A maple syrup extract alters lipid metabolism in obese type 2 diabetic model mice
Some polyphenols are known to improve the symptoms of diabetes. In the present study, we investigated the effects of a polyphenol-rich extract of maple syrup (MSx) on a diabetic mouse model.
KK-Ay mice were fed a normal or 0.05% MSx-supplemented diet for 42 days. Body weight, food intake, serum biochemical parameters, and fecal total bile acid were measured. Gene expression of liver and epididymal white adipose tissue (WAT) and cecal microbiota were analyzed. Data were analyzed with an unpaired two-tailed Student’s t test or Welch’s t test according to the results of the F test.
Serum low-density lipoprotein cholesterol levels were significantly reduced in mice that consumed MSx. Hepatic genes related to fatty acid degradation and cholesterol catabolism were upregulated in mice that consumed MSx. In contrast, the expression of genes related to lipid metabolism in WAT was unaffected by the intake of MSx. There were no significant differences between the two groups in terms of total bile acid level in the feces and the relative abundance of bacteria in the cecum.
Our results primarily indicate that MSx can help alleviate one of the symptoms of dyslipidemia.
KeywordsDiabetes Lipid metabolism Maple syrup Polyphenol
Central Institute for Experimental Animals
Database for Annotation, Visualization and Integrated Discovery
Differentially expressed gene
False discovery rate
Homeostasis model assessment of insulin resistance
Kyoto Encyclopedia of Genes and Genomes
Maple syrup extract
Nonesterified fatty acid
Operational taxonomic unit
Tumor necrosis factor
terminal restriction fragment length polymorphism
Universal Protein Resource Knowledgebase
Very low-density lipoprotein
White adipose tissue
The increasing prevalence of diabetes is a global problem . Diabetes mellitus is characterized by chronic hyperglycemia due to defects in insulin production and/or the response to insulin. Diabetes is classified as type 1 or 2 according to the underlying cause of these defects: type 1 results from the destruction of pancreatic β cells, whereas type 2 is due to environmental factors such as obesity, stress, and lack of exercise as well as genetics. Diabetes patients often have dyslipidemia, which is a risk factor for developing atherosclerotic cerebrovascular and cardiovascular diseases. Dyslipidemia is a pathology that promotes triglyceride degradation in adipose tissue and very low-density lipoprotein (VLDL) synthesis in the liver and suppresses VLDL catabolism in the blood due to insulin resistance. Some polyphenols are known to improve the symptoms of diabetes . Brown alga polyphenols reduced nonfasting blood glucose levels in KK-Ay mice, a model for type 2 diabetes accompanied by obesity, and acacia polyphenols improved dyslipidemia and insulin resistance in KK-Ay mice [3, 4].
Maple syrup is a sweetener made from the sap of the sugar maple tree, Acer saccharum, which contains a large number of polyphenols . A butanol extract of maple syrup polyphenols was shown to inhibit α-amylase and α-glucosidase activities in vitro . Inhibiting these carbohydrate hydrogenases is expected to prevent an increase in blood glucose levels by delaying the absorption of carbohydrates. Additionally, the ethanol extract suppressed the production of nitric oxide and prostaglandin E2, a substance that induces inflammation, in RAW 264.7 cells activated by lipopolysaccharides . In our previous study, we reported the effect of the ethanol extract on hepatic gene expression in C57BL/6 J mice fed a high-fat diet . Maple sugar, which is produced by boiling and drying maple syrup, was shown to elevate blood glucose levels to a lesser degree than sucrose in Otsuka Long-Evans Tokushima Fatty rats, a type 2 diabetes model .
Based on the above reports, we speculated that maple syrup, particularly its polyphenol-enriched extract, could alleviate chronic hyperglycemia in individuals with diabetes. In the present study, we used KK-Ay mice and fed them an ethanol extract of maple syrup (MSx) containing 15.02% polyphenols as gallic acid equivalents for 42 days. Then, serum biochemical parameters related to nutrient metabolism, gene expression profiles of liver and epididymal white adipose tissue (WAT), and composition of intestinal bacteria were evaluated to determine the effects of MSx on nutrient metabolism, with the results demonstrating that MSx alters lipid metabolism.
MSx prepared from Canadian maple syrup (Canada No. 2/Amber) by SiliCycle Inc. (Quebec, QC, Canada) was purchased by the Federation of Quebec Maple Syrup Producers (FPAQ; Longueuil, QC, Canada) . Briefly, maple syrup was diluted with deionized water and was applied to an Amberlite XAD16 column (Sigma-Aldrich, St. Louis, MO, USA). The non-adsorbed fraction was eluted with deionized water and discarded. The adsorbed fraction was eluted with ethanol and evaporated in vacuo in a rotary evaporator. MSx refers to this dried fraction.
Animals and diets
AIN-93G mineral mix
AIN-93 vitamin mix
Eight serum biochemical parameters, including glucose, glycated albumin, total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein cholesterol (HDL), triglycerides, nonesterified fatty acids (NEFA), and total ketone bodies, were measured on a 7180 Clinical Analyzer (Hitachi High-Technologies, Tokyo, Japan) by Oriental Yeast Co. Serum insulin and tumor necrosis factor (TNF)-α levels were measured with a Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science, Kanagawa, Japan) and a Mouse TNF-α Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA), respectively. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula:
HOMA-IR = insulin (ng/ml) × 26 μIU/ml × glucose (mg/dl).
Total cholesterols were isolated as total lipids from the liver according to the Folch method  and its levels were measured with the Cholesterol E-Test Wako (Wako Pure Chemical Industries, Osaka, Japan). The total bile acid level in the feces was measured with the Total Bile Acid Test Wako (Wako Pure Chemical Industries) as follows. Feces were lyophilized and ground with a mortar. A 5-fold volume of ethanol was added to the feces prior to heating at 70 °C for 1 h and centrifugation at 3500×g for 15 min, setting the supernatant aside. The same volume of ethanol was again added to the pellets and centrifuged as described above to set the supernatant aside. This procedure was repeated twice. The three supernatants were pooled for measurement.
Data were analyzed with the unpaired two-tailed Student’s t test or Welch’s t test according to the results of the F test. Differences were considered significant at P < 0.05.
Analysis of gene expression in the liver and WAT using DNA microarray
Total RNA was isolated from the liver and WAT with TRIzol Reagent (Thermo Fisher Scientific Inc.) and was purified with an RNeasy Mini Kit and RNase-Free DNase Set (Qiagen, Venlo, the Netherlands). Total RNA concentration was measured on a spectrophotometer. RNA integrity was evaluated with an Agilent RNA 6000 Nano Kit and on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA Integrity Number was confirmed greater than 8.0. cRNA was prepared from purified 100 ng total RNA with the GeneChip 3′ IVT PLUS Reagent Kit and hybridized to a GeneChip Mouse Genome 430 2.0 Array (Thermo Fisher Scientific Inc.). The array was stained using a GeneChip Hybridization, Wash and Stain Kit and a GeneChip Fluidics Station 450 (Thermo Fisher Scientific Inc.). The fluorescence signals of the probes were scanned with a GeneChip Scanner 3000 7G (Thermo Fisher Scientific Inc.) and converted to an intensity value with Affymetrix GeneChip Command Console software (Thermo Fisher Scientific Inc.).
The intensity values of probe sets were normalized by the distribution-free weighted method in the case of liver and by the quantile normalization factor analysis for robust microarray summarization method in the case of WAT [12, 13]. Data were compared between the control and MSx groups using the rank products method . Probe sets with a false discovery rate (FDR) < 0.001 in the liver were considered to show significant differences in expression. The threshold of FDR in the WAT was set to < 0.02 to obtain an approximately equal number of probe sets in the liver. Probe sets sorted as both up- and downregulated were excluded, and the genes for the remaining probe sets were treated as differentially expressed genes (DEGs). Normalization and between-group comparisons were performed with R v.3.2.2  and Bioconductor v.3.1 . DEGs were annotated according to biological process in Gene Ontology (GO) terms, which were enriched using the Database for Annotation, Visualization and Integrated Discovery v.6.7 (DAVID) . Overrepresented GO terms were evaluated according to a modified Fisher’s exact test p value . FDR was calculated from the p value using the Benjamini and Hochberg method . GO terms in the liver and WAT with FDR < 0.01 were regarded as significantly enriched. The hierarchical structure of GO terms was determined with QuickGO . DEGs annotated with GO terms related to lipid metabolism were mapped to metabolic pathways by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in the DAVID browser. The detailed functions of DEGs annotated with GO terms related to the immune system were searched with Universal Protein Resource Knowledgebase (UniProtKB) . Ensemble IDs were assigned to DEGs with the annotation file Mouse 430 2 Annotations Release 35 .
Quantification of expression level of hepatic genes related lipid metabolism by qRT-PCR
Total RNA (1 μg) isolated from the liver as described above was reverse transcribed with a SuperScript IV VILO Master Mix (Thermo Fisher Scientific Inc.). The synthesized cDNA (1 ng) was amplified in a 10-μL reaction volume with a PowerUp SYBR Green Master Mix (Thermo Fisher Scientific Inc.) on a CFX Connect Real-Time PCR Detection System with CFX Maestro 1.1 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) under the following conditions: 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Primers were designed with Primer3web v.4.1.0 (http://primer3.ut.ee/) and these sequences are shown in Additional file 1. The primer specificity was confirmed using dissociation curve. Expression level was measured using the calibration curve and that of each gene was normalized by that of actin, beta (Actb) whose expression showed the smallest differences between the two groups among 5 housekeeping genes. PCR reactions were performed with 3 technical replicates for each gene. DNA contamination was not detected.
Analysis of cecal bacterial composition
The cecal microbiota was evaluated by the Central Institute for Experimental Animals (CIEA; Kanagawa, Japan) using a modified terminal restriction fragment length polymorphism (T-RFLP) method . Briefly, genomic DNA was isolated from cecal contents and amplified with fluorophore-labeled primer sets designed for amplifying 16S rDNA. The amplified product was digested with a restriction enzyme, HpyCH4III, and analyzed on an ABI PRISM 310 Genetic Analyzer with GeneScan software (Thermo Fisher Scientific Inc.). Fragment length was assigned to an operational taxonomic unit (OTU), which is specific to bacteria in a microbiota database constructed by the CIEA. The OTU area was regarded as the number of bacteria.
Physical and biochemical parameters
Physical and biochemical parameters of mice fed a normal or MSx-supplemented diet
Total food intake (g)
292.3 ± 7.1
293.3 ± 9.6
Final body weight (g)
36.9 ± 0.5
37.0 ± 0.5
Liver weight (g)
1.6 ± 0.0
1.6 ± 0.0
Serum biochemical parameters
Glycated albumin (%)
8.3 ± 0.5
7.6 ± 0.5
77.0 ± 17.0
87.0 ± 11.0
2.1 ± 0.4
1.5 ± 0.2
10.0 ± 2.6
8.8 ± 2.1
Total cholesterol (mg/dl)
117.0 ± 5.0
114.0 ± 5.0
LDL cholesterol (mg/dl)
9.0 ± 1.0
6.0 ± 1.0*
HDL cholesterol (mg/dl)
64.0 ± 4.0
67.0 ± 3.0
98.0 ± 10.0
105.0 ± 14.0
619.0 ± 69.0
750.0 ± 99.0
Total ketone body (μmol/l)
452.0 ± 69.0
567.0 ± 61.0
11.5 ± 2.5
7.4 ± 0.4
Liver biochemical parameters
Total cholesterol (mg/g liver)
5.5 ± 0.3
6.0 ± 0.3
Fecal biochemical parameter
Total bile acid (μmol/g feces)
1.6 ± 0.1
1.6 ± 0.1
Changes in the gene expression of the liver
GO terms annotated to genes differentially expressed in the liver
Number of genes
Steroid metabolic process
Fatty acid metabolic process
└ Inflammatory response
└ Acute inflammatory response
└ Acute-phase response
Response to wounding
Inflammation leads to the aggravation of diabetes symptoms by activating the immune system . To examine the effect of MSx on the immune system, the biological function of the DEGs related to the immune system was examined using UniProtKB. Acute phase proteins encoded by orosomucoid 1/ 2/ 3 (Orm1/ 2/ 3) and serum amyloid A 1/ 2/ 4 (Saa1/ 2/ 4) are synthesized in the liver in response to inflammatory cytokines . These DEGs were downregulated in the MSx group (Additional file 3). This result suggests that the inflammatory response was influenced by the intake of MSx.
Changes in the gene expression of the WAT
GO terms annotated to genes differentially expressed in WAT
Number of genes
└ Inflammatory response
Response to wounding
Response to organic substance
Bacterial composition of cecal contents in mice fed a normal or MSx-supplemented diet
OTU area (%)
16.13 ± 1.58
19.00 ± 1.30
11.80 ± 1.28
10.09 ± 1.28
47.89 ± 1.83
47.17 ± 3.21
7.14 ± 1.91
6.76 ± 3.12
66.84 ± 1.18
64.02 ± 1.01
1.83 ± 0.07
1.75 ± 0.11
0.37 ± 0.08
0.31 ± 0.12
2.83 ± 0.29
2.45 ± 0.26
12.02 ± 0.53
12.48 ± 0.60
17.04 ± 0.78
16.98 ± 0.74
Various parameters (e.g. insulin, NEFA, and total ketone body in serum) in this study showed the large variance, which may be derived from an individual difference of KK-Ay mice. The results of qRT-PCR analysis were also affected by an individual difference, especially C1 in the control group. The effects of MSx intake on lipid metabolism of KK-Ay mice will be accurately revealed by increasing sample size.
In the context of diabetes, including in KK-Ay mice, the catabolism of lipoproteins such as VLDL and LDL is suppressed due to insulin resistance, resulting in the elevation of these lipoprotein cholesterol levels in blood , which is one of the pathologies of dyslipidemia. Our finding that serum LDL cholesterol level was lowered by MSx intake suggests that MSx can help alleviate one of the symptoms of dyslipidemia.
Using gene expression analysis of the liver and WAT, this study showed the possibility that MSx intake may affect the immune system. However, the results demonstrating the effect of MSx intake at the protein level could not be shown. Thus, further study is required to understand the effect of MSx intake on the immune system.
The present study examined the effect of MSx intake in obese diabetic mice. The intake of MSx does not influence chronic hyperglycemia, but it reduces the LDL cholesterol level. Hepatic gene expression analysis suggested that the intake of MSx promotes cholesterol catabolism, although further studies are required to identify the detailed mechanism underlying the reduction in LDL cholesterol levels. Our findings provide evidence that MSx contributes to alleviating one of the symptoms of dyslipidemia.
We gratefully acknowledge the work of past and present members of our laboratory.
KA, AK, and TT formulated the research question. SO, TI, and TT designed the study. TI and TT performed the experiments. TT analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.
This study was supported in part by Ministère de l’Agriculture, des Pêcheries et de l’Alimentation of Quebec through the “Soutien aux strategies sectorielles de développement Volet 1: Appui au développement sectorial” program with the participation of the FPAQ. MSx was provided by the FPAQ from this program. Our study was also supported by a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (26242007 and 15 K12334). These bodies had no role in the design, analysis, or writing of this manuscript.
Protocols for the animal experiment were approved by the Animal Use Committee of the Faculty of Agriculture, The University of Tokyo (permit no: P15–6).
Consent for publication
The authors declare that they have no competing interests.
- 4.Ikarashi N, Toda T, Okaniwa T, Ito K, Ochiai W, Sugiyama K. Anti-obesity and anti-diabetic effects of acacia polyphenol in obese diabetic KKAy mice fed high-fat diet. Evidence-based Complement Altern Med. 2011;2011.Google Scholar
- 15.The Comprehensive R Archive Network. https://cran.r-project.org/. .
- 16.Bioconductor. http://www.bioconductor.org/. .
- 17.DAVID Bioinformatics Resources 6.7. https://david-d.ncifcrf.gov/. .
- 19.Benjamini Y, Hochberg Y. Controlling the false discovery rate : a practical and powerful approach to multiple testing. J R Stat Soc Ser B. 1995;57(1):289–300.Google Scholar
- 20.QuickGO. http://www.ebi.ac.uk/QuickGO/. .
- 21.UniProt. http://www.uniprot.org/. .
- 22.GeneChip Array Annotation Files. http://www.affymetrix.com/support/technical/annotationfilesmain.affx. .
- 24.Kitada M, Ogura Y, Monno I, Koya D. Sirtuins and type 2 diabetes: role in inflammation, oxidative stress, and mitochondrial function. Front Endocrinol. 2019;10(March):1–12.Google Scholar
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