A Polysaccharide from Ganoderma atrum Improves Liver Function in

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A polysaccharide from Ganoderma atrum improves liver function in type 2 diabetic rats via antioxidant action and short-chain fatty acids excretion Ke-xue Zhu, Shao-Ping Nie, Lehe Tan, Chuan Li, Deming Gong, and M. Y. Xie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06103 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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Journal of Agricultural and Food Chemistry

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A polysaccharide from Ganoderma atrum improves liver function in

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type 2 diabetic rats via antioxidant action and short-chain fatty acids

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excretion

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Ke-Xue Zhu,†,

‡

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Ming-Yong Xie†

Shao-Ping Nie,*,

†

Le-He Tan,‡ Chuan Li,†,

§

De-Ming Gong,†,

#

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†

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Nanchang, Jiangxi 330047, China.

State Key Laboratory of Food Science and Technology, Nanchang University,

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‡

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Sciences, Wanning, Hainan 571533, China

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§

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570228, China

14

#

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Auckland, New Zealand

Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural

College of Food Science and Technology, Hainan University, Haikou, Hainan

School of Biological Sciences, The University of Auckland, Private Bag 92019,

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* To whom correspondence should be addressed. E-mail: [email protected]

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(Professor Shao-Ping Nie, PhD); Tel. & Fax: +86-791-88304452; Address: State Key

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Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East

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Road, Nanchang, Jiangxi 330047, China.

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ACS Paragon Plus Environment


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Abstract

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The present study was to evaluate the beneficial effect of polysaccharide isolated from

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Ganoderma atrum (PSG-1) on liver function in type 2 diabetic rats. Results showed

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that PSG-1 decreased the activities of serum aspartate aminotransferase (AST) and

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alanine aminotransferase (ALT), while increased hepatic glycogen levels. PSG-1

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also exerted strong antioxidant activities, together with upregulated mRNA

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expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), glucose

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transporter-4 (GLUT4), phosphoinositide 3-kinase (PI3K) and phosphorylated-Akt

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(p-Akt) in the liver of diabetic rats. Moreover, the concentrations of short-chain fatty

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acids (SCFA) were significantly higher in the liver, serum and faeces of diabetic rats

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after treating with PSG-1 for 4 weeks. These results suggest that the improvement of

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PSG-1 on liver function in type 2 diabetic rats may be due to its antioxidant effects,

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SCFA excretion in the colon from PSG-1, and regulation of hepatic glucose uptake

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by inducing GLUT4 translocation through PI3K/Akt signaling pathways.

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Keywords: Polysaccharide; Ganoderma atrum; Type 2 diabetes; Liver function;

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Short-chain fatty acids

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Introduction

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Diabetic mellitus is a chronic metabolic disorder characterized by hyperglycemia

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and mainly affects carbohydrate, lipid and protein metabolism.1 Liver, as a

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‘bifunctional’ organ, plays a major role in glucose homeostasis including glycolysis,

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gluconeogenesis and glycogen metabolism, and is severely affected during diabetes

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mellitus.2 Recent studies provided more insight into the relationship between liver

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disease and diabetes mellitus.3,4 Chiang et al.5 reported that obesity and elevated

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fasting glucose are major risk factors for the pathogenesis of the liver disease. Harris6

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found that type 2 diabetic patients had a higher incidence of hepatic injury, including

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elevated

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aminotransferase (ALT).

concentrations

of

aspartate

aminotransferase

(AST)

and

alanine

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Numerous studies have shown that polysaccharides isolated from natural sources

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exerted beneficial effects on diabetes mellitus and its complications through

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protecting against oxidative stress damage and/or improving insulin sensitivity. Liu et

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al.7 reported that Sarcandra glabra polysaccharide exhibited hypoglycemic activity

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by relieving insulin resistance, reducing postprandial blood glucose levels, and

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ameliorating lipid metabolism, oxidative stress in type 2 diabetic mice. A

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polysaccharide extract from mulberry leaves improved hepatic glucose metabolism

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and insulin resistance by inhibiting the expression of protein tyrosine phosphatase-1B

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(PTP1B), activating the PI3K-Akt pathway and mitigating oxidative stress in liver of 3



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type 2 diabetic rats.8

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Ganoderma, as popular medicinal fungi, have been used as functional food and

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medicine in many Asian countries for centuries. Polysaccharides from Ganoderma

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were reported to exhibit diverse bioactivities including immunomodulatory, antitumor,

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antioxidant, hepatoprotective and anti-hypertensive activities.9 Ganoderma atrum is

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one member of Ganoderma, belongs to the polyporaceae family of Basidiomycota.

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Our previous study found that polysaccharide isolated from Ganoderma atrum

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(PSG-1) exerted hypoglycemic and hypolipidemic effects through inhibiting

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expression of Bax and improving the expression of Bcl-2 protein in pancreatic tissues

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of type 2 diabetic rats.10 Moreover, PSG-1 protected against diabetes-induced

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endothelial dysfunction in aorta of type 2 diabetic rats through up-regulating

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expression of PI3K, p-Akt, eNOS, NO and Bcl-2 levels and down-regulating Bax

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expression.11 However, there is a lack of knowledge about the beneficial effect of

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PSG-1 on hepatic function in type 2 diabetes.

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The aim of this work was to evaluate the beneficial effects of PSG-1 on hepatic

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function of type 2 diabetic rats. The liver index, liver glycogen, liver function,

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antioxidant activities, and short-chain fatty acids (SCFA) concentrations in the liver,

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serum and faeces of type 2 diabetic rats were determined after treating with PSG-1 for

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4 weeks.

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Materials and Methods

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Materials and Reagents. PSG-1 was isolated from the fruiting bodies of 4


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Ganoderma atrum by our research group in the State Key Laboratory of Food Science

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and Technology, Nanchang University, China. The purified PSG-1 (purity ≥ 99.8%)

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was composed of glucose, mannose, galactose and galacturonic acid in a molar ratio

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of 4.91: 1: 1.28: 0.71, with the molecular weight of 1,013 kDa.12

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All chemicals and reagents used in this study were of analytical grade or higher.

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Animals and Experimental Design. Sixty male Wistar rats (180-200 g) were

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obtained from Shanghai Slaccas Laboratory Animal Company (Certificate Number

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SCXK (hu) 2007-0005, Shanghai, China). Before starting the experiments, all the

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animals were acclimatized to the ambient temperature of 25 ± 2 ºC, 12/12 h of

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light-dark cycle for 1 week. After acclimation, ten rats fed a standard normal chow

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diet (SND) consisting of 12% fat, 60% carbohydrate and 28% protein (as a percentage

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of total kcal), the others were fed a high fat diet (HFD) consisting of 40% fat, 42%

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carbohydrate and 18% protein. After 8 weeks of dietary manipulation, all rats were

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fasted for 12 h (with free access to water). Rats fed on HFD were injected with a

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single low dose of streptozotocin (STZ, 30 mg/kg body weight (BW) in 154 mmol/L

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isotonic saline, pH 7.2, Sigma Chemical Co., St. Louis, Missouri, USA) into the tail

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vein to induce type 2 diabetes according to our previous method.11 Metformin (1,

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1-dimethylbiguanide hydrochloride) used as positive control was obtained from

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Sigma Chemical Co. (St. Louis, Missouri, USA). The experimental design was shown

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in Figure 1.

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All animals used in this study were cared in accordance with the Care and Use of 5



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Laboratory Animals Guidelines published by the United States National Institute of

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Health (NIH Publication 85-23, 1996). All experimental procedures involving the use

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of animals were approved by the Animal Care and Use Committee of Medical College

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of Nanchang University.

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Measurement of Liver Index, Liver Glycogen and Liver Function. At the end

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of experimental period, all the experimental rats were fasted for 12 h and anesthetized

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with 10% chloralic hydras intraperitoneally. Blood samples were then collected under

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anaesthetized conditions and centrifuged at 1000 ×g for 10 min. The serum was

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collected to study the biochemical parameters of liver function.

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The liver samples were collected under anaesthetized conditions to calculate the

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liver index (liver weight as a percentage of BW). The content of liver glycogen was

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determined with a commercial kit purchased from Nanjing Jiancheng Bioengineering

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Institute (Nanjing, Jiangsu, China).

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The activities of serum aspartate aminotransferase (AST) and alanine

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aminotransferase (ALT), as well as levels of serum total protein (TP) and albumin

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(ALB) were measured by an enzymatic method, using the corresponding Flex

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reagents on a Dimension RxL Max analyzer from Dade-Behring Diagnostics

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(Marburg, Hesse, Germany).

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Measurement of Activities of SOD, GPx and CAT, and MDA Level in Liver.

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The liver samples were rinsed in ice-cold saline to remove the blood cells. Then 10%

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(w/v) liver homogenate was prepared in saline using glass homogenizer and 6


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centrifuged at 2000 ×g for 10 min at 4 °C. The supernatant was collected for the

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measurements of activities of SOD, GPx and CAT, and level of MDA in the liver

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samples following the manufacturer's instructions of commercial kits purchased from

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Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

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Analyses of mRNA Expression of PPAR-γ, GLUT4, PI3K and p-Akt in Liver.

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RT-PCR analyses of mRNA expression of peroxisome proliferator-activated

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receptor-γ (PPAR-γ), glucose transporter-4 (GLUT4), phosphoinositide 3-kinase

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(PI3K) and phosphorylated-Akt (p-Akt) in liver from different groups were performed

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according to the method by Li et al.13 The primers as follows were sequenced by

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Invitrogen Co. Ltd (Beijing, China):

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β-actin: F: 5′-TGTTGTCCCTGTATGCCTCT-3′, R: 5′-TAATGTCACGCACGATTTCC-3′

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PPAR-γ: F: 5′-TCAGGGCTGCCAGTTTCG-3′, R: 5′-GCTTTTGGCATACTCTGTGATCTC-3′

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GLUT4: F: 5′-GACATTTGGCGGAGCCTAAC-3′, R: 5′-TAACTCCAGCAGGGTGACACAG-3′

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PI3K: F: 5′-CAAAGCCGAGAACCTATTGC-3′, R: 5′-GGTGGCAGTCTTGTTGATGA-3′

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p-Akt: F: 5′-CCGCTATTATGCCATGAAGAT-3′, R: 5′-TGTGGGCGACTTCATCCT-3′

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GAPDH: F: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′,

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R: 5′-AGCCTTCTCCATGGTGGTGAAGAC-3’

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The PCR amplification of different genes was performed in a 50 µL reaction

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mixture containing 1 x Easy Taq PCR SuperMix obtained from TransGen Biotech

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(Beijing, China), 0.4 µM forward and reverse primers and 5 ng of template cDNA.

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Then PCR products were separated on 1.5% agarose gels, stained with Goldview

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obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China) and

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photographed under UV light using Molecular Imager® system, Bio-Rad 7



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Laboratories, Inc. (Hercules, California, USA).

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Measurement of SCFA Level. At the end of experimental period, the liver,

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serum and faeces samples were collected from different groups to study the effect of

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PSG-1 on SCFA production. 10% (w/v) samples homogenate were prepared by

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adding 9 times ultra-pure water to each tube contained experimental sample and

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mixed intermittently on a vortex mixer for 2 min. The supernatant was collected after

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centrifuging at 4800 ×g for 20 min, and then analyzed by injection into a

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chromatographic system.

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Chromatographic analysis was carried out using an Agilent 6890N GC system

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equipped with a flame ionization detector (FID) and an N10149 automatic liquid

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sampler (Palo Alto, California, USA). The chromatography parameters were provided

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in Table 1 according to the method described by Hu et al.14 Briefly, Nitrogen was

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supplied as the carrier gas at a flow rate of 19.0 mL/min with a split ratio of 1:10. The

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initial oven temperature was 100 °C and was kept there for 0.5 min and then raised to

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180 °C by 4 °C/min. The temperatures of the FID and injection port were 240 °C. The

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flow rates of hydrogen and air were 30 and 300 mL/min, respectively. The injected

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sample volume for GC analysis was 0.2 µL.

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Calibration curves were made in the range 2-80 mmol/L for acetic acid ((100%

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purity, Merck, Darmstadt, Hessen, Germany), 1.5-60 mmol/L for propionic acid

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(100% purity, Janssen Chimica, Beerse, Antwerpen, Belgium), 1-50 mmol/L for

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n-butyric acid (100% purity, Sigma Chemical Co, St. Louis, Missouri, USA), and by

8


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adding 4-methylvaleric acid (99.9% purity, Sigma Chemical Co, St. Louis, Missouri,

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USA) as internal standard. The independently replicated determinations were

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performed three times for standard solutions and each sample.

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Statistical Analysis. Data were expressed as the mean ± standard error of the

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mean (SEM). One-way analysis of variance (ANOVA) followed by the multiple

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comparisons test of Dunnett to assess the statistical differences using SPSS 13.0

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software (Statistical Package for Social Sciences, SPSS Inc, Chicago). A value of P

A Polysaccharide from Ganoderma atrum Improves Liver Function in

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