Administration of a maple syrup extract to mitigate their hepatic inflammation induced by a high-fat diet: a transcriptome analysis

Abstract

Effects of the administration of maple syrup extract (MSX) on hepatic gene expression were investigated in mice fed a high-fat diet. Gene annotation enrichment analysis based on gene ontology revealed some changes in the expression of genes related to lipid metabolism and the immune response in MSX-fed mice. Detailed analysis of these data indicated that MSX ingestion mitigates hepatic inflammation.

Key words:

• maple syrup
• liver
• inflammation
• immune response
• lipid metabolism

Maple syrup as a concentrated sap of the sugar maple, Acer saccharum, is a popular sweetener consumed worldwide. We have shown that maple syrup has a significant liver-ameliorating function in male Wistar rats.¹⁾ In detail, rats have been fed a modified AIN93G diet in which sucrose powder (10%) and β-cornstarch (10%) are substituted with sucrose solution (20%) as a control and also fed maple syrup instead of the sucrose solution for comparison. Our conclusion is that the maple syrup diet compared with the standard diet can significantly reduce the levels of the serum–organ impairment markers AST and LDH. Hepatic gene expression profiles analyzed by DNA microarray have indicated that the decreases in the levels of these markers are probably caused by a regulation of the amino acid metabolism to produce ammonium as a hepatocyte toxin. This may be the first report that have shown a health benefit of maple syrup found to confer a liver-protecting effect in animals.¹⁾ Nagai et al.²⁾ have reported that oral administration of maple sugar results in lowering plasma glucose levels in 30- and 60-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Apostolidis et al.³⁾ showed that a butanol extract of maple syrup inhibits α-glucosidase in vitro, possibly by preventing elevation of plasma glucose levels in maple syrup-fed OLETF rats. Recent studies have also reported that maple syrup contains many antioxidative phenolic compounds,^4–7) such as quebecol.⁸⁾ Dietary polyphenols have various functional effects, including anti-obesity⁹⁾ and cancer prevention.¹⁰⁾ These studies suggest that phenolic compounds in maple syrup ameliorate various metabolic pathways. In our present work, we evaluated effects of a maple syrup extract (MSX, containing 1.61% polyphenols) on mice fed a high-fat diet (HFD).

MSX was prepared by NutraCanada (Quebec, Canada) according to previously published protocols.⁶⁾ Briefly, maple syrup (grade C-66°Brix; provided by the Federation of Maple Syrup Producers of Quebec, Canada) was diluted with deionized water and loaded to a resin column (XAD-16 Amberlite, Sigma-Aldrich, St. Louis, MO, USA). The column was then eluted with 90% ethanol and the eluate was concentrated by vacuum evaporation and the material was spray-dried. A total of 200 g of MSX was prepared from 50 L of maple syrup. The polyphenol concentration of MSX was measured as previously described,³⁾ with the result that it contained 1.61% polyphenols as gallic acid equivalents. Then, 3-week-old male C57BL/6J mice were purchased from Charles River, Japan (Kanagawa, Japan) and housed in a room maintained at 23 ± 1 °C and 49 ± 16% humidity with a 12-h light/dark cycle (light 08:00–20:00; dark 20:00–08:00). For a 1 week acclimation period after purchase, all the mice were fed a low-fat diet (LFD) (10 kcal% fat). Then, they were randomly divided into three dietary groups. The first group were fed a low-fat diet, (n = 8; 10 kcal% fat); the second group were fed a HFD, (n = 10; 45 kcal% fat); and the third were fed an HFD plus 0.06% MSX (n = 10). The composition of the LFD was as follows: casein, 18.96%; l-cystine, 0.28%; cellulose, 4.74%; lard, 4.27%; AIN93G mineral mixture, 4.27%; AIN93 vitamin mixture, 0.95%; choline bitartrate, 0.19%; sucrose, 33.18%; α-cornstarch, 3.32%; and β-cornstarch, 29.86%. The HFD contained the following ingredients: lard, 23.60%; sucrose, 20.14%; α-cornstarch, 11.65%; and β-cornstarch, 8.48%; the other ingredients were energetically made matched to the LFD. The mice were fed ad libitum for 8 weeks and then starved for 16 h prior to being sacrificed by anesthetic for dissection. All of the animal experimental protocols were approved by the Animal Use Committee of the Faculty of Agriculture at the University of Tokyo. Between-diet group differences were investigated for statistical significance using Tukey’s test. No significant between-diet difference was found in total energy intake, while body weight gain was significantly increased in the HFD group than in the LFD group (Table 1). Total RNA was extracted from the liver and DNA microarray analysis was performed on seven randomly selected samples from each group with a GeneChip^® 3′ IVT Express Kit and GeneChip^® Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA, USA), as previously described.¹¹⁾ All microarray data were submitted to the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/, GEO Series ID: GSE65667). The DNA microarray data (CEL files) were normalized by Factor Analysis for Robust Microarray Summarization,¹²⁾ and a between-diet comparison was performed by rank products.¹³⁾ Probe sets with significant gene expression changes (false discovery rate (FDR) < 0.01 each) were extracted according to expression patterns; specifically, those with expression changes in the HFD group compared with the LFD group that were reversed by MSX group were identified. In summary, 177 probe sets were up-regulated in the HFD group compared with the LFD group and down-regulated in the MSX group compared with the HFD group, and 171 were inversely correlated. These data sets were evaluated with the Database for Annotation, Visualization, and Integrated Discovery¹⁴⁾ to identify over-represented Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and in parallel with gene ontology (GO) terms in the biological process category for these genes. KEGG pathway with FDR¹⁵⁾-corrected p-values of less than 0.05, which was considered to be significantly enriched, was biosynthesis of unsaturated fatty acid (mmu01040). GO terms with FDR-corrected p-values of less than 0.05 were applied to QuickGO¹⁶⁾ to map the terms hierarchically (Table 2). The genes involved in the GO terms in the deepest in the hierarchy included lipid metabolic process (GO:0006629) and immune response (GO:0006955), are listed Table 3.

Table 1. Body weights, serum biochemical parameters, and hepatic biochemical parameters for the three groups, LFD, HFD, and MSX^a.

	LFD	HFD	MSX
Final body weight (g)	29.01 ± 0.70^a	32.66 ± 1.31^b	31.88 ± 0.72^ab
Body weight gain (g)	13.18 ± 0.85^a	16.46 ± 0.99^b	15.90 ± 0.57^ab
Liver weight (g)	1.113 ± 0.032	0.998 ± 0.104	1.058 ± 0.025
Serum biochemical parameters
Triacylglycerol (mg/dL)	25.75 ± 7.95^a	71.60 ± 9.08^b	73.2 ± 10.38^b
Total cholesterol (mg/dL)	124.3 ± 4.2	147.2 ± 7.5	145.2 ± 7.1
Glucose (mg/dL)	177.5 ± 16.8	185.0 ± 14.1	188.0 ± 8.3
Glycoalbumin (%)	3.14 ± 0.17	3.54 ± 0.07	3.43 ± 0.12
Hepatic biochemical parameters
Triacylglycerol (mg/g wet liver)	79.30 ± 12.02	93.5 ± 13.97	76.36 ± 4.41
Total cholesterol (mg/g wet liver)	4.08 ± 0.19^a	2.79 ± 0.19^b	2.66 ± 0.07^b

^a LFD: low-fat diet (n = 8), HFD: high-fat diet (n = 10), and MSX: MSX-added high-fat diet (n = 10). Each value is the mean ± SEM. Values not sharing a common letter are significantly different at p < 0.05.

Table 2. GO terms significantly enriched (FDR-corrected p < 0.05) among the 177 probe sets that were up-regulated in the HFD group compared with the LFD group and down-regulated in the MSX group compared with the HFD group, 171 of which were inversely correlated.

Table 3. Genes related to the GO terms of lipid metabolic process and immune response.

Affymetrix ID	RefSeq transcript ID	Gene name
(A) Down-regulated in the HFD group compared with the LFD group and up-regulated in the MSX group compared with the HFD group
Lipid metabolic process
1422996_at	NM_134188	Acyl-CoA thioesterase 2
1422925_s_at	NM_134246	Acyl-CoA thioesterase 3
1417130_s_at	NM_020581	Angiopoietin-like 4
1442277_at	NM_001025566 /// NM_013490	Choline kinase alpha
1418780_at	NM_018887	Cytochrome P450, family 39, subfamily a, polypeptide 1
1424943_at	NM_001252539 /// NM_201640	Cytochrome P450, family 4, subfamily a, polypeptide 31
1417403_at	NM_130450	ELOVL family member 6, elongation of long-chain fatty acids (yeast)
1416021_a_at	NM_010634 /// XR_105655	Fatty acid-binding protein 5, epidermal /// predicted gene 3601
1423680_at	NM_146094	Fatty acid desaturase 1
1449325_at	NM_019699	Fatty acid desaturase 2
1421011_at	NM_053262	Hydroxysteroid (17-beta) dehydrogenase 11
1456156_at	NM_001122899 /// NM_010704 /// NM_146146	Leptin receptor
1418288_at	NM_001130412 /// NM_015763 /// NM_172950	Lipin 1
1424715_at	NM_026159	Retinol saturase (all-trans-retinol 13,14-reductase)
1415964_at	NM_009127	Stearoyl-coenzyme A desaturase 1
1448506_at	NM_007618	Serine (or cysteine) peptidase inhibitor, clade A, member 6
Immune response
1417761_at	NM_007468	Apolipoprotein A-IV
1421818_at	NM_009744	B-cell leukemia/lymphoma 6
1453851_a_at	NM_011817	Growth arrest and DNA-damage-inducible 45 gamma
1421653_a_at	NM_001024700	Immunoglobulin heavy chain 2 (serum iga) /// immunoglobulin heavy chain (J558 family) /// similar to immunoglobulin mu-chain
1420515_a_at	NM_021319	Peptidoglycan recognition protein 2
1429399_at	NM_026301	Ring finger protein 125
(B) Up-regulated in HFD group compared with LFD group and down-regulated in MSX group compared with HFD group
Lipid metabolic process
1457403_at	NM_001033819	RIKEN cDNA 9130409I23 gene
1456081_a_at	NM_030210	Acetoacetyl-CoA synthetase
1418601_at	NM_011921	Aldehyde dehydrogenase family 1, subfamily A7
1417257_at	NM_009885	Carboxyl ester lipase
1415805_at	NM_025469	Colipase, pancreatic
1423804_a_at	NM_145360	Isopentenyl-diphosphate delta isomerase
1449385_at	NM_013786	Hydroxysteroid (17-beta) dehydrogenase 6
1427377_x_at	NM_001012306 /// NM_001161742 /// NM_001161743 /// NM_001161744 /// NM_001161745	Hydroxy-delta-5-steroid dehydrogenase, 3-beta- and steroid delta-isomerase 3
1451737_at	NM_001024955 /// NM_001077495	Phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 (p85 alpha)
1437015_x_at	NM_011107	Phospholipase A2, group IB, pancreas
1433431_at	NM_026925	Pancreatic lipase
1415777_at	NM_018874	Pancreatic lipase-related protein 1
1437438_x_at	NM_011128	Pancreatic lipase-related protein 2
1419075_s_at	NM_009117	Serum amyloid A 1
1423078_a_at	NM_025436	Sterol-C4-methyl oxidase-like
1418940_at	NM_019878	Sulfotransferase family 1B, member 1
Immune response
1417867_at	NM_013459	Complement factor D (adipsin)
1419697_at	NM_019494 /// NR_038116	Chemokine (C-X-C motif) ligand 11
1418652_at	NM_008599	Chemokine (C-X-C motif) ligand 9
1436562_at	NM_172689	DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
1420549_at	NM_010259 /// XM_003086912	Guanylate-binding protein 1 /// interferon-induced guanylate-binding protein 1-like
1435906_x_at	NM_010260	Guanylate-binding protein 2
1418392_a_at	NM_018734	Guanylate-binding protein 3
1438676_at	NM_194336	Guanylate-binding protein 6
1434380_at	NM_001083312 /// NM_145545	Guanylate-binding protein 7
1425477_x_at	NM_207105	Histocompatibility 2, class II antigen A, beta 1
1426276_at	NM_001164477 /// NM_027835	Interferon induced with helicase C domain 1
1418825_at	NM_008326	Immunity-related GTPase family M member 1
1424339_at	NM_145209	2′-5′ oligoadenylate synthetase-like 1
1420603_s_at	NM_009016 /// NM_009017 /// NM_009018 /// NM_020030 /// NM_198193 /// XM_001006217	Retinoic acid early transcript 1, alpha /// retinoic acid early transcript beta /// retinoic acid early transcript gamma /// retinoic acid early transcript delta /// retinoic acid early transcript 1E
1424923_at	NM_009251	Serine (or cysteine) peptidase inhibitor, clade A, member 3G
1449009_at	NM_001145164 /// NM_011579	T cell-specific GTPase 1 /// T cell-specific GTPase 2
1422701_at	NM_009539	Zeta-chain (TCR) associated protein kinase

For the GO term lipid metabolism, genes involved in fatty acid desaturation and elongation, acyl-CoA thioesterases (Acot2 and Acot3), stearoyl-coenzyme A desaturase 1 (Scd1), fatty acid desaturases (Fads1 and Fads2), ELOVL family member 6, elongation of long-chain fatty acids (yeast) (Elovl6) were down-regulated in the HFD group and up-regulated in the MSX group (Table 3A). These genes are also included in KEGG pathway of biosynthesis of unsaturated fatty acid. Because SCD1 is the rate-limiting enzyme in the synthesis of oleate, usable to produce triacylglycerol,¹⁷⁾ its up-regulation indicates that the accumulation of triacylglycerol was accelerated in the livers of the mice fed the MSX diet. However, the gene expression of glycerol-3-phosphate acyltransferase, mitochondrial (Gpam), members of 1-acylglycerol-3-phosphate O-acyltransferase (Agpat), and members of diacylglycerol O-acyltransferase (Dgat), which are related to the de novo synthesis of triacylglycerol, did not change in MSX group. Also, the hepatic triacylglycerol level did not change in the MSX group (Table 1). These data indicate that the up-regulation of Scd1 was not for the de novo synthesis of triacylglycerol, while it would reduce the levels of saturated fatty acids which may have cytotoxicity.¹⁸⁾

A total of 17 genes assigned to the GO term for immune response were up-regulated in the HFD group and down-regulated in the MSX group, and six of these genes were related to protection against infection (Table 3B). These include members of the guanylate-binding protein family (Gbp1, Gbp2, Gbp3, Gbp6, and Gbp7),^19,20) immunity-related GTPase family M member 1 (Irgm1),²¹⁾ 2′-5′ oligoadenylate synthetase-like 1 (Oasl1).²²⁾ The other three of genes assigned to the immune response GO term, including chemokine (C-X-C motif) ligand 9 (Cxcl9),²³⁾ chemokine (C-X-C motif) ligand 11 (Cxcl11),²³⁾ and immunity-related GTPase family M member 1 (Irgm1),^24,25) are involved in inflammation. These data indicated that MSX ingestion mitigated liver inflammation induced by HFD consumption. Also, other inflammation markers, including serum amyloid A 1, 2, and 3 (Saa1, Saa2, and Saa3)²⁶⁾ and orosomucoid 2 (Orm2)²⁷⁾ which were not annotated with the immune response GO term, were down-regulated in the MSX group.

All these data suggest that MSX is effective in mitigating the hepatic inflammation induced by the consumption of a HFD.

Author contribution

A.K., S.A., and K.A. designed the experiments. A.K., Y.W., and F.S. performed the experiments. A.K. performed gene expression analysis. The manuscript was written by A.K. All authors participated in the discussion of the data and in the production of the final version of the manuscript.

Disclosure statement

All the authors do not have any potential conflict of interest.

Additional information

Funding

This project was supported by Ministère de l’Agriculture, des Pêcheries et de l’Alimentation of Quebec (MAPAQ) through the “Soutien aux strategies sectorielles de développement Volet 1: Appui au développement sectorial” program with the participation of the Federation of Quebec Maple Syrup Producers (FPAQ); Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries”; Regional Innovation Strategy Support Program.

Notes

Abbreviations: MSX, maple syrup extract; qFARMS, Factor Analysis for Robust Microarray Summarization; FDR, false discovery rate; RP, rank products; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, gene ontology.