Orally Administered Baker's Yeast Î²-Glucan Promotes Glucose and

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Orally Administrated Baker’s Yeast beta-Glucan Promotes Glucose and Lipid Homeostasis in the Livers of ob/ob Mice Yan Cao, Ying Sun, Siwei Zou, Mengxia Li, and Xiaojuan Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03782 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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Journal of Agricultural and Food Chemistry

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Orally Administrated Baker’s Yeast β-Glucan Promotes Glucose and Lipid

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Homeostasis in the Livers of ob/ob Mice

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Yan Cao, Ying Sun, Siwei Zou, Mengxia Li, and Xiaojuan Xu*

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College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.

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*

Corresponding author: Xiaojuan Xu, Tel/Fax: + 86 27 68754188, E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT: Baker’s yeast glucan (BYG) has been reported to be anti-diabetic. Herein, further study

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on the effect of orally administrated BYG on glucose and lipid homeostasis in the livers of ob/ob mice

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was performed. It was found that BYG decreased the blood glucose and the hepatic glucose and lipid

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disorders. Western blotting analysis revealed that BYG up-regulated p-AKT and p-AMPK, and

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down-regulated p-Acc in the liver. Furthermore, RNA-Seq analysis indicated that BYG down-regulated

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genes responsible for gluconeogenesis (G6pase and Got1), fatty acid biosynthesis (Acly, Acc, Fas, etc.),

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glycerolipid synthesis (Gpam and Lipin1/2) and cholesterol synthesis (Hmgcr, Fdps, etc.). Additionally,

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BYG decreased glucose transporters SGLT1 and GLUT2, fat emulsification, and adipogenic genes/

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proteins in the intestine to decrease glucose and lipid absorption. All these findings demonstrated that

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BYG is benefit for regulate glucose and lipid homeostasis in diabetic mice, and thus has potential

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applications in anti-diabetic foods or drugs.

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KEYWORDS: Yeast β-glucan, type 2 diabetes, liver, blood glucose, lipid metabolism

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INTRODUCTION

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Diabetes, characterized by metabolic disorders and various complications such as cardiovascular

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risks, renal disease and blindness, causes severe morbidity and mortality.1 According to the statistics

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from International Diabetes Federation, the number of diabetic cases is predicted to reach approximately

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642 million by 2040, that is, one person will suffer from diabetes every 10 persons.2 The severe situation

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is driving the anti-diabetic pharmaceutical frenzy. Moreover, many synthetic anti-diabetic drugs have

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aroused public concerns over their side effects and adverse events.3 The interest in safe anti-diabetic

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natural products is increasing.

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Polysaccharides, as the third biomacromolecules, have long been underappreciated by the scientific

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community compared with proteins and nucleic acids. As is well known, polysaccharides have been

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found to show various bioactivities such as anti-tumor, anti-inflammation, and so forth.4, 5 And more and

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more natural polysaccharides have been reported to be potential anti-diabetic agents. During the period

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of 2011-2015, 114 types of polysaccharides from 78 kinds of natural sources such as plants, fungi, algae,

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bacteria, etc., were found to show anti-diabetic effect.6 Actually, in China, several polysaccharide

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products such as astragalus, ginseng and pumpkin polysaccharides have been used as the alternative

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medicines to treat diabetes in clinic.7 Hence, polysaccharides are one of potential candidates as an

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anti-diabetic component. However, the vast majority of anti-diabetic polysaccharides have not been fully

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scientifically validated and utilized due to their complex structure and undefined mechanisms. Therefore,

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more intensive researches on the anti-diabetic mechanism of polysaccharides are extremely urgent for

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rational use. 3



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The liver is one of the most important organs for glucose and lipid metabolism. Very recently, we

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have demonstrated that the purified Baker’s yeast β-(1→3)-glucan, herein denoted as BYG, shows

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significant

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streptozotocin-HFD co-induced type 2 diabetic mice.8 Interestingly, BYG promotes glycogen synthesis

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and inhibits fat accumulation in the liver remarkably. Therefore, in this study, a more comprehensive

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study on the glucose and lipid homeostasis in the liver was carried out in the classic animal models of

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obesity and type 2 diabetes (T2D) , ob/ob mice, which have similar disease characteristics to those in

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human T2D,9, 10 to further explore the possible hypoglycemic mechanism of BYG. In particular, the

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RNA-Seq technology, a powerful method for profiling the global transcriptome, was performed to

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inspect transcription profile alterations initiated by BYG in the livers of ob/ob mice. This work provides

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more detailed information at the protein and gene levels concerning what happened in the liver after oral

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administration of BYG in ob/ob mice.

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anti-hyperglycemic

effect

in

high-fat

diet

(HFD)-induced

obese

mice

and

MATERIALS AND METHODS

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Samples. BYG used in this work was the same glucan as the one in our recently reported work,

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which is a water-insoluble linear β-(1→3)-glucan with purity of 99% and viscosity-average molecular

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weight (Mη) of ∼25 kD from Baker’s yeast.8

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Animals and Experimental Protocols. Male ob/ob mice (C57BLKS.B6.V-Lepob/Nju, 11~12

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weeks old, 48~51 g) were purchased from Nanjing Biomedical Research Institute of Nanjing University.

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The mice were fed with free intake of water and maintenance diets (Beijing HFK Bioscience Co. Ltd) as

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shown in supplemental Table S1. All animal protocols were approved by the Animal Research 4


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Committee of Zhongnan Hospital of Wuhan University (Hubei Province, China).

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The ob/ob mice were divided into two groups with 7 mice in each group randomly. The

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BYG-treated mice (denoted as BYG group) were orally given 25 mg/kg/d BYG, and the untreated ob/ob

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mice were given equal volume of water (denoted as the control group or ob/ob group). The fasting blood

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glucose (FBG) levels after starvation from 8 a.m. to 14 p.m. were measured at the indicative time. 4~5

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weeks later, ob/ob mice were sacrificed by cervical dislocation after starvation overnight, and the liver

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and intestine tissues were kept at −86℃ before use.

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Oral Glucose Tolerance Test (OGTT). OGTT was carried out on day 15 after BYG treatment. The detailed description was provided in Supplemental Materials.

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Western Blotting Analysis. The protein expression of glucose transporter 2 (GLUT2),

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phosphorylated (at Ser473) protein kinase B (p-AKT), phosphorylated adenosine 5’-monophosphate

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(AMP)-activated protein kinase (p-AMPK, α1 at Thr183 and α2 at Thr172), phosphorylated acetyl-CoA

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carboxylase (p-Acc), fatty acid synthase (Fas), sodium glucose co-transporter 1 (SGLT1) and β-actin in

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L02 cells treated with 100 µg/mL of BYG for 12 h , the livers and jejunal segments from ob/ob mice

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treated with 25 mg/kg/d BYG for 4~5 weeks were analyzed by western blotting. The detailed

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description was provided in Supplemental Materials.

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Quantitative Real Time RT-PCR Assay. The detailed description of the quantitative RT-PCR

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assay for the liver and jejunal segments was provided in Supplemental Materials, and the sequences of

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primers used were listed in supplemental Table S2.

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RNA-Seq Analysis. Total RNA was extracted from livers of mice in the ob/ob and BYG groups

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(two mice in each group). The transcriptome sequencing and bioinformatics analysis were carried out in 5



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BGI Co., Ltd (China). The mRNA was purified and used for cDNA library preparation. Sequencing was

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performed on the BGISEQ-500RS developed by BGI. The raw RNA-seq data were filtered into clean

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reads, followed by mapping to the mouse reference genome (mm10) using HISAT. The gene

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quantification was analyzed using RSEM quantification tool, and the expression level of each gene was

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calculated in fragments per kilobase of exon per million fragments mapped. The NOISeq method was

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used to screen the differentially expressed genes (DEGs) between the control and BYG groups

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according to the criteria of fold change ≥ 2 and diverge probability ≥ 0.8. The gene ontology (GO)

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annotation mapped all DEGs to GO terms in the database (http://www.geneontology.org/), and GO terms

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fulfilling Qvalue (the corrected Pvalue) ≤ 0.05 were defined as significantly enriched ones in DEGs. The

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public KEGG database was used to perform the pathway enrichment analysis, and the pathway terms

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fulfilling Qvalue ≤ 0.05 were defined as significantly enriched ones.

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Pyruvate Tolerance Test (PTT) in ob/ob Mice. Male ob/ob mice (7~8 weeks old, 29~34 g) on

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maintenance diet were orally administrated with BYG (25 mg/kg/d, BYG group, n = 7) or equal volume

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of water (ob/ob group, n = 7). After treatment for 25 days, ob/ob mice were fasted for 16 h and were

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intraperitoneally injected (i.p.) with pyruvate at a dose of 1.5 g/kg. Blood glucose levels were measured

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before the injection (as 0 min) and at 15, 30 and 60 min after the injection.

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Fat Tolerance Test. Male Sprague Dawley (SD) rats (7 weeks old, 250 g) were purchased from

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Hubei Research Center of Laboratory Animals and fed with a normal chow diet. After starvation for 6 h,

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BYG solution (50 mg/kg, BYG group, n = 5) or water (equal volume, the control group, n = 5) were

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given orally. 0.5 h later, 1.5 mL lards were given via gavage. Blood samples were collected before the

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gavage (0 h) and at 1 and 3 h after the gavage of lard, and serum triglycerides were determined using 6


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Triglyceride Assay Kit (Nanjing Jiancheng bioengineering institute) according to the manufacturer’s

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instructions.

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Glucose Uptake and Lipid Accumulation in L02 Hepatocytic Cells. L02 hepatocyte cells were

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obtained from China Center for Type Culture Collection (Wuhan, China) and maintained in DMEM

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(glutamine, high glucose) supplemented with penicillin (100 units/mL), streptomycin (100 µg/mL) and

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10% heat-inactivated fetal bovine serum at 56℃ for 30 min. Subculturing was done by dislodging the

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cells with trypsin (0.25%) and EDTA⋅2Na⋅2H2O (0.02%), followed by seeding at the desired cell density

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and incubation at 37℃ under a humidified atmosphere of 95% air and 5% CO2. BYG used in the cell

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experiments was suspended in PBS (5 mg/mL) and sterilized at 121℃ for 30 min before use.

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For the glucose uptake analysis, BYG at the final concentration of 100 µg/mL and 200 µg/mL

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(denoted as BYG100 and BYG200, respectively) were added into the medium when L02 cells

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confluence reached 80%. Equal volumes of PBS were added in the control groups (coded as CT100 and

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CT200, respectively). 12 h and 18 h later, the medium was collected, and the remaining glucose levels in

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the medium were analyzed using Glucose Assay Kit (Nanjing Jiancheng Bioengineering Institute)

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according to the manufacturer’s instructions.

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For the lipid accumulation analysis, 100 µg/mL and 200 µg/mL BYG were co-incubated with L02

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cells in the presence or absence of 0.2 mM palmitic acid (PA). Equal volumes of PBS were added as the

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control groups (coded as CT100 and CT200, respectively). 12 h later, the cells were washed with PBS

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for 3 times and lysed. And triglyceride (TG) was extracted with 50 mM NaOH followed by incubation at

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60℃ for 10 min and centrifuged at 2810×g for 5 min. The TG level in lysate was analyzed by

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Triglyceride Assay Kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s 7



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instructions.

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Bile Acid Binding Capacity and Effects on Emulsification. A cholic acid (CA) solution (20 mL)

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was prepared with 10 mg of CA, 0.47 mL of 0.1 M NaOH and distilled water. The concentrations of test

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solutions containing cellulose (negative control), cholestyramine (positive control, Nangjing Lifecare

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Pharmaceutical Co., Ltd) and BYG, respectively, were fixed at 2.5 mg/mL. Then 100 µL of CA solution

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was mixed with 100 µL of the test solution. The mixtures were stirred at room temperature for 2 h, then

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transferred into PALL Nanosep centrifuge tube (3k molecular cut off) and centrifuged at 8000×g for 20

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min. The remaining CA in the resulting solution was measured by Total Bile Acid Assay Kit (Nanjing

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Jiancheng Bioengineering Institute) on Spark 10M microplate reader (Tecan).

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The edible oil (final concentration 5% w/v) was added into the mixture solution of CA (final

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concentration 0.25 mg/mL) and polysaccharides (BYG or cellulose, 2.5 mg/mL). After being mixed

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thoroughly, 10 µL of emulsion solution was dropped on the slide and covered with the coverslip,

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followed by observation with the microscope.

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Data and Statistical Analysis. The statistical analysis for RNA-seq was described in the

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“RNA-Seq Analysis” section. All other data were expressed as means ± SD and analyzed using software

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SPSS to determine statistically significant differences (p < 0.05) by Student’s t-test.

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RESULTS

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BYG Decreases the Blood Glucose and Improves the Hepatic Glucose Homeostasis. As

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reported, ob/ob mouse model is one of well-studied models of obesity and diabetes, which is considered

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as a model for the pre-diabetic state with moderate hyperglycemia, high adiposity and lifelong insulin 8


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resistance; they become hyperglycemic and the glucose level reaches a maximum at the age of 3–5

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months; then the glucose level starts to decrease and becomes nearly normal at old age.10 In this work,

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ob/ob mice at the age of 11∼12 weeks were used, and BYG treatment for 5 weeks was carried out; that

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is, ob/ob mice were killed at the age of 4∼5 months. According to the above statement, ob/ob mice

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should be hyperglycemic and diabetic. As shown in Figure 1A, FBG levels were higher than 7.0 mmol/L,

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suggesting that ob/ob mice used herein were diabetic according to the 2006 World Health Organization

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recommendations.2 Interestingly, BYG decreased the FBG levels, especially after long-term oral

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administration. It is worth noting that BYG had no obvious effect on food intake of ob/ob mice (Figure

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S1A) and the body weights of ob/ob mice were slightly suppressed by BYG (Figure S1B). Moreover,

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BYG significantly improved the glucose tolerance and decreased the blood glucose level characterized

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by the area under curve (AUC) in OGTT (Figure 1B). These findings clearly suggested that BYG

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possessed the capacity to reduce both fasting and postprandial blood glucose levels in diabetic ob/ob

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mice.

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In viewing that the liver plays an important role in maintaining the blood glucose level11 and is

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suggested to be an important target organ for anti-diabetic polysaccharides,6 we analyzed the glucose

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homeostasis in livers of ob/ob mice. The western blotting results show that BYG markedly enhanced

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expression of p-AKT, which is closely associated with the insulin signaling pathway,12 as well as the

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p-AMPK level in ob/ob mice (Figure 1 C and D), suggesting that BYG was involved in the regulation of

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glucose metabolism.13 Furthermore, the RT-PCR results indicate that the mRNA level of

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glucose-6-phosphatase (G6pase), an important enzyme catalyzing the hydrolysis of glucose-6-phosphate

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to glucose which is the last step in gluconeogenesis and glycogenolysis in the liver, was significantly 9



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inhibited by BYG (Figure 1E). All these data suggest that oral administration of BYG inhibited

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gluconeogenesis and improved glucose metabolism in ob/ob mice livers, possibly contributing to the

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decreased blood glucose level.

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BYG Reduces Lipid Accumulation in ob/ob Mice Livers. In addition to hyperglycaemia, lipid

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metabolic dysfunctions also characterize obesity and T2D.14 We therefore analyzed the effects of BYG

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on hepatic lipid homeostasis. As a result, BYG decreased the protein expression of p-Acc, which is

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responsible for fatty acid synthesis (Figure 1C and D). The hematoxylin and eosin (HE) staining of

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livers from BYG-treated mice exhibited more intact cell morphology and less lipid droplet cavity in

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contrast to those from ob/ob mice, and the oil red O (OR) staining of lipids showed that BYG

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significantly reduced hepatic lipid accumulation (Figure 1F). To gain deep insight into the decreased

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lipid accumulation, the levels of critical lipogenic genes were measured by RT-PCR. Clearly, BYG

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evidently inhibited the mRNA of Acc, Fas and sterol regulatory element-binding protein-1c (Srebp-1c)

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in contrast to the untreated ob/ob mice (Figure 1E). In other words, BYG inhibited lipid synthesis via

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down-regulating the lipogenic gene levels, leading to the reduced lipid accumulation in ob/ob mice

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livers.

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BYG Changes Transcriptional Profiles in ob/ob Mice Livers. The results above indicate that

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BYG affected glucose and lipid metabolisms obviously in the liver. To gain more comprehensive

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profiles of the hepatic glucose and lipid metabolisms at the transcriptome level, RNA‑seq and DEGs

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screening analysis were performed. Consequently, BYG altered a total of 90 genes including 56

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up-regulated genes and 34 down-regulated genes (Figure 2A), and 23 genes were changed significantly

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in metabolism pathways with 4 DEGs in carbohydrate metabolism and 16 DEGs in lipid metabolism 10


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(Figure 2B). The top 20 KEGG pathways and the associated DEGs are shown in Table S3. Of these, 17

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pathways were closely related with energy metabolism, such as fatty acid metabolism and biosynthesis,

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bile acid biosynthesis and bile secretion, pyruvate metabolism, insulin signaling pathway, AMPK

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signaling pathway, and so forth. Interestingly, all the DEGs associated with glucose and lipid

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metabolism were significantly down-regulated in BYG group as shown in Table 1. Overall, BYG

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significantly changed transcriptional profiles in ob/ob mice livers, which was described intensively in

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the following.

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BYG Down-Regulates Gluconeogenesis-Associated Genes Level. Gluconeogenesis is glucose

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synthesis from non-carbohydrate precursors such as amino acids, etc. Some amino acids can be

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catalyzed by aspartate aminotransferase (Got1)15 and eventually be converted into glucose via several

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enzymes such as G6pase mentioned above, etc. Herein, BYG decreased the mRNA expression of Got1

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(Table 1) as well as G6pase (Figure 1E), suggesting the suppressed gluconeogenesis occurred, and BYG

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was a potential inhibitor of gluconeogenesis for maintaining the glucose level in ob/ob mice as sketched

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in supplemental Figure S2A.

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Besides, BYG decreased mRNA expression of pyruvate kinase gene (Pklr) (Table 1), which

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converts phosphoenolpyruvate (PEP) into pyruvate, and pyruvate converges into tricarboxylic acid cycle

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to form acetyl-CoA, the major precursor for the synthesis of fatty acids.16 The reduced Pklr mRNA

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expression may lead to decrease of acetyl-CoA, possibly contributing to the less lipid accumulation in

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livers of BYG group.

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BYG Depresses Gene Levels of Fatty Acids Biogenesis and Elongation. As reported, the hepatic

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fatty acid synthesis increases in T2D.17 ATP citrate lyase (Acly) is a lipogenic enzyme that catalyzes the 11



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conversion of cytosolic citrate into acetyl-CoA, and acetyl-CoA is then catalyzed into malonyl-CoA by

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Acc, which is the rate limiting step in the synthesis of fatty acid.18 Malonyl-CoA as the C2 donor is

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consumed by Fas in de-novo synthesis of fatty acids (n ≤ 16). As shown in Table 1, BYG decreased the

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genes of Acly, Fas and Acc, suggesting that BYG inhibited biosynthesis of fatty acids. Then the

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Acyl-CoA (n ≥ 16) can be further elongated by ELOVL family member 6 (Elovl6) to form Acyl-CoA

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(n+2), which can be hydrolyzed by Acyl-CoA thioesterase (Acot) to form free long chain fatty acid.19

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Herein, BYG decreased the mRNA expression of genes Elovl6 and Acot3, suggesting the decreased

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elongation of fatty acids. Therefore, BYG down-regulated some key genes responsible for fatty acids

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biogenesis and elongation, which was sketched in supplemental Figure S2B.

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BYG Reduces the Gene Expressions of Glycerolipid Biosynthesis. Fatty acids can be esterified

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into triglycerides and diglycerides, which are accumulated in obese and diabetic livers.

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Glycerol-3-phosphate acyltransferase 1 (Gpam) catalyzes the rate-limiting step in the hepatic

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triglyceride biosynthesis.20 Lipin is also the crucial regulator in triglyceride synthesis, catalyzing the

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dephosphorylation of phosphatidic acid to form diacylglycerol.21 In our findings, BYG reduced the

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mRNA expression of genes Gpam and Lipin1/2 (Table 1), indicating that BYG inhibited the triglyceride

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biosynthesis (Figure S2C).

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BYG Decreases Gene Expressions of Cholesterol Biosynthesis and Alters Metabolism of Bile

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Acids. Acetyl-CoA is not only involved in fatty acid synthesis, but also in cholesterol biosynthesis by

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generating 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA). Reduction of HMG-CoA by the

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rate-limiting enzyme HMG-CoA reductase (Hmgcr) gives intermediates which are finally catalyzed by

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farnesyl-diphosphate synthase (Fdps) to give (E, E)-farnesyl-PP.22 Through being catalyzed by several 12


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cholesterogenic enzymes such as methylsterol monooxygenase (Msmo1) and sterol-C5-desaturase

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(Sc5d), farnesyl-PP is finally converted into cholesterol. As shown in Table 1, BYG decreased the

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cholesterogenic genes of Hmgcr, Fdps, Msmo1 and Sc5d, suggesting that BYG reduced cholesterol

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biosynthesis, as sketched in supplemental Figure S2D. Cholesterol 7 alpha-monooxygenase (Cyp7a1) is

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a liver-specific rate-limiting enzyme responsible for the conversion of cholesterol into bile acids, and

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sterol 12-alpha-hydroxylas (Cyp8b1) is required for synthesis of cholic acid.23 Aquaporin 8 (Aqp8) is

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involved in the ratio of bile acids and it’s secretion.24 Clearly, BYG decreased the mRNA expression of

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Cyp7a1, Cyp8b1 and Aqp8 genes (Table 1), implying BYG altered the bile acid conversion from

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cholesterols in ob/ob mice livers.

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Confirmation of Transcriptional Regulation by BYG. To confirm the mRNA expression

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changes mentioned above from the RNA-seq analysis, some representative DEGs in gluconeogenesis

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and lipid metabolism pathways were chosen for further analysis by RT-PCR. Consequently, BYG

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significantly down-regulated the mRNA expression of Got1, Acly, Elovl6, Acot3, Gpam, Hmgcr and

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Cyp7a1 (Figure 3A), consistent with the RNA-seq results. We also performed the PTT to confirm the

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effect of BYG on gluconeogenesis. Pyruvate is the substrate of gluconeogenesis, and BYG decreased the

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sharp increase of blood glucose induced by the gluconeogenesis of pyruvate (Figure 3B). Therefore,

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these results offered powerful evidences that BYG really contributed to improvement of diabetes

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mellitus through decreasing gluconeogenesis and lipid synthesis in the liver.

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Effect of BYG on Glucose-Lipid Metabolism in L02 Hepatocytic Cells. In our previous work,

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BYG was found to be distributed in livers after oral administration.8 To examine whether BYG affected

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the hepatic glucose and lipid metabolisms directly or indirectly, L02 hepatocytes were stimulated by 13



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BYG directly in vitro. And it was found that BYG did not increase glucose uptake into hepatic cells

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(Figure 4A). Fatty acids such as palmitic acid (PA) are reported to increase lipid accumulation in hepatic

250

cells,25 and BYG decreased lipid accumulation neither in L02 cells nor in PA-stimulated L02 cells

251

(Figure 4B). The expressions of critical glucose and lipid metabolic proteins were also evaluated in L02

252

cells, and it was found that the 12-h incubation with BYG showed no effect on the expression of p-AKT,

253

p-AMPK, p-Acc and Fas proteins. Therefore, the beneficial effects in the liver induced by BYG was

254

possibly indirect.

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BYG Decreases Glucose and Lipid Absorption in the Intestine. We have demonstrated

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previously that the most of orally administrated BYG was attached to the intestine and excreted in

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stools.8 The intestine is the major place where glucose and fat are absorbed, and there should be an

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intimate relationship between the gut and liver. Therefore, we isolated mice jejunum of ob/ob mice and

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extracted mucosal protein for western blotting analysis of SGLT1 and GLUT2, which are the major

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glucose transporters responsible for the glucose transportation from the lumen into blood.26 As a result,

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the expression of SGLT1 and GLUT2 were reduced to 74.3% and 25.7%, respectively, in BYG-treated

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group (Figure 5A). AMPK is an important glucose sensor in enterocytes, and plays an important role in

263

the regulation of SGLT1 and GLUT2. Indeed, BYG induced an obvious decrease of p-AMPK in the

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intestine (Figure 5A). Moreover, BYG down-regulated the protein expression of p-Acc and Fas in the

265

intestines of ob/ob mice (Figure 5A). The intestinal genes involved in the lipid synthesis were also

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decreased in BYG group (Figure 5B).

267

To further assess the effect of BYG treatment on the intestinal lipid absorption, SD rats were

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subjected to an oral fat challenge. And it was found that BYG decreased the serum TG level 14


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significantly (Figure 5C), suggesting suppression of the intestinal lipid absorption. CA is an essential

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constituent of bile that facilitates dietary lipid absorption and cholesterol catabolism in the intestine after

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food ingestion. The binding of BYG to CA in vitro, representing the binding of BYG and bile acids to

272

some extent,27 was also assessed. As shown in Figure 5D, although the CA binding ability of BYG was

273

considerably weaker than the positive control of cholestyramine, a bile acid-binding resin used as a

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cholesterol-reducing drug, BYG remarkably bound CA as compared with the negative control of

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cellulose. Moreover, the oil droplets in water can be emulsified into smaller size oil droplets by CA, and

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BYG weakened the emulsification effect of CA (Figure 5E), further demonstrating that BYG bound CA

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leading to the weak fat emulsification and then the decreased intestinal lipid absorption.

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DISCUSSION

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With the high prevalence of diabetes worldwide, much efforts have gone into the study on

280

anti-diabetic drug and food development. Carbohydrates as the third class of biomacromolecules, have

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been proposed as the next frontier in pharmaceutical research and hold a wealth of hope for the future of

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medicine.28 In our previous work, we have found that the linear β-(1,3)-D-glucan from the crude baker’s

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yeast polysaccharides shows significant anti-diabetic effect in diet-induced obese/diabetic mice.8 To

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further confirm this effect, ob/ob mice, the gene-induced obese and type 2 diabetic mice, were used in

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this work. Likewise, oral administration of BYG decreased both fasting and postprandial blood glucose

286

levels in ob/ob mice (Figure 1A and B), which was partly ascribed to the down-regulation of glucose

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transporters SGLT1 and GLUT2 in the intestinal mucosa (Figure 5A). These findings were essentially

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consistent with our previous study in diet-induced obese/diabetic mice,8 demonstrating the fact that 15



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orally administrated yeast β-glucan can really reduce the high blood glucose level in obese/diabetic mice

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through decreasing glucose adsorption via inhibition of SGLT1 and GLUT2 in the intestine as the first

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step.

292

As well known, the liver plays a major role in maintaining the blood glucose in normal level. When

293

the blood glucose concentration rises, the glucose is transported into hepatocytes to be stored as

294

glycogen;29 when the blood glucose is relative scare, the liver releases glucose to the blood by glucose

295

production metabolic pathways such as gluconeogenesis. Then we focused on the molecular events

296

happened in the liver after oral administration of BYG. Herein, BYG sharply enhanced the expressions

297

of hepatic p-AKT and p-AMPK (Figure 1C and D), which are reported to contribute to glycogen

298

synthesis and lower blood glucose.30, 31 Our previous work has shown that BYG really promotes the

299

synthesis of glycogen in the liver of diet-induced obsess/diabetic mice.8 We thus conclude that BYG

300

promoted the hepatic glycogen synthesis. As reported, gluconeogenesis is excessive in metabolic

301

diseases such as obese, diabetes, etc.,32 accelerating glucose release to the blood. In our findings, BYG

302

markedly decreased the mRNA levels of G6pase and Got1 genes (Figure 1E, Table 1), both of which are

303

closely associated with the gluconeogenesis in the liver,33 suggesting that BYG suppressed the glucose

304

production through gluconeogenesis pathways in the liver, which was further confirmed by PTT results

305

(Figure 3B). Collectively, BYG decreased the hyperglycaemia through promoting glycogen storage, as

306

well as suppressing glucose output from gluconeogenesis in livers of ob/ob mice.

307

In addition to hyperglycaemia, ob/ob mice are also characterized by hyperlipidaemia.17 Fatty acid,

308

triacylglycerol and cholesterol are three crucial indexes in hyperlipidaemia, and lipogenic enzymes are

309

substantially up-regulated in the liver of obese subjects and ob/ob mice.34 As reported, a 5-fold induction 16


Page 17 of 31


310

in the expression of Fas mRNA occurs in ob/ob mice,35 Acly gene is elevated in diabetic mice livers,36

311

and Elovl6 expression is positively correlated with lipid droplet size and steatosis in the liver.37 In our

312

findings, BYG markedly down-regulated the mRNA expression of Acly, Acc, Fas, Elovl6 and Acot3 in

313

the fatty acid biosynthesis pathway (Table 1, Figure 1E). Therefore, it can be deduced that BYG

314

inhibited the genes responsible for fatty acid biosynthesis to hold the lipid homeostasis in livers of ob/ob

315

mice. It has been reported that hepatic Lipin 1/2 expression is enhanced in the insulin resistant mice.38 In

316

this work, BYG suppressed the triglycerides synthesis pathway indicated by the decreased mRNA of

317

Gpam and Lpin1/2 (Table 1), implying that BYG tended to alleviate the disorders in diabetic mice

318

through inhibiting triglyceride biosynthesis. Besides, in the cholesterol and bile acid metabolism

319

pathways, BYG decreased the mRNA levels of Hmgcr, Fdps, Msmo1, Sc5d, Cyp7a1 and Cyp8b1 to

320

suppress hepatic cholesterol and alter bile acid biosynthesis (Table 1). It has been reported that the

321

decreased Fdps is associated with the hypocholesterolaemic and hypotriglyceridaemic effects,39 and the

322

basal expression of Cyp7a1 is elevated in genetically obese type 2 diabetic ob/ob mice.40 Our results

323

suggest the possible beneficial effect of BYG on the cholesterol and bile acid metabolism. Taken

324

together, BYG restrained the lipid accumulation in the diabetic liver (Figure 1F) via down-regulating

325

lipogenic enzymes biosynthesis.

326

AMPK plays an important role in the regulation of glucose and lipid metabolisms, and it is usually

327

the target molecule of many anti-diabetic synthetic drugs such as metformin. Activation of AMPK in the

328

liver stimulates glucose uptake, while inhibits gluconeogenesis, fatty acid and cholesterol synthesis.41

329

Ob/ob mice are associated with a decrease in AMPK activity in the liver.42 As reported, all the mRNAs

330

of G6pase, Fas, Acc, Hmgcr, and Srebp-1c can be repressed by the activated AMPK.41 Srebp-1c, the 17



Page 18 of 31

331

transcription factor, is a master regulator of lipogenesis and is necessarily required for the transcriptional

332

activation of the genes involved in the fatty acid biosynthesis43 and triacylglycerol formation. The leptin

333

deficient ob/ob mice produce a 40% increase in Srebp-1c mRNA,35 and over expression of Srebp-1c

334

gives rise to large increases in both fatty acid and cholesterol synthesis in the liver.41 In our findings,

335

BYG significantly enhanced AMPK activation (Figure 1, C and D), and remarkably down-regulated the

336

Srebp-1c related genes (Figure 1E and Table 1), consistent with the observations that numerous

337

anti-diabetic polysaccharides activate AMPK and inhibit Srebp-1c in livers to elicit metabolic benefits.41

338

Additionally, the fat emulsification as well as the expression of some lipogenic proteins and genes

339

(Figure 5) in the intestine were decreased by BYG, suggesting that down-regulation of the lipid level

340

was partly ascribed to the decreased adsorption of lipid through the intestine. Similar to glucose

341

metabolism, BYG first reduced the lipid adsorption in the intestine, and then suppressed lipid

342

accumulation through inhibiting fatty acids and cholesterol synthesis via activating AMPK and

343

inhibiting Srebp-1c in livers (Figure 6).

344

Undoubtedly, the above analysis suggests that the disordered hepatic glucose and lipid biosynthesis

345

fluxes, contributing to hyperglycaemia and hyperlipidemia in diabetes, were at least partially recovered

346

in BYG-treated ob/ob mice. We have reported that some of the green fluorescence-labeled yeast

347

β-glucans were distributed in the livers of mice after oral administration.8 To check if BYG directly

348

promoted the glucose uptake and reduced lipid accumulation in the liver, the in vitro tests against human

349

L02 hepatocytic cells were performed. Interestingly, BYG was unable to promote glucose uptake and

350

lower lipid accumulation in L02 cells in vitro, and the expressions of proteins related with glucose and

351

lipid metabolisms were unchanged in BYG-stimulated L02 cells (Figure 4), suggesting that the changes 18


Page 19 of 31


352

in the liver was not directly triggered by the distribution of BYG. As reported in our previous work,

353

BYG significantly changed the gut microbiota of Akkermansia in diet-induced obese mice.8 Similarly,

354

BYG also increased proportion of Akkermansia and improved the microbial diversity distribution in

355

ob/ob mice (data not shown). It is thus deduced that BYG promoted the glucose/lipid hemostasis in the

356

liver was possibly associated with the improved gut microbiota by BYG. In combination of above

357

analysis, we thus suggest that the orally administrated BYG reduced glucose and lipid absorption in the

358

intestine, leading to the decreased blood glucose and the improved glucose/lipid metabolism in the liver.

359

Accumulating studies have proposed that water-soluble β-glucans from oats and barley with mixed

360

linkages of (1→3)- and (1→4)-β-D-glucans show hypoglycemic and hypolipidemic effects partly due to

361

the increased viscosity in the gastrointestinal tract induced by the intake of soluble dietary fiber.44

362

Obviously, BYG is a linear (1→3)-β-D-glucan with water-insolubility, and high viscosity is impossible

363

after oral intake in the gastrointestinal tract. The major reason should be ascribed to the adsorption

364

inhibition of glucose (via down-regulating GLUT2 /SGLT1 and p-AMPK) and lipid (through decreasing

365

fat emulsion and down-regulating Fas and p-Acc) in the intestine, as well as the improvement of glucose

366

homeostasis and inhibition of lipid accumulation in the liver (Figure 6).

367

In conclusion, the hyperglycemia and hyperlipidemia in ob/ob mice were reduced by the orally

368

administrated BYG with improvement of hepatic glucose/lipid metabolism. In detail, BYG significantly

369

promoted glucose uptake, and down-regulated some critical proteins and enzyme genes responsible for

370

gluconeogenesis, fatty acid, glyceride and cholesterol biogenesis in the liver. Our results confirmed that

371

the insoluble β-glucan is one of the bioactive components responsible for the beneficial effects of

372

anti-diabetic natural polysaccharides. This provides valuable information for the research and rational 19



Page 20 of 31

373

use of natural anti-diabetic polysaccharides in areas of functional food and pharmacology.

374

375

Corresponding Author

376

*Phone: 86-27-68754188. Fax: 86-27-68754188. E-mail: [email protected]

377

ORCID

378

Xiaojuan Xu: 0000-0002-1404-5088

379

Funding

380

This research was supported by National Key R&D Program of China (2016YFD0400202), National

381

Natural Science Foundation of China (21574102, 21274114 and 21334005), and New Century Excellent

382

Talents Program of Education Ministry (NCET-13-0442).

383

Notes

384

The authors have declared no conflict of interest.

385

386

Acc, acetyl-CoA carboxylase; Acly, ATP citrate lyase; Acot3, acyl-coenzyme A thioesterase 1/2/4; AKT,

387

protein kinase B; AMPK, adenosine 5’-monophosphate -activated protein kinase; Aqp8, aquaporin 8;

388

AUC, area under curve; BYG, baker’s yeast glucan; CA, cholic acid; Cyp7a1, cholesterol 7

389

alpha-monooxygenase; Cyp8b1, sterol 12-alpha-hydroxylas; DEGs, differentially expressed genes;

390

Elovl6, ELOVL family member 6; Fas, fatty acid synthase; FBG, fasting blood glucose; Fdps, farnesyl

391

diphosphate synthetase; GLUT2, glucose transporter 2; GO, gene ontology; Got1, aspartate

AUTHOR INFORMATION

ABBREVIATIONS USED

20


Page 21 of 31


392

aminotransferase,

cytoplasmic;

393

glucose-6-phosphatase;

394

3-hydroxy-3-methyl glutaryl coenzyme A; Hmgcr, HMG-CoA reductase; Msmo1, methylsterol

395

monoxygenase 1; OGTT, oral glucose tolerance test; OR, oil red O; PA, palmitic acid; Pklr, pyruvate

396

kinase; PTT, pyruvate tolerance test; Sc5d, sterol-C5-desaturase; SGLT1, sodium glucose co-transporter

397

1; Srebp-1c, sterol regulatory element-binding protein-1c; TG, triglyceride; T2D, type 2 diabetes

398

399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423

(1) Seshasai, S. R. K.; Kaptoge, S.; Thompson, A.; Di Angelantonio, E.; Gao, P.; Sarwar, N.; Whincup, P. H.; Mukamal, K. J.;

HE,

Gpam,

hematoxylin

glycerol-3-phosphate and

eosin;

HFD,

acyltransferase; high-fat

diet;

G6pase, HMG-CoA,

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23



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Figure captions

501

Figure 1. BYG ameliorated glucose and lipid metabolism in the livers of ob/ob mice. (A) BYG

502

decreased fasting blood glucose in ob/ob mice. BYG (25 mg/kg/d, BYG group) or equal volume of

503

water (ob/ob group) were orally given to ob/ob mice. The FBG levels were measured after starvation

504

from 8 a.m. to 14 p.m. (B) BYG decreased blood glucose in OGTT. The ob/ob mice were fasted for 6 h,

505

then the blood glucose was determined at 0, 15, 30, 60, and 120 min after 1 g/kg glucose oral

506

administration. The inset indicates the calculated AUC of glucose. Date represent mean ± SD of 7 mice

507

per group. *p < 0.05 versus the ob/ob group. (C-D) BYG promoted the expressions of protein p-AKT

508

(Ser473), p-AMPK, and decreased p-Acc protein in the livers of ob/ob mice. The optical density of the

509

target proteins was normalized to that of β-actin before analysis. Date represent mean ± SD of 4 mice

510

per group. *p < 0.05 versus the ob/ob group. (E) BYG decreased the mRNA expressions of G6pase and

511

lipogenic genes (Srebp-1c, Acc and Fas) in the livers of ob/ob mice. Data represent mean ± SD of 3

512

mice. *p < 0.05 versus ob/ob group. (F) Hepatic microscope images of HE and oil red O staining of the

513

ob/ob mice, scale bar = 50 µm.

514 515

Figure 2. BYG changed transcriptional profiles in ob/ob mice livers. (A) Scatter plots of all expressed

516

genes in pairwise of the BYG versus the ob/ob group. X-axis and Y-axis present gene expression level.

517

Blue means down-regulated genes, orange means up-regulated genes and brown means unchanged

518

genes. (B) KEGG pathway classification on DEGs for the BYG versus the ob/ob groups. X axis

519

represents second KEGG pathway terms, and Y axis means the number of DEGs. All second pathway 24


Page 25 of 31


520

terms are grouped in top pathway terms indicated in different color. Qvalue ≤ 0.05 was defined as

521

significantly enriched pathway terms.

522 523

Figure 3. Confirmation of transcriptional regulation by BYG. (A) The decreased expression of hepatic

524

mRNA responsible for lipid metabolism in the BYG group confirmed by RT-PCR. Data represent mean

525

± SD of 3 mice. *p < 0.05 versus the ob/ob group. (B) The decreased gluconeogenesis in the BYG group

526

confirmed by pyruvate tolerance test. On day 25 after BYG (25 mg/kg/d) treatment, ob/ob mice were

527

fasted for 16 h and injected with pyruvate (i.p., 1.5 g/kg). Blood glucose levels were measured at 0, 15,

528

30, 60 min. Data represent mean ± SD of 7 mice per group. *p < 0.05 versus the ob/ob group.

529

530

Figure 4. BYG had no effect on glucose and lipid metabolism in L02 cells. (A) Effect of BYG on

531

glucose uptake in L02 cells. L02 cells were treated with 100 and 200 µg/mL of BYG for 12 h and 18 h.

532

Then the remaining glucose levels in media were analyzed. Equal volumes of PBS were added as the

533

control. (B) Effect of BYG on lipid accumulation in L02 cells. L02 cells stimulated with or without 0.2

534

mM PA were incubated with 100 and 200 µg/mL of BYG for 12 h. Then the TG in cells was extracted

535

and analyzed. (C) Glucose-lipid metabolic protein expressions in L02 cells treated with 100 µg/mL of

536

BYG for 12 h. Data represent mean ± SD of 3 independent experiments.

537 538

Figure 5. BYG decreased glucose and lipid absorption in the intestine. (A) BYG depressed expression

539

of SGLT1, GLUT2, p-AMPK and p-Acc and Fas in the intestine from ob/ob mice. Date represent mean 25



Page 26 of 31

540

± SD, n = 4 mice per group. *p < 0.05 versus the ob/ob group. (B) BYG decreased mRNA of lipid

541

metabolism in the intestine from ob/ob mice. Date represent mean ± SD of 3 mice. *p < 0.05 versus the

542

ob/ob group. (C) Fat tolerance test in SD rats. After a 6-h fasting, SD rats were orally given 50 mg/kg

543

BYG (BYG group) or equal volume of water (CT group). 0.5 h later, 1.5 mL lard were given orally and

544

serum triglycerides were determined at 0, 1 and 3 h. Date represent mean ± SD of 5 rats per group. *p

Orally Administered Baker's Yeast Î²-Glucan Promotes Glucose and

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