Alleviative effect of sodium butyrate on high-fructose-diet-induced hepatic accumulation of triacylglycerol is associated with reduced expression levels of fructolytic and fatty acid synthetic enzymes in rats

Abstract

Sodium butyrate (NaB) intake reduces high-sucrose diet-induced hepatic expression of genes and proteins related to fatty acid synthesis and the accumulation of triacylglycerols (TAGs) in rats. In this study, we examined the effects of NaB intake on the hepatic accumulation of TAGs and the expression of genes and proteins related to lipid metabolism, including fructolysis induced by excessive ingestion of fructose, a more potent inducer of lipogenesis than sucrose. Dietary supplementation with 5 % NaB decreased the high-fructose-induced hepatic levels of TAG and the expression levels of genes and proteins related to fructolysis and fatty acid synthesis, but not those related to β-oxidation. These results suggest that NaB intake alleviates high-fructose-induced non-alcoholic fatty liver disease by suppressing a wide range of pathways, including those from fructolysis to fatty acid synthesis.

Introduction

In the liver, fructose is rapidly phosphorylated to fructose-1-phosphate by ketohexokinase (KHK). Fructose-1-phosphate is subsequently cleaved by aldolase B (ALDOB) into dihydroxyacetone phosphate and glyceraldehyde. Glyceraldehyde is phosphorylated by triokinase/FMN cyclase (TKFC) to form glyceraldehyde-3-phosphate (Mayes, 1993). Triose phosphates derived from the fructolytic pathway are used in the oxidative, gluconeogenic, and de novo lipogenic pathways (Sun and Empie, 2012). Compared with glucose, fructose bypasses the rate-limiting step of glycolysis and rapidly induces hepatic de novo lipogenesis; thus, excessive consumption of fructose leads to nonalcoholic fatty liver disease (NAFLD) and insulin resistance (Herman and Samuel, 2016).

Animal studies, including ours, have shown that the expression of genes related to fructolysis (e.g., Khk, Aldob) and fatty acid synthesis [e.g., acetyl-CoA carboxylase alpha (Acaca) and fatty acid synthase (Fasn)] is increased in the triacylglycerol (TAG)-accumulated liver of rats fed a high-fructose diet (Koo et al., 2008; Shimada et al., 2017). In addition, Khk-knockout mice exhibited decreased hepatic accumulation of TAGs when exposed to a high-fat/high-fructose diet (Miller et al., 2018). Therefore, reducing the expression of both fructolytic and fatty acid synthesis enzymes may prevent the onset and development of NAFLD induced by high fructose levels.

Butyrate is a short-chain fatty acid produced by the bacterial fermentation of dietary fibers in the large intestine (Donohoe et al., 2011). It exerts beneficial effects on metabolic diseases such as NAFLD and obesity by regulating the metabolism of glucose and lipids (Canfora et al., 2015). In addition to butyrate derived from intestinal bacterial fermentation, oral intake of sodium butyrate (NaB) improved high-fat diet-induced NAFLD (Den Besten et al., 2015; Matheus et al., 2017; Li et al., 2018). Furthermore, we recently reported that dietary supplementation with NaB reduced high-sucrose diet-induced hepatic TAG accumulation, accompanied by a decrease in the expression of fatty acid synthesis enzymes (Hattori et al., 2022). Sucrose is composed of two monosaccharides, glucose and fructose; the fructose component of sucrose is thought to be the major factor responsible for the accumulation of TAG in the liver (Stanhope, 2016). An in vivo study showed that NaB supplementation nominally suppressed hepatic TAG accumulation in Western-style diet-induced obese mice drinking fructose (Beisner et al, 2021). However, it remains unclear whether NaB intake affects the hepatic expression of genes and proteins related to fructolysis and fatty acid synthesis, as well as TAG accumulation induced by the excessive ingestion of fructose, which is a more potent inducer of hepatic lipogenesis than sucrose.

In this study, we examined the effects of dietary supplementation with NaB on hepatic TAG levels and the expression of genes and proteins related to lipid metabolism, including fructolysis, in rats fed a high-fructose diet for 15 days.

Materials and Methods

Animals and diets  Four-week-old male Wistar rats were purchased from Japan SLC Inc. (Shizuoka, Japan). After a 5-day acclimation period being fed a laboratory chow diet (MF; Oriental Yeast, Tokyo, Japan), the rats received a control, high-fructose, or high-fructose diet supplemented with 5 % NaB (Tokyo Chemical Industry, Tokyo, Japan) for 15 days (n = 6 per group). The animals were housed individually in a metal cage at a stable temperature (22 ± 2 °C) under a 12-h light/dark cycle (lights on: 07:00–19:00). Food and water were provided ad libitum throughout the acclimation and feeding periods.

The control diet contained 20.0 % (w/w) casein, 64.9 % α-cornstarch, 5.0 % corn oil, 5.0 % cellulose, 3.5 % AIN93G mineral mixture, 1.0 % AIN93 vitamin mixture, 0.3 % L-cystine, and 0.3 % choline bitartrate. The high-fructose diet was prepared by replacing 50.0 % α-cornstarch in the control diet with the same amount of fructose (50.0 %). Then, 5.0 % NaB was added to the high-fructose diet to replace the same amount of α-cornstarch (5.0 %). The purity of NaB was greater than 98.0 %.

Our previous study reported that feeding rats with a 61.9 % high-sucrose diet supplemented with 3 % NaB for 21 days decreased hepatic TAG levels (Hattori et al., 2022). The integer ratio of fructose to NaB in the high-sucrose diet containing NaB was 10 : 1. In addition, it was shown that dietary supplementation with 5 % NaB reduced hepatic lipogenesis without affecting food intake in mice fed a high-fat diet for 12 weeks (Den Besten et al., 2015). Thus, we added 5 % NaB to the 50 % high-fructose diet.

The intake of fructose derived from the high-sucrose diet throughout the experimental period (21 days) was estimated to be approximately 101 g per rat. In our preliminary study, the fructose intake was estimated to be approximately 6.92 g per rat per day when the rats were fed a 50 % high-fructose diet for 10 days. If the rats ingested a 50 % high-fructose diet for 14 days, the fructose intake throughout the experimental period (14 days) was estimated to be approximately 96.9 g per rat. Thus, to ingest equal or greater amounts of fructose throughout the experimental period, as described in our previous study, rats must be fed a 50 % high-fructose diet for more than 14 days. Therefore, we determined the feeding period to be 15 days.

After 15 days, the non-fasting rats were euthanized by decapitation under isoflurane anesthesia. Serum and liver tissue samples were collected for subsequent analyses. All animal care and experimental procedures were approved by the Animal Care and Use Committee of Gifu University, Gifu, Japan (Approval Number: 2019-120).

Hepatic and serum parameter assays  Hepatic total lipids were extracted with approximately equal amounts of 0.1 M KCl, chloroform, and methanol, based on the Bligh and Dyer method with some modifications (Shimada et al., 2017). The extracted lipids were dried and dissolved in 2-propanol for the TAG measurements. Serum and hepatic TAG levels were measured using commercial kits (Triglyceride E-test, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Serum glucose, insulin, and glucagon-like peptide-1 (GLP-1) levels were determined using commercial kits (Glucose CII-test Wako, LBIS Rat Insulin ELISA Kit, FUJIFILM Wako Pure Chemical Corporation, and Multi Species GLP-1 Total ELISA, EMD Millipore Corporation, Burlington, MA, USA).

Real-time quantitative PCR  Hepatic total RNA was extracted and converted to cDNA using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and an ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). A total of 20 ng of cDNAs were subjected to real-time quantitative PCR using a mixture consisting of TB Green Premix Ex Taq with ROX reference dye (Takara-Bio Inc., Shiga, Japan) and 0.2 μM of each primer on a StepOnePlus system (Applied Biosystems, Waltham, MA, USA), as described previously (Shimada et al., 2017). The primers were:

•  Khk, 5′-CACCTTCAATGCCTCTGTCA-3′, 5′- CCTG CAAGCCACACTTCTT-3′;
•  Aldob, 5′-TTGCCAATGGGAAGGGTA-3′, 5′-ATCCT CTGTAGGCGGTTTCC-3′;
•  Tkfc, 5′-CTCTGTGACTGGAAGGAATCG-3′, 5′-GCA CAAGTGTCATCTGCTTTG-3′;
•  acetyl-CoA carboxylase alpha (Acaca), 5′-TGGCTGT CATACATGCCTAAGA-3′, 5′-TGTGGGAACAAACTCGA TGA-3′;
•  fatty acid synthase (Fasn), 5′-GGATATCCCAGTTG CTGAAGAC-3′, 5′-TAGTGGTCAGATGACTCGGAAC-3′;
•  stearoyl-coenzyme A desaturase (Scd), 5′-CTGACCT GAAAGCTGAGAAGC-3′, 5′-GATGAAGCACATGAGCA GGA-3′;
•  MLX interacting protein (Mlxipl), 5′-CTGCTACTGC CAGAGACAACAA-3′, 5′-GGAAGGTTCCAGTACTTGTT CA-3′;
•  sterol regulatory element binding transcription factor 1 (Srebf1), 5′-GAGCCATGGATTGCACATT-3′, 5′-AGCA TAGGGGGCATCAAATAG-3′;
•  carnitine palmitoyltransferase 1a (Cpt1a), 5′-GGAGC GACTCTTCAATACTTCC-3′, 5′-TGCCTGCTGTCCTTGA TATG-3′;
•  Cpt2, 5′-CTTGGATGACAGCCAGTTCA-3′, 5′-TTAT CCTGAGCAAGCAGGTG-3′;
•  acyl-coenzyme A oxidase 1 (Acox1), 5′-TGGTGGAT GCCTTTGACTTT-3′, 5′-GTGCTTGTGGTAAGATTCATG G-3′;
•  peroxisome proliferator-activated receptor alpha (Ppa ra), 5′-CGCAGGAAAGACTAGCAACA-3′, 5′-TGGCAGC AGTGGAAGAATC-3′; and
•  ribosomal protein lateral stalk subunit P0 (Rplp0), 5′ -CGAGAAGACCTCTTTCTTCCAA-3′, 5′-AGTCTTTATC AGCTGCACATCG-3′.

Relative mRNA levels were determined by calculating the ΔΔCt value, with Rplp0 as the normalization control.

Immunoblotting  Hepatic protein lysates were prepared using RIPA buffer (FUJIFILM Wako Pure Chemical Corporation) containing a protease inhibitor cocktail (cOmplete Mini; Roche Diagnostics, Mannheim, Germany). The lysates containing 30 μg of protein were subjected to immunoblotting as described previously (Shimada et al., 2017). As the primary antibodies, anti-KHK, anti-FASN antibody (GeneTex, Irvine, CA, USA), and anti-α-tubulin (as a loading control; Cell Signaling Technologies, Danvers, MA, USA) were used at dilutions of 1 / 10 000, 1 / 20 000, and 1 / 5 000, respectively. Anti-rabbit IgG and horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were used at a dilution of 1 / 5 000. The signals were detected using enhanced chemiluminescence (ECL) (Immunostar LD; FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer’s instructions and scanned using an ECL scanner (C-DiGit Blot Scanner; LI-COR, Lincoln, NE, USA).

Statistical analyses  Values are expressed as the mean ± standard error of the mean. Differences between the three groups were evaluated using a post-hoc Tukey’s test following a one-way analysis of variance. Pearson’s correlation coefficient (r) was used to assess the association between the hepatic expression levels of Mlxipl and fructolysis/fatty acid synthesis enzyme genes. p < 0.05 indicated statistical significance.

Results and Discussion

Body weight gain, food intake, and serum levels of glucose and insulin did not differ between rats fed the control, high-fructose, or high-fructose diets supplemented with NaB. In contrast, the hepatic TAG, serum TAG, and GLP-1 levels were substantially higher in rats fed the high-fructose diet than in the control diet group. However, hepatic TAG levels were substantially reduced by NaB supplementation (Table 1).

Table 1. Parameters of rats fed a control diet, high-fructose diet, or high-fructose diet supplemented with 5 % NaB for 15 d.

	Control	Fructose	Fructose + NaB
Body weight gain (g)	63.0 ± 3.0	63.9 ± 4.7	60.0 ± 2.9
Food intake (g/15 d)	212 ± 7	205 ± 7	195 ± 5
Liver
TAG (mg/g liver)	37.9 ± 3.9c	132 ± 11a	86.0 ± 12.7 b
Serum
TAG (mg/100 mL)	83.5 ± 13.2b	186 ± 22a	154 ± 24ab
Glucose (mg/100 mL)	129 ± 4	135 ± 3	136 ± 2
Insulin (ng/mL)	0.626 ± 0.391	0.538 ± 0.135	0.333 ± 0.095
GLP-1 (pM)	40.5 ± 3.3 b	69.9 ± 5.5 a	66.6 ± 12.2 ab

Values are presented as means ± standard error of the mean (n = 6). Values not sharing a common superscript letter are significantly different (p < 0.05, one-way analysis of variance followed by post hoc Tukey’s test). Control, rats fed a control diet; Fructose, rats fed a high-fructose diet; Fructose + NaB, rats fed a high-fructose diet supplemented with 5 % NaB. NaB, sodium butyrate; TAG, triacyglycerol; GLP-1, glucagon-like peptide-1

Hepatic expression levels of fructolysis enzyme genes (Khk, Aldob, Tkfc), fatty acid synthesis enzyme genes (Acaca, known as ACC1; Fasn; Scd), and Mlxipl, which encodes a lipogenic transcription factor, carbohydrate-responsive element-binding protein (ChREBP), were substantially increased in rats fed the high-fructose compared to the control diet. However, dietary supplementation with NaB decreased the high-fructose-diet-induced hepatic expression levels of these genes, especially Khk, Tkfc, Fasn, and Scd (p < 0.05) (Fig. 1A). Similar to the changes in the expression levels of these genes, NaB supplementation reduced the high-fructose-diet-induced hepatic expression levels of KHK and FAS (Fig. 1B). In contrast, the hepatic expression levels of Srebf1, which encodes another lipogenic transcription factor, sterol regulatory element-binding protein 1c (SREBP-1c); β-oxidation enzyme genes (Cpt1a; Cpt2; Acox1, known as ACO); and Ppara, which encodes a transcription factor (PPARα) that targets genes involved in β-oxidation, did not differ substantially among the three groups (Fig. 1A and 1C).

Fig. 1 Effects of dietary supplementation with sodium butyrate on hepatic expression of genes and proteins related to fructolysis, fatty acid synthesis, and fatty acid β-oxidation in rats fed a high-fructose diet. Hepatic expression of (A) genes related to fructolysis and fatty acid synthesis, (B) proteins related to fructolysis and fatty acid synthesis, and (C) genes related toβ-oxidation. Values are expressed as the mean ± standard error of the mean; n = 6 for (A) and (C), n = 5 for (B). Values not sharing a common superscript are significantly different (p < 0.05, Tukey’s test based on one-way analysis of variance). Control, rats fed a control diet; Fructose, rats fed a high-fructose diet; Fructose + NaB, rats fed a high-fructose diet supplemented with 5% sodium butyrate.

Although NaB supplementation has been reported to alleviate hepatic TAG accumulation in obese mice fed a Western diet drinking fructose (Beisner et al., 2021), this constitutes the first evidence that dietary supplementation with NaB reduces high-fructose-diet-induced hepatic accumulation of TAGs and the expression levels of genes and proteins related to both fructolysis and fatty acid synthesis pathways in rats. However, the mechanism through which NaB supplementation alleviated high-fructose-diet-induced TAG accumulation remains unclear.

ChREBP is generally activated in response to glucose efflux and upregulates the expression of genes related to fatty acid synthesis, such as Acaca and Fasn (Dentin et al., 2005). Studies, including ours, have reported that ChREBP is activated in response to fructose/sucrose signals rather than glucose/starch signals, and its activation is parallel to the upregulated expression of fatty acid synthesis enzyme genes (Acaca, Fasn, Scd) in rat livers (Janevski et al., 2012; Shimada et al., 2021). In addition, studies have shown that the high-fructose-induced expression of fructolysis enzyme genes (Khk, Aldob, Tkfc) was downregulated in the livers of rats with liver-specific ChREBP knockdown (Erion et al., 2013) and in the small intestines of ChREBP-knockout mice (Oh et al., 2018).

In the current study, the expression level of Mlxpil, which encodes ChREBP, was positively correlated with that of Khk, Aldob, Tkfc, Acaca, Fasn, and Scd (Pearson’s r = 0.651, r = 0.871, r = 0.762, r = 0.874, r = 0.730, and r = 0.812, respectively;p < 0.01). In addition, a recent study showed that high-sucrose diet-induced hepatic expression of both Mlxpil and fatty acid synthesis enzyme genes, such as Acaca and Fasn, was suppressed by NaB supplementation (Hattori et al., 2022). Therefore, a decrease in the high-fructose-induced expression of both fructolytic and fatty acid synthesis enzyme genes induced by NaB may be involved in the reduction of ChREBP activity. Further studies are needed to investigate whether fructose-induced ChREBP translocation into the nucleus and ChREBP binding to the promoters of both fructolytic and fatty acid synthesis enzyme genes are suppressed by NaB supplementation.

In vivo studies have demonstrated that treatment with NaB enhances β-oxidation, including increased expression levels and activity of CPT1 in mice with high-fat-diet-induced NAFLD, likely via the action of NaB as a histone deacetylase inhibitor (Den Besten et al., 2015; Sun et al., 2018). In contrast, we previously reported that dietary supplementation with NaB did not affect the hepatic expression levels of β-oxidation enzyme genes in rats fed a high-sucrose diet (Hattori et al., 2022). Similarly, in the present study, NaB supplementation did not substantially increase the expression levels of genes related to β-oxidation in the livers of rats fed a high-fructose diet. It has been reported that treatment with NaB decreases the expression levels of various genes and histone acetylation in human hepatocyte HepG2 cells (Rada-Iglesias et al., 2007). Our data support the abovementioned findings, and it is likely that the alleviative effect of NaB on hepatic TAG accumulation induced by high fructose/sucrose diets contributes to the reduced expression of fructolytic/fatty acid synthetic enzyme genes, accompanied by reduced acetylation of histones associated with these genes. Therefore, further studies are needed to investigate the relationship between NaB supplementation and the hepatic histone acetylation of fructolytic/fatty acid synthetic enzyme genes in an animal model of high-fructose diet-induced NAFLD.

Conclusions

We showed that dietary supplementation with NaB decreased the high-fructose-diet-induced hepatic accumulation of TAG and the expression of genes and proteins related to both fructolysis and fatty acid synthesis without affecting the expression levels of genes related to β-oxidation. These results suggest that NaB alleviates high-fructose diet-induced NAFLD by repressing a wide range of fructose pathways and subsequent fatty acid synthesis.

Acknowledgements  This work was supported by JSPS KAKENHI Grant Numbers JP21K02073, JP24K00351.

Conflict of interest  There are no conflicts of interest to declare.

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