measured using an automatic chemistry analyzer (Hitachi). The levels of hepatic TG and total cholesterols (TC) of the mice were quantified using a TG assay kit and a TC assay kit, respectively, according to the manufacturer’s protocols (Jiancheng, Nanjing, China).

Histology and immunohistochemistry

To evaluate hepatic fat deposition, frozen liver sections and AML-12 cells were stained with Oil Red O (Sigma) according to the standard protocols. The paraffin blocks from formalin-fixed livers were cut into 4-μm sections. The liver sections were stained with hematoxylin and eosin (H&E) for histological examination. To assess macrophage infiltration, liver sections were stained for F4/80 expression using a rabbit antibody (Santa Cruz). The quantitative analysis of Sirius-Red-positive areas and Oil Red O staining in liver sections was performed using ImageJ software with six fields per slide. The livers of four to six mice per group were examined, and three parallel experiments were conducted.

Cell cytotoxicity assays

An enhanced cell counting kit 8 assay (Beyotime) was used to determine cell cytotoxicity (Shi et al. 2018). AML-12 and RAW 264.7 cells were seeded at a density of 5 × 103 per well onto flat-bottom 96-well culture plates (Corning) and treated as mentioned earlier. The absorbance values of viable cells were finally determined at 450 nm using a microplate spectrophotometer (BioTek). The cell inhibitory rates were calculated using the following formula: cell inhibition rate (%) = (1 − A450 (sample)/A450 (control)) × 100.

Western blot analysis

Frozen liver tissues and cultured cells were prepared for Western blot analysis as described previously (Luo et al. 2018). The membranes were incubated with primary antibodies: phosphorylated AMPK alpha (P-AMPKa), AMPKa, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphorylated c-Jun N-terminal protein kinase (P-JNK), JNK, phosphorylated nuclear factor kappa B (NFkB) p65 (Pp65), p65 (CST, Danvers, MA, USA), and/or PPARa (Abcam). After incubation with a goat anti-rabbit horseradish peroxidase–conjugated secondary antibody (Beyotime) at a dilution of 1:10,000 for 1 h, the proteins were visualized using a Luminata Forte Enhanced Chemiluminescence Kit (Millipore) and the band intensities were analyzed using the QuantityOne analysis software.

Quantitative real-time polymerase chain reaction

Total RNA was extracted using TRIzol Reagents (Austin, TX, USA) and subjected to revers transcription using a







PrimeScript RT-PCR kit (TaKaRa). Quantitative RT-PCR was carried out with SYBR Premix Ex Taq (TaKaRa) using the Light-Cycler 480 Software (Roche Diagnostics GmbH) (Guo et al. 2013). The primers were synthesized by Sangon Biotech (Shanghai, China). The fluorescence data of target genes were analyzed by the 2−ΔΔCt method for relative quantification using Actin or Gapdh as a control.

Statistical analyses

Numeric data were presented as mean ± s.e.m.. Statistical significance was determined using the one-way ANOVA followed by LSD post-tests with SPSS 22.0. Differences were considered significant at a two-tailed P value less than 0.05.

Results

SCFAs ameliorated MCD diet-induced hepatic dysfunction

During and/or after the feeding period, MCD-fed C57BL/6L mice revealed significant decreases in food intake, body weight, and liver weight compared

with chow diet-fed mice. These decreases were not significantly altered by SCFA treatment (Fig. 1A, B and C). Compared with chow diet, the MCD diet induced significantly increased serum levels of ALT and AST, indicating hepatocellular damage associated with NASH (Srivastava et al. 2007). These elevations were markedly decreased in response to the treatment with each of the three major SCFAs in a dose-dependent manner. Specifically, the ALT levels in mice treated with a high dose of SCFA were 58.2% (NaP-H), 53.7% (NaA-H), and 46.9% (NaB-H) lower than their respective levels in the MCD group (Fig. 1D). The AST levels in mice treated with a high dose of SCFA were 31.1% (NaB-H), 28.3% (NaA-H), and 26.3% (NaP-H) lower than their respective levels in the MCD group (Fig. 1E). These results indicated that SCFA treatment improved MCD diet-induced liver dysfunction without affecting MCD diet-induced decreases in food intake, body weight, and liver weight.

SCFAs ameliorated MCD diet-induced hepatic steatosis

The liver sections of MCD group showed obvious vacuolated hepatocytes and severe micro- and macro-vesicular steatosis with marked inflammatory cell

Figure 1Feeding an MCD diet induced NASH in mice. Male C57BL/6J mice, at 10 weeks of age, were fed a standard chow diet or methionine-and-choline deficient (MCD) diet for 6 weeks. Mice were divided into 8 groups as detailed in the ‘Materials and methods’ section. (A) Effects of SCFAs on food intake. (B) Effects of SCFAs on body weight. (C) Effects of SCFAs on liver weight. (D) Plasma levels of ALT. (E) Plasma levels of AST. For all bar graphs, data are the mean ± s.e.m. (n = 6–10). #P < 0.05 and ##P < 0.01 vs the control group, and *P < 0.05, **P < 0.01 vs the MCD group. The significant difference was assessed using the one-way ANOVA followed by LSD post-tests. CON, control; MCD, group model mice fed with a MCD diet; NaA-L, low dose of sodium acetate; NaA-H, high dose of sodium acetate; NaP-L, low dose of sodium propionate; NaP-H, high dose of sodium propionate; NaB-L, low dose of sodium butyrate; NaB-H, high dose of sodium butyrate. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0018.







429

Research


infiltration. In contrast, SCFA supplementation significantly ameliorated the severity of hepatic steatosis, revealing decreased degrees of macro-vesicular and mixed vesicular steatosis (Fig. 2A). Consistently, SCFA treatment decreased hepatic lipid deposition and the accumulation of lipid droplets as indicated by the liver sections stained with Oil Red O. A high dose of acetate (NaA-H) generated the most potent efficacy at decreasing hepatic lipid accumulation relative to propionate and butyrate (Fig. 2B).

The hepatic levels of TG and TC increased significantly in the MCD group (7.8-fold and 1.9-fold

higher than their respective levels in mice fed a chow diet), and this elevation was decreased by treatment with SCFAs. The TG content of the NaA-H group reduced to 49.2%, followed by 67.5% (NaB-H) and 67.8% (NaP-H) compared with the MCD group (Fig. 2C). In contrast, the TC content of the NaA-H group decreased to 44.7%, followed by 54.5% (NaP-H) and 55.4% (NaB-H) compared with the MCD group (Fig. 2D). The results indicated that SCFA treatment ameliorated MCD diet-induced hepatic steatosis, and the sodium acetate treatment group revealed the greatest reduction in TG and TC contents.

Figure 2SCFAs ameliorated MCD diet-induced liver steatosis. (A) H&E staining of liver sections (magnification: 100×, scale bar: 500 μm). (B) Oil Red O staining of liver sections (magnification: 100×, scale bar: 500 μm). Bar graph displays percentages of lipid accumulation (n = 6–10). (C) The levels of hepatic triglycerides. (D) The levels of hepatic cholesterol. For all bar graphs, data are the mean ± s.e.m. (n = 6–10). #P < 0.05 and ##P < 0.01 vs the control group, and *P < 0.05, **P < 0.01 vs the MCD group. +P < 0.05 and ++P < 0.01. The significant difference was assessed using the one-way ANOVA followed by LSD post-tests. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0018.








NaA attenuated hepatic steatosis in a manner involving altered expression of genes for liver lipogenesis and FA oxidation

To gain insights of NaA alleviation of MCD diet-induced hepatic steatosis, we examined the effects of NaA on the expression of genes related to lipid metabolism. Compared with control mice, MCD-fed mice revealed markedly decreased expression of lipogenesis-related genes, including sterol regulatory element–binding protein 1c (Srebp1c), stearoyl-Coenzyme A desaturase 1 (Scd1), and acetyl-Coenzyme A carboxylase alpha (Acc1) (Fig. 3A, B, C and D), which may be a compensatory change. However, NaA treatment still reduced liver mRNA levels of fatty acid synthase (Fasn) in a dose-dependent manner compared with control treatment in MCD-fed mice (Fig. 3C); although NaA did not significantly alter

the expression of Srebp1c, Scd1, and Acc1 (Fig. 3A, B, C and D). With regard to fat oxidation-related genes, MCD-fed mice revealed markedly decreased mRNA levels of carnitine palmitoyltransferase 1a (Cpt1a) compared with mice fed a chow diet. Moreover, treatment with NaA, at either a low or high dose, significantly ameliorated MCD-induced decrease in Cpt1a mRNAs (Fig. 3E).

Next, the levels of proteins related to lipid metabolism were assessed. In MCD-fed mice, hepatic amount of PPARa, one of the main fatty acid oxidation enzymes, was significantly decreased compared with that in chow diet-fed mice. This decrease, however, was not significantly altered in response to NaA treatment (Fig. 3F). Interestingly, MCD-fed mice revealed decreased phosphorylation states of AMPKa. This decrease was reversed, to certain degrees, in response to NaA supplementation in a dose-dependent manner. The levels of activated AMPKa were 2.3 times

Figure 3NaA attenuated hepatic steatosis and altered events related to hepatic lipogenesis and FA oxidation. (A, B, C, D, and E) Hepatic expression of genes related to lipogenesis and FA oxidation was examined using RT-PCR (n = 6–10). (F) Protein levels of PPARα and GAPDH in the indicated groups were shown. Bar graph displays quantification of blots (n = 3). (G) NaA enhanced AMPK phosphorylated levels in MCD diet-induced NASH. Bar graph displays quantification of blots (n = 3). For all bar graphs, data are the mean ± s.e.m. #P < 0.05 and ##P < 0.01 vs the control group, and *P < 0.05, **P < 0.01 vs the MCD group. The significant difference was assessed using the one-way ANOVA followed by LSD post-tests. AU, arbitrary unit; FA, fatty acid.






431

Research


in the NaA-L group and 3.3 times in the NaA-H group compared with those in the MCD group (Fig. 3G).

NaA inhibited MCD diet-induced hepatic inflammation

To assess the effects of SCFAs on MCD diet-induced hepatic inflammation, we measured the degree of liver macrophage infiltration upon staining liver sections for the expression of F4/80. Compared with those of chow diet-fed mice, liver sections of mice in the MCD group exhibited aggregations of F4/80-positive cells. However, liver sections of mice in all SCFA treatment groups revealed significantly decreased aggregations of F4/80

positive cells, indicating reduced macrophage infiltration (Fig. 4A). Liver sections of mice treated with a high dose of NaA displayed 26.2% of F4/80 positive cells relative to those in the MCD group, indicating that NaA-H generated the highest efficacy at reducing liver macrophage infiltration among the three SCFA treatment groups. MCD feeding also significantly elevated liver mRNA levels of Tnfa, whereas both low and high doses of NaA reduced liver Tnfa levels compared with the MCD group (Fig. 4B). Moreover, the expression of phosphorylated JNK p46 and NFkB p65 in the NaA treatment groups was significantly lower than that in the MCD group (Fig. 4C and D). These results suggested that NaA treatment decreased MCD diet-induced hepatic inflammation.

Figure 4NaA supplementation decreased MCD diet-induced hepatic inflammation. (A) Liver sections were stained for F4/80 expression. Bar graph displays percentages of macrophages (n = 6–10, magnification: 100×, scale bar: 500 μm). (B) Liver mRNA levels were examined using RT-PCR (n = 6–10). (C and D) Liver lysates were examined for inflammatory signaling using Western blot analysis. Bar graphs display quantification of blots (n = 3). For all bar graphs, data are the mean ± s.e.m. #P < 0.05 and ##P < 0.01 vs the control group, and *P < 0.05, **P < 0.01 vs the MCD group. The significant difference was assessed using the one-way ANOVA followed by LSD post-tests. NaA, sodium acetate; AU, arbitrary unit. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0018.









NaA decreased the effect of MCD-mimicking medium on increasing hepatocyte fat deposition

To examine the direct effect of NaA on hepatocyte responses, we sought to treat AML-12 hepatocytes with NaA in the presence of MCD-mimicking medium as previously described (Sahai et al. 2006). The viability of AML-12 cells was not significantly reduced by incubating with either 0–10 mM NaA (Fig. 5A) or MCD-mimicking medium for 24 h (date not shown). Incubation of AML-12 hepatocytes with MCD-mimicking medium resulted in a marked increase in hepatocyte fat deposition, and this increase was significantly reduced upon treatment with NaA at various concentrations (0.05, 0.5, and/or 5 mM) (Fig. 5B).

Next, we sought to examine changes in the expression of genes related to lipid metabolism. Compared with control medium, MCD-mimicking medium markedly decreased the mRNA levels of Acc1 without significantly

altering the levels of Srebp1c, Scd1, and Fasn (Fig. 5C, D, E and F). NaA treatment (5 mM) decreased Acc1 mRNA levels in the treated cells to 46.4% of those in cells treated with MCD-mimicking media (Fig. 5F). Also, treatment with NaA (5 mM) caused a significant decrease in the mRNA levels of Fasn compared with each of other treatments (Fig. 5E). Interestingly, treatment with NaA at a dose of 0.05 or 0.5 mM increased Srebp1c and Scd1 levels without significantly altering the levels of Fasn and Acc1 compared with MCD-mimicking medium in the absence of NaA (Fig. 5C, D, E and F). With regard to the expression of genes for fat oxidation, treatment with NaA at a dose of 5 mM significantly upregulated the mRNA levels of Cpt1a (1.9 times) compared with control treatment in MCD-mimicking medium (Fig. 5G). These results, together with the findings presented in Figs 2 and 3, strongly indicated that a high dose of NaA was capable of improving MCD-mimicking medium-induced hepatocyte fat deposition through regulating lipid metabolism, especially fat oxidation.

Figure 5NaA decreased hepatocyte fat deposition. AML-12 cells were treated as detailed in the ‘Materials and methods’ section. (A) NaA cytotoxic effects. After serum starvation for 24 h, a CCK8 assay was performed for AML-12 cells treated with NaA at doses of 0–10 mmol/L for 24 h. (B) Oil Red O staining of AML-12 cells. Bar graph displays percentages of lipid accumulation (n = 3, magnification: 400×, scale bar: 100 μm). (C, D, E, F, and G) The expression of genes related to lipogenesis and FA oxidation was examined using RT-PCR (n = 3). (B-I) AML-12 cells were treated as detailed in the ‘Materials and methods’ section. For all bar graphs, data are the mean ± s.e.m. #P < 0.05 and ##P < 0.01 vs the control group, and *P < 0.05, **P < 0.01 vs the MCD-induced group. +P < 0.05 and ++P < 0.01. The significant difference was assessed using the one-way ANOVA followed by LSD post-tests. NaA, sodium acetate; NaA-L, the low dose of NaA group; NaA-M, the medium dose of NaA group; NaA-H, the high dose of NaA group; AU, arbitrary unit. A full color version of this figure is available at https://doi.org/10.1530/JOE-20-0018.








433

Research


NaA alleviated MCD-mimicking medium-induced proinflammatory responses in hepatocytes and macrophages

We sought to examine the direct effects of NaA on MCD-mimicking medium-induced proinflammatory responses in hepatocytes and macrophages. The viability of RAW264.7 cells was not reduced by incubating with either 0–10 mM NaA (Fig. 6A) or MCD-mimicking medium for 24 h (data not shown). When the inflammatory signaling was analyzed, treatment of cultured hepatocytes (AML-12) with MCD-mimicking medium for 24 h did not bring about significant changes in LPS-induced phosphorylation states of JNK p46 and NFkB p65 compared with the control medium (Fig. 6B), suggesting that MCD-mimicking medium had a limited effect on altering hepatocyte proinflammatory responses. In contrast, incubation of macrophages (RAW264.7) with MCD-mimicking medium significantly increased LPS-induced phosphorylation states of NFkB p65 (Fig. 6C). These results suggest that MCD-mimicking medium differentially altered the proinflammatory responses in macrophages and hepatocytes.

NaA treatment (5 mM) significantly reduced MCD/LPS-induced phosphorylation states of NFkB p65 but not JNK p46 in hepatocytes (Fig. 6B). In macrophages, NaA treatment significantly decreased the phosphorylation states of both JNK p46 and NFkB p65 stimulated by MCD/LPS (Fig. 6C). For proinflammatory genes, 0.5 and/or 5 mM NaA treatment significantly reduced the mRNA levels of Tnfa and/or Il6 stimulated by supplementation of MCD-mimicking medium with LPS in hepatocytes (Fig. 6D and E). Similarly, in macrophages, 0.5 and/or 5 mM NaA treatment markedly decreased the increases in Tnfa and Ilb levels induced by supplementation of MCD-mimicking medium with LPS (Fig. 6F and G). The results indicated that NaA treatment reduced proinflammatory responses stimulated by MCD/LPS in both hepatocytes and macrophages. Additionally, macrophages revealed much stronger responses to MCD/LPS and NaA treatment, relative to hepatocytes, and appeared to play a more important role in responding to NaA treatment.

Discussion

Acetate, propionate, and butyrate are the most abundant SCFAs in the human gut (den Besten et al. 2015, Oliphant & Allen-Vercoe 2019). After absorption, acetate is at the highest abundance in the liver and reaches the highest peripheral concentrations (19–160 μM) compared with

Figure 6NaA alleviated MCD-induced inflammatory responses in macrophages. AML-12 and RAW264.7 cells were treated as detailed in the ‘Materials and methods’ section. (A) NaA cytotoxic effects on RAW264.7 cells. A CCK8 assay was performed for RAW264.7 cells treated with NaA at a dose range of 0–10 mmol/L for 24 h. (B and C) Hepatocytes and macrophages were examined for inflammatory signaling using Western blot analysis. Bar graphs display quantification of blots (n = 3). (D, E, F, and G) The expression of hepatocyte (D and E) and macrophage (F and G) proinflammatory genes was examined using RT-PCR (n = 3). For all bar graphs, data are the mean ± s.e.m. #P < 0.05 and ##P < 0.01 vs the control group, and *P < 0.05, **P < 0.01 vs the LPS-induced MCD group. +P < 0.05 and ++P < 0.01. The significant difference was assessed using the one-way ANOVA followed by LSD post-tests. NaA, sodium acetate; AU, arbitrary unit.







propionate (1–13 μM) and butyrate (1–12 μM), as revealed by human studies (Bloemen et al. 2009, Canfora et al. 2015). Several studies suggested that high levels of SCFAs were related to obesity and excess fat deposition (Duarte et al. 2019). In contrast, orally administering propionate and butyrate for 4 weeks significantly decreased the severity of high-fat diet-induced weight gain and insulin resistance (Lin et al. 2012). Also, oral supplementation of butyrate improved hepatic inflammation through inhibiting the toll-like receptor 4 signaling pathway and production of inflammatory cytokines such as Il6 and Tnfa (Wu et al. 2017). These reports point to the importance of SCFAs in alleviating factors that contribute to the development and progression of NASH. More significantly, orally administering acetic acid markedly improved dyslipidemia and decreased hepatic expression of lipogenic genes such as Acc1 and Fasn in a type 2 diabetes model (Yamashita 2016). Mechanistically, acetate, propionate, or butyrate, in the intestine, appears to activate GPR41 and GPR43 and promote PYY and GLP1 release by L cells, which in turn affects satiety and energy intake (Samuel et al. 2008, Tolhurst et al. 2012). In addition, SCFAs are absorbed into the portal vein and directly affected the lipid metabolism in the liver. While three SCFAs are closely involved in host metabolism, there are clear differences in their specific metabolic ways. For example, acetate and butyrate are incorporated into lipid metabolism, whereas propionate is mainly involved in gluconeogenesis (den Besten et al. 2015). Currently, the evidence showing how exogenous SCFAs directly act on hepatic metabolism is still lacking (Canfora et al. 2015). The present study provided the primary data concerning the direct effects of acetate, propionate, and/or butyrate on alleviating NASH phenotype in MCD diet-fed mice. While validating the strongest efficacy of NaA at alleviating hepatic steatosis and inflammation, this study further demonstrated that NaA is capable of decreasing hepatocyte fat deposition and suppressing macrophage proinflammatory responses. As such, supplementation of SCFA(s) is beneficial for managing NASH.

Abnormal hepatic fat deposition and inflammation are the major characteristics of NASH (Nugent & Younossi 2007, Estes et al. 2018). In the present study, supplementation of SCFA(s) attenuated MCD diet-induced hepatic injury and steatosis. As supporting evidence, three major SCFAs (acetate, propionate, and butyrate) significantly decreased plasma levels of ALT and AST, which are the diagnostic markers of MCD diet-induced hepatic injury (Srivastava et al. 2007, Larter et al. 2008). Moreover, supplementation of SCFA(s) decreased liver TG

and TC contents, which were the major risk events for the development of NASH. These results were consistent with previous findings (Lin et al. 2012, Yamashita 2016, van der Beek et al. 2018). Notably, supplementation of each of the three major SCFAs ameliorated hepatic lipid accumulation to various degrees; although the three SCFAs revealed similar impacts on NASH-related hepatic injury and inflammation. Specifically, acetate displayed the greatest efficacy at decreasing liver steatosis. This is consistent with the previous report, in which acetate appeared to play an important role in affecting lipid deposition in the liver (Yamashita 2016).

Acetate had greater efficacy at improving steatosis in vivo and was selected for in vitro study with hope to gain mechanistic insights. Several mechanisms likely account for acetate actions. First, higher abundance of acetate is detected in the liver compared with propionate or butyrate after absorption (Bloemen et al. 2009, Koh et al. 2016). This may enable acetate to act as a metabolic substrate or signal molecule to regulate the lipid metabolism (Yamashita 2016, Dangana et al. 2019). In this study, decreasing liver steatosis appeared to be attributable to NaA actions on increasing the expression of genes for FA oxidation. In support of this, NaA improved the MCD-induced reduction in Cpt1a expression central to FA oxidation. Secondly and interestingly, activated AMPK, but not PPARa, was involved in NaA regulation of gene expression. As an energy sensor, AMPK regulates a number of enzymes involved in lipid homeostasis. As it is well documented, AMPK inhibits FA synthesis through inactivating acetyl-CoA carboxylase by phosphorylation and increases FA oxidation through decreasing intracellular malonyl CoA (Cool et al. 2006, Yamashita 2016). Previous studies confirmed that acetate altered lipid metabolism through activating AMPK and PPARa in a high-fat model (Hattori et al. 2010, Canfora et al. 2015, Yamashita 2016). In this study, NaA treatment restored MCD feeding-inactivated AMPK, but not PPARa in mice. Moreover, NaA stimulation of AMPK activation is dose-dependent, and low concentrations of acetate seem to have limited effects on AMPK. These findings are in agreement with the data by a previous study (Koh et al. 2016). Additional to AMPK, complex metabolic network including hormonal and nutritional status, transcription factors, and others also critically regulate FA oxidation-related genes (Shin et al. 2006, Tobita et al. 2018). For instance, GPCRs and PPARs are participating in FA oxidation (Hong et al. 2005, Canfora et al. 2015). How these AMPK-independent mechanisms account for acetate actions warrant future investigations. Nonetheless, it is conceivable that NaA improvement of MCD diet–induced






435

Research


hepatic steatosis is attributable to increased hepatic FA oxidation through a mechanism involving AMPK activation. In addition to altering FA oxidation, NaA also altered Fasn expression in the liver, which is consistent with the finding by Yamashita in supporting that acetate treatment inactivates carbohydrate-responsive element-binding protein (ChREBP) and thus decreases transcription of lipogenic genes via activating AMPK (Yamashita 2016). To be noted, in vitro, low doses of NaA (0.05 and 0.5 mM) increased the transcription of Srebp1c and Scd1 genes, which was opposite to the results of NaA at a dose of 5 mM. The possible explanation is that low concentrations of NaA are not sufficient enough to activate AMPK (Hong et al. 2005, Yamashita 2016).

Inflammation is key to the pathogenesis of NASH, which is characterized by inflammatory damage and increased proinflammatory responses (Kodama et al. 2009, Huang et al. 2010). In the present study, liver sections of mice in the MCD-fed group revealed increased liver proinflammatory status, indicated by massive macrophage aggregations and increased liver Tnfa mRNA levels and phosphorylation states of JNK p46 and NFkB p65. These results were consistent with the findings of previous studies and confirmed the successful establishment of an MCD model of NASH (Luo et al. 2018). Upon supplementation of SCFA(s), the severity of MCD diet-induced hepatic inflammation was significantly decreased. As supporting evidence, supplementation of each of the three SCFAs resulted in decreased hepatic macrophage aggregation. In particular, supplementation of acetate displayed the greatest decreases in MCD-induced macrophage aggregations and proinflammatory responses (Tnfa, JNK p46, and NFkB p65). In terms of NASH pathogenesis, both macrophages and hepatocytes critically determine the development and progression of liver inflammation (Kodama et al. 2009, Cai et al. 2018). For this purpose, the present study examined the effects of MCD-mimicking medium on the proinflammatory responses of macrophages and/or hepatocytes. One of the notable findings is that MCD-mimicking medium enhanced LPS-induced proinflammatory responses in macrophages but not in hepatocytes, although MCD-mimicking media increased basal proinflammatory responses in both hepatocytes and macrophages. In response to NaA treatment, macrophages revealed a greater decrease in LPS-stimulated proinflammatory responses, although both hepatocytes and macrophages revealed decreased proinflammatory responses. Taken together, these results suggest that macrophages appeared

to play a more important role in responding to MCD diet-induced proinflammatory signaling and likely were a primary target for NaA treatment to reduce MCD diet-induced hepatic inflammation. While exactly how SCFA(s) decrease macrophage and hepatocyte proinflammatory responses remains to be elucidated, SCFAs are shown to decrease the expression of inflammatory cytokines and the migration and recruitment of immune cells through the combined effects on altering cell surface receptors and inhibiting intracellular enzyme activity (histone acetylation) (Li et al. 2018b). Also, AMPK activation has been shown to suppress NFkB phosphorylation and Tnfa production in multiple cell types (Chen & Vitetta 2018, Wu et al. 2018). In the present study, treatment with NaA increased hepatic AMPK. Considering this, NaA suppression of hepatic inflammation appeared to involve AMPK activation. This view, however, needs to be further studied.

In conclusion, the present study demonstrated that supplementation of each of the three SCFAs (acetate, propionate, or butyrate) alleviated MCD diet-induced NASH and that acetate supplementation displayed the strongest efficacy at decreasing MCD diet-induced hepatic steatosis and inflammation. Mechanistically, NaA reduction of hepatic steatosis was associated with increased events related to FA oxidation and decreased events related to lipogenesis in a manner involving increased activation of AMPK. At the cellular levels, NaA treatment attenuated the proinflammatory responses of macrophages, which appears to account for NaA alleviation of MCD-induced hepatic inflammation. As such, the present study validated supplementation of SCFAs as a viable approach for alleviating NASH.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis study was funded by Beijing Natural Science Foundation (Grant No. 6182024) and the ‘111’ Project from the Education Ministry of China (Grant No. B18053).

Author contribution statementM D, F Q, L C, and C L carried out most experiments involving mice. M D carried out most experiments involving cells. M D and F Q acquired data







and analyzed data. L Z and C W came up with the study concept. M Z, F R, H G, H Z, S G, L Z, and C W contributed to the scientific discussion. M D wrote the manuscript. L Z and C W designed the study and revised the manuscript.

ReferencesBloemen JG, Venema K, van de Poll MC, Olde Damink SW, Buurman WA

& Dejong CH 2009 Short chain fatty acids exchange across the gut and liver in humans measured at surgery. Clinical Nutrition 28 657–661. (https://doi.org/10.1016/j.clnu.2009.05.011)

Cai Y, Li H, Liu M, Pei Y, Zheng J, Zhou J, Luo X, Huang W, Ma L, Yang Q, et al. 2018 Disruption of adenosine 2A receptor exacerbates NAFLD through increasing inflammatory responses and SREBP1c activity. Hepatology 68 48–61. (https://doi.org/10.1002/hep.29777)

Canfora EE, Jocken JW & Blaak EE 2015 Short-chain fatty acids in control of body weight and insulin sensitivity. Nature Reviews: Endocrinology 11 577–591. (https://doi.org/10.1038/nrendo.2015.128)

Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, Harrison SA, Brunt EM & Sanyal AJ 2018 The diagnosis and management of nonalcoholic fatty liver disease: practice guidance from the American Association for the Study of Liver Diseases. Hepatology 67 328–357. (https://doi.org/10.1002/hep.29367)

Chambers ES, Byrne CS, Rugyendo A, Morrison DJ, Preston T, Tedford C, Bell JD, Thomas L, Akbar AN, Riddell NE, et al. 2019 The effects of dietary supplementation with inulin and inulin-propionate ester on hepatic steatosis in adults with non-alcoholic fatty liver disease. Diabetes, Obesity and Metabolism 21 372–376. (https://doi.org/10.1111/dom.13500)

Chen J & Vitetta L 2018 Inflammation-modulating effect of butyrate in the prevention of colon cancer by dietary fiber. Clinical Colorectal Cancer 17 e541–e544. (https://doi.org/10.1016/j.clcc.2018.05.001)

Cool B, Zinker B, Chiou W, Kifle L, Cao N, Perham M, Dickinson R, Adler A, Gagne G, Iyengar R, et al. 2006 Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome. Cell Metabolism 3 403–416. (https://doi.org/10.1016/j.cmet.2006.05.005)

Dangana EO, Omolekulo TE, Areola ED, Olaniyi KS, Soladoye AO & Olatunji LA 2019 Sodium acetate protects against nicotine-induced excess hepatic lipid in male rats by suppressing xanthine oxidase activity. Chemico-Biological Interactions 316 108929.

den Besten G, Bleeker A, Gerding A, van Eunen K, Havinga R, van Dijk TH, Oosterveer MH, Jonker JW, Groen AK, Reijngoud DJ, et al. 2015 Short-chain fatty acids protect against high-fat diet-induced obesity via a PPARγ-dependent switch from lipogenesis to fat oxidation. Diabetes 64 2398–2408. (https://doi.org/10.2337/db14-1213)

Duarte SMB, Stefano JT & Oliveira CP 2019 Microbiota and nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH). Annals of Hepatology 18 416–421. (https://doi.org/10.1016/j.aohep.2019.04.006)

Estes C, Razavi H, Loomba R, Younossi Z & Sanyal AJ 2018 Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease. Hepatology 67 123–133. (https://doi.org/10.1002/hep.29466)

Guo X, Li H, Xu H, Halim V, Thomas LN, Woo SL, Huo Y, Chen YE, Sturino JM & Wu C 2013 Disruption of inducible 6-phosphofructo-2-kinase impairs the suppressive effect of PPARgamma activation on diet-induced intestine inflammatory response. Journal of Nutritional Biochemistry 24 770–775. (https://doi.org/10.1016/j.jnutbio.2012.04.007)

Hattori M, Kondo T, Kishi M & Yamagami K 2010 A single oral administration of acetic acid increased energy expenditure in C57BL/6J mice. Bioscience, Biotechnology, and Biochemistry 74 2158–2159. (https://doi.org/10.1271/bbb.100486)

Hong YH, Nishimura Y, Hishikawa D, Tsuzuki H, Miyahara H, Gotoh C, Choi KC, Feng DD, Chen C, Lee HG, et al. 2005 Acetate and propionate short chain fatty acids stimulate adipogenesis via GPCR43. Endocrinology 146 5092–5099. (https://doi.org/10.1210/en.2005-0545)

Huang W, Metlakunta A, Dedousis N, Zhang P, Sipula I, Dube JJ, Scott DK & O’Doherty RM 2010 Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance. Diabetes 59 347–357. (https://doi.org/10.2337/db09-0016)

Jin CJ, Sellmann C, Engstler AJ, Ziegenhardt D & Bergheim I 2015 Supplementation of sodium butyrate protects mice from the development of non-alcoholic steatohepatitis (NASH). British Journal of Nutrition 114 1745–1755. (https://doi.org/10.1017/S0007114515003621)

Kodama Y, Kisseleva T, Iwaisako K, Miura K, Taura K, De Minicis S, Osterreicher CH, Schnabl B, Seki E & Brenner DA 2009 C-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice. Gastroenterology 137 1467.e5–1477.e5. (https://doi.org/10.1053/j.gastro.2009.06.045)

Koh A, De Vadder F, Kovatcheva-Datchary P & Backhed F 2016 From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell 165 1332–1345. (https://doi.org/10.1016/j.cell.2016.05.041)

Kohli R, Pan X, Malladi P, Wainwright MS & Whitington PF 2007 Mitochondrial reactive oxygen species signal hepatocyte steatosis by regulating the phosphatidylinositol 3-kinase cell survival pathway. Journal of Biological Chemistry 282 21327–21336. (https://doi.org/10.1074/jbc.M701759200)

Larter CZ, Yeh MM, Williams J, Bell-Anderson KS & Farrell GC 2008 MCD-induced steatohepatitis is associated with hepatic adiponectin resistance and adipogenic transformation of hepatocytes. Journal of Hepatology 49 407–416. (https://doi.org/10.1016/j.jhep.2008.03.026)

Li M, van Esch BCAM, Wagenaar GTM, Garssen J, Folkerts G & Henricks PAJ 2018a Pro- and anti-inflammatory effects of short chain fatty acids on immune and endothelial cells. European Journal of Pharmacology 831 52–59. (https://doi.org/10.1016/j.ejphar.2018.05.003)

Li Y, Zhang S, Wang Y, Peng J, Fang F & Yang X 2018b MLH1 enhances the sensitivity of human endometrial carcinoma cells to cisplatin by activating the MLH1/c-Abl apoptosis signaling pathway. BMC Cancer 18 1294. (https://doi.org/10.1186/s12885-018-5218-4)

Lin HV, Frassetto A, Kowalik Jr EJ, Nawrocki AR, Lu MM, Kosinski JR, Hubert JA, Szeto D, Yao X, Forrest G, et al. 2012 Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms. PLoS ONE 7 e35240. (https://doi.org/10.1371/journal.pone.0035240)

Liu T, Li J, Liu Y, Xiao N, Suo H, Xie K, Yang C & Wu C 2012 Short-chain fatty acids suppress lipopolysaccharide-induced production of nitric oxide and proinflammatory cytokines through inhibition of NF-kappaB pathway in RAW264.7 cells. Inflammation 35 1676–1684. (https://doi.org/10.1007/s10753-012-9484-z)

Luo X, Li H, Ma L, Zhou J, Guo X, Woo SL, Pei Y, Knight LR, Deveau M, Chen Y, et al. 2018 Expression of STING is increased in liver tissues from patients with NAFLD and promotes macrophage-mediated hepatic inflammation and fibrosis in mice. Gastroenterology 155 1971.e4–1984.e4. (https://doi.org/10.1053/j.gastro.2018.09.010)

Mridha AR, Wree A, Robertson AAB, Yeh MM, Johnson CD, Van Rooyen DM, Haczeyni F, Teoh NC, Savard C, Ioannou GN, et al. 2017 NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. Journal of Hepatology 66 1037–1046. (https://doi.org/10.1016/j.jhep.2017.01.022)

Nugent C & Younossi ZM 2007 Evaluation and management of obesity-related nonalcoholic fatty liver disease. Nature Clinical Practice: Gastroenterology and Hepatology 4 432–441. (https://doi.org/10.1038/ncpgasthep0879)




https://doi.org/10.1016/j.clnu.2009.05.011

https://doi.org/10.1002/hep.29777

https://doi.org/10.1038/nrendo.2015.128


https://doi.org/10.1111/dom.13500

https://doi.org/10.1111/dom.13500

https://doi.org/10.1016/j.clcc.2018.05.001

https://doi.org/10.1016/j.cmet.2006.05.005

https://doi.org/10.2337/db14-1213

https://doi.org/10.1016/j.aohep.2019.04.006

https://doi.org/10.1016/j.aohep.2019.04.006


https://doi.org/10.1016/j.jnutbio.2012.04.007

https://doi.org/10.1016/j.jnutbio.2012.04.007

https://doi.org/10.1271/bbb.100486

https://doi.org/10.1210/en.2005-0545

https://doi.org/10.2337/db09-0016

https://doi.org/10.1017/S0007114515003621

https://doi.org/10.1017/S0007114515003621

https://doi.org/10.1053/j.gastro.2009.06.045


https://doi.org/10.1016/j.cell.2016.05.041

https://doi.org/10.1016/j.cell.2016.05.041

https://doi.org/10.1074/jbc.M701759200

https://doi.org/10.1074/jbc.M701759200

https://doi.org/10.1016/j.jhep.2008.03.026

https://doi.org/10.1016/j.ejphar.2018.05.003

https://doi.org/10.1016/j.ejphar.2018.05.003

https://doi.org/10.1186/s12885-018-5218-4

https://doi.org/10.1371/journal.pone.0035240

https://doi.org/10.1371/journal.pone.0035240

https://doi.org/10.1007/s10753-012-9484-z


https://doi.org/10.1016/j.jhep.2017.01.022

https://doi.org/10.1038/ncpgasthep0879




437

Research


Oliphant K & Allen-Vercoe E 2019 Macronutrient metabolism by the human gut microbiome: major fermentation by-products and their impact on host health. Microbiome 7 91. (https://doi.org/10.1186/s40168-019-0704-8)

Papathanasopoulos A & Camilleri M 2010 Dietary fiber supplements: effects in obesity and metabolic syndrome and relationship to gastrointestinal functions. Gastroenterology 138 65.e1–72.e1. (https://doi.org/10.1053/j.gastro.2009.11.045)

Sahai A, Pan X, Paul R, Malladi P, Kohli R & Whitington PF 2006 Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium. American Journal of Physiology: Gastrointestinal and Liver Physiology 291 G55–G62. (https://doi.org/10.1152/ajpgi.00360.2005)

Sahuri-Arisoylu M, Brody LP, Parkinson JR, Parkes H, Navaratnam N, Miller AD, Thomas EL, Frost G & Bell JD 2016 Reprogramming of hepatic fat accumulation and ‘browning’ of adipose tissue by the short-chain fatty acid acetate. International Journal of Obesity 40 955–963. (https://doi.org/10.1038/ijo.2016.23)

Samuel BS, Shaito A, Motoike T, Rey FE, Backhed F, Manchester JK, Hammer RE, Williams SC, Crowley J, Yanagisawa M, et al. 2008 Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41. PNAS 105 16767–16772. (https://doi.org/10.1073/pnas.0808567105)

Sanyal AJ 2005 Mechanisms of disease: pathogenesis of nonalcoholic fatty liver disease. Nature Clinical Practice: Gastroenterology and Hepatology 2 46–53. (https://doi.org/10.1038/ncpgasthep0084)

Shi H, Bi H, Sun X, Dong H, Jiang Y, Mu H, Liu G, Kong W, Gao R & Su J 2018 Antitumor effects of Tubeimoside-1 in NCI-H1299 cells are mediated by microRNA-126-5p-induced inactivation of VEGF-A/VEGFR-2/ERK signaling pathway. Molecular Medicine Reports 17 4327–4336. (https://doi.org/10.3892/mmr.2018.8459)

Shin ES, Cho SY, Lee EH, Lee SJ, Chang IS & Lee TR 2006 Positive regulation of hepatic carnitine palmitoyl transferase 1A (CPT1A) activities by soy isoflavones and L-carnitine. European Journal of Nutrition 45 159–164.

Srivastava AR, Kumar S, Agarwal GG & Ranjan P 2007 Blunt abdominal injury: serum ALT-A marker of liver injury and a guide to assessment of its severity. Injury 38 1069–1074. (https://doi.org/10.1016/j.injury.2007.04.019)

Tobita H, Sato S, Yazaki T, Mishiro T, Ishimura N, Ishihara S & Kinoshita Y 2018 Alogliptin alleviates hepatic steatosis in a mouse model of nonalcoholic fatty liver disease by promoting CPT1a expression

via Thr172 phosphorylation of AMPKalpha in the liver. Molecular Medicine Reports 17 6840–6846.

Tolhurst G, Heffron H, Lam YS, Parker HE, Habib AM, Diakogiannaki E, Cameron J, Grosse J, Reimann F & Gribble FM 2012 Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2. Diabetes 61 364–371. (https://doi.org/10.2337/db11-1019)

van der Beek CM, Canfora EE, Kip AM, Gorissen SHM, Olde Damink SWM, van Eijk HM, Holst JJ, Blaak EE, Dejong CHC & Lenaerts K 2018 The prebiotic inulin improves substrate metabolism and promotes short-chain fatty acid production in overweight to obese men. Metabolism: Clinical and Experimental 87 25–35. (https://doi.org/10.1016/j.metabol.2018.06.009)

Wu JL, Zou JY, Hu ED, Chen DZ, Chen L, Lu FB, Xu LM, Zheng MH, Li H, Huang Y, et al. 2017 Sodium butyrate ameliorates S100/FCA-induced autoimmune hepatitis through regulation of intestinal tight junction and toll-like receptor 4 signaling pathway. Immunology Letters 190 169–176. (https://doi.org/10.1016/j.imlet.2017.08.005)

Wu W, Wang S, Liu Q, Wang X, Shan T & Wang Y 2018 Cathelicidin-WA attenuates LPS-induced inflammation and redox imbalance through activation of AMPK signaling. Free Radical Biology and Medicine 129 338–353. (https://doi.org/10.1016/j.freeradbiomed.2018.09.045)

Yamashita H 2016 Biological function of acetic acid-improvement in obesity and glucose tolerance by acetic acid in type 2 diabetic rats. Critical Reviews in Food Science and Nutrition 56 (Supplement 1) S171–S175. (https://doi.org/10.1080/10408398.2015.1045966)

Yamashita H, Fujisawa K, Ito E, Idei S, Kawaguchi N, Kimoto M, Hiemori M & Tsuji H 2007 Improvement of obesity and glucose tolerance by acetate in Type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Bioscience, Biotechnology, and Biochemistry 71 1236–1243. (https://doi.org/10.1271/bbb.60668)

Ye J, Lv L, Wu W, Li Y, Shi D, Fang D, Guo F, Jiang H, Yan R, Ye W, et al. 2018 Butyrate protects mice against methionine-choline-deficient diet-induced non-alcoholic steatohepatitis by improving gut barrier function, attenuating inflammation and reducing endotoxin levels. Frontiers in Microbiology 9 1967. (https://doi.org/10.3389/fmicb.2018.01967)

Younossi Z, Anstee QM, Marietti M, Hardy T, Henry L, Eslam M, George J & Bugianesi E 2018 Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nature Reviews: Gastroenterology and Hepatology 15 11–20. (https://doi.org/10.1038/nrgastro.2017.109)

Received in final form 12 March 2020Accepted 9 April 2020Accepted Manuscript published online 9 April 2020




https://doi.org/10.1186/s40168-019-0704-8

https://doi.org/10.1186/s40168-019-0704-8



https://doi.org/10.1152/ajpgi.00360.2005

https://doi.org/10.1152/ajpgi.00360.2005

https://doi.org/10.1038/ijo.2016.23

https://doi.org/10.1073/pnas.0808567105


https://doi.org/10.3892/mmr.2018.8459

https://doi.org/10.1016/j.injury.2007.04.019

https://doi.org/10.1016/j.injury.2007.04.019

https://doi.org/10.2337/db11-1019

https://doi.org/10.2337/db11-1019

https://doi.org/10.1016/j.metabol.2018.06.009

https://doi.org/10.1016/j.metabol.2018.06.009

https://doi.org/10.1016/j.imlet.2017.08.005

https://doi.org/10.1016/j.freeradbiomed.2018.09.045

https://doi.org/10.1016/j.freeradbiomed.2018.09.045

https://doi.org/10.1080/10408398.2015.1045966

https://doi.org/10.1271/bbb.60668

https://doi.org/10.3389/fmicb.2018.01967

https://doi.org/10.3389/fmicb.2018.01967

https://doi.org/10.1038/nrgastro.2017.109

https://doi.org/10.1038/nrgastro.2017.109

scfas alleviated steatosis and inflammation in mice with

Documents