Deacetylated Konjac Glucomannan Is Less Effective in Reducing

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Deacetylated Konjac Glucomannan is Less Effective in Reducing Dietary-induced Hyperlipidemia and Hepatic Steatosis in C57BL/6 Mice



Meiying Li, Guanping Feng, Hong Wang, Ruili Yang, Zhenlin Xu, and Yuanming Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05320 • Publication Date (Web): 07 Feb 2017



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Journal of Agricultural and Food Chemistry

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Deacetylated Konjac Glucomannan is Less Effective in Reducing

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Dietary-induced Hyperlipidemia and Hepatic Steatosis in C57BL/6

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Mice

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Mei-ying Li† , Guan-ping Feng†, Hong Wang,† Rui-li Yang,† Zhenlin Xu, † Yuan-Ming

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Sun,†,*

6" 7" 8" 9" 10"

† Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food

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Science, South China Agricultural University, Guangzhou 510642, China

12" 13"

* Corresponding author. No.483, Wushan Road, Tianhe District, Guangzhou,

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510642, China. Tel.: +86 20 8528 3448; Fax: +86 20 8528 0270

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E-mail: [email protected] (Y.-M. Sun);

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Short title: Deacetylated Konjac Glucomannan is Less Effective Than Native

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Glucomannan

19" 20" 21"

1" "


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ABSTRACT:

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Konjac gel foods that mainly consist of deacetylated konjac glucomannan (Da-KGM)

24"

are considered to have the same health benefits as native konjac glucomannan

25"

(KGM); however, no definitive data support this notion. The objective of this study

26"

was to compare the effects of Da-KGM and KGM on the hyperlipidemia and liver

27"

steatosis induced by high-fat diet feeding and to investigate the underlying

28"

molecular mechanisms. C57BL/6 mice were fed (1) normal chow diet; (2) high-fat diet;

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(3) HFD with KGM; (4) HFD with Da-KGM for 10 weeks. KGM, but not Da-KGM,

30"

showed decreased fat accumulation, improved blood and liver lipid profiles, and

31"

prevention of liver lipid droplet deposition compared with HFD. Compared with Da-

32"

KGM, KGM increased the outputs of fecal bile acid (KGM 22.5 ± 2.34 mg/g vs. Da-

33"

KGM 19.3 ± 1.87 mg/g), fat (KGM 5.56 ± 0.68 mg/g vs. Da-KGM 4.42 ± 0.57 mg/g) and

34"

cholesterol (KGM2.67 ± 0.43 mg/g vs. Da-KGM 1.78 ± 0.28 mg/g), fecal concentrations

35"

of total short-chain fatty acids (KGM 103 ± 14.8 μmol/g vs. Da-KGM 74.5 ± 8.49 μmol/g),

36"

and improved hepatic antioxidant status, and upregulated CYP7A1 and LDLR gene

37"

expression. These findings suggest that deacetylation of KGM negatively affects its

38"

fermentation characteristics and its inhibition of lipid absorption, thereby reducing

39"

Da-KGM’s health benefits.

40"

KEYWORDS: konjac glucomannan, deacetylated konjac glucomannan, lipid

41"

metabolism, high-fat diet , konjac gel foods

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2" "



43"

! INTRODUCTION

44"

Amorphophallus konjac (konjac) originates in South East Asia and has long

45"

been used in China, Japan and South East Asia as a food source. Konjac

46"

glucomannan (KGM) is a water-soluble, fermentable hydrocolloid obtained from

47"

konjac. The main chain of KGM is composed of D-mannose and D-glucose

48"

connected by β-1,4 linkages with a reported ratio of 1.6:1. Acetyl groups are located

49"

along the glucomannan backbone on average every 9 to 19 sugar units.1, 2 KGM is one

50"

of the most viscous water-soluble dietary fibers known. With its unique rheological

51"

and gelling properties, KGM has been widely used as an emulsifier and stabilizer in

52"

drinks and foods,3 and has been approved by the U.S. Food and Drug Administration

53"

as a food additive since 1994. KGM has a number of desirable nutritional and health

54"

characteristics. Preliminary evidence suggests that KGM may markedly suppressed

55"

development of atopic dermatitis by suppresses immunoglobulin E production in

56"

mice.4 The β-1,4 linkages of KGM are resistant to human digestive enzymes, so KGM

57"

reaches the colon unchanged and is fermented by gut microbiota. In addition, KGM

58"

may act as a prebiotic, because it exerts beneficial effects on the intestinal mucosal

59"

barrier and gut microflora by adding to the intestinal short-chain fatty acid (SCFA)

60"

content.5,6 Lipid-lowering and anti-obesity effects are the functions most commonly

61"

attributed to KGM, and these have been demonstrated in many studies.7-9 A meta-

62"

analysis of randomized controlled trials of KGM concluded that KGM can

63"

significantly lower blood total cholesterol (TC), triglycerides (TG) and low-density 3" "


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lipoprotein cholesterol (LDL-C).10 Based on these findings, konjac gel foods made

65"

with KGM have been promoted in many newspapers, magazines and TV programs

66"

as having the potential to treat obesity and obesity-related dyslipidemia. Konjac

67"

foods have been adopted worldwide as functional foods in the form of noodles, tofu

68"

and snacks, and continue to gain in popularity.11 However, to date there are no

69"

definitive data to support these claims.

70"

The production of konjac traditional foods is based on the chemical process of

71"

thermo-irreversible gel forming. In an alkaline environment, the acetyl groups of

72"

KGM are replaced by hydroxyl groups under heating, which enhances hydrogen

73"

bonding between KGM molecules and results in the formation of a thermo-

74"

irreversible gel (Figure 1). On basis of this gel formation, KGM can be made into

75"

various traditional foods, such as konjac tofu in China and konnyaku or shirataki

76"

noodles in Japan. Preliminary research shows that the acetyl groups of KGM are

77"

removed during gel formation,12 so that the main component of konjac foods is

78"

deacetylated konjac glucomannan (Da-KGM) rather than KGM. However, the acetyl

79"

groups of KGM are considered to have functional importance, contributing to its

80"

solubility properties, molecular chain morphology, intramolecular hydrogen

81"

bonding interactions and gel properties.13 Most research to date on Da-KGM has

82"

focused on its physicochemical and rheological properties,14 and there have been no

83"

studies on the effects of Da-KGM. The physicochemical and rheological properties of

84"

dietary fiber are proposed to be important mechanistic factors in reducing plasma 4" "



85"

lipid.15 Our previous studies showed that Da-KGM has poor lipid binding capacity

86"

compared with KGM in gastrointestinal-simulating experiments in vitro.16 These

87"

results indicate that KGM and Da-KGM may have different effects on the intestinal

88"

absorption of lipids, which may result in different lipid-lowering effects. To our

89"

knowledge, there have been no studies on the lipid-lowering effects of Da-KGM to

90"

date.

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In the present study, we investigate the effects of KGM and Da-KGM on weight

92"

gain, blood lipid levels and hepatic steatosis in high-fat diet-fed C57BL/6 mice. We

93"

then determine the influence of KGM and Da-KGM on food intake, fecal lipid

94"

excretion, SCFA profiles, antioxidant capacity and expression of genes that regulate

95"

lipid metabolism i.e. cytochrome P450 7A1 (CYP7A1), low-density lipoprotein

96"

receptor (LDLR), scavenger receptor class B type I receptor (SRBI) and farnesoid X

97"

receptor (FXR), to investigate the mechanism for any difference in the effects of KGM

98"

and Da-KGM on lipid metabolism.

99" 100"

! MATERIALS AND METHODS

101"

Chemicals."KGM was purchased from Shiyan Flower Fairy Konjac Productions

102"

Co., Ltd (Hubei, China). Da-KGM was laboratory-made according to a traditional

103"

konjac food production process. The acetyl groups of Da-KGM were verified to be

104"

completely removed by FT-IR (see Supporting information). Assay kits for TC, TG,

105"

total bile acid (TBA), high-density lipoprotein cholesterol (HDL-C) and LDL-C were 5" "


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purchased from Biosino Bio-Technology and Science Co., Ltd (Beijing, China). Assay

107"

kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST),

108"

glutathione peroxidase (GPx), superoxide dismutase (SOD) and malondialdehyde

109"

(MDA) and were purchased from Strong Biotechnologies (Beijing, China).

110"

TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix and

111"

TransStart Tip Green qPCR SuperMix were purchased from Transgen (Beijing,

112"

China). Other solvents or chemicals were analytical grade.

113"

Animals and experimental design. Four-week-old C57BL/6 male mice (initial

114"

body weight 13-15 g) were purchased from the Medical Experimental Animal Center

115"

of Guangdong Province (Qualification no. 44007200014783). They were housed in

116"

plastic cages in a laboratory animal room maintained on a standardized 12 h light-

117"

dark cycle at 23 ± 2 °C and a relative humidity of 50-70%. All experimental procedures

118"

were conducted in compliance with the guidelines of Animal Care and Use of South

119"

China Agricultural University. Mice were randomly assigned to one of the following

120"

four groups (n=16 per group): (1) normal chow diet (NCD); (2) high-fat diet (HFD); (3)

121"

high-fat diet with 100 g/kg diet KGM (KGM); (4) high-fat diet with 100 g/kg diet Da-

122"

KGM (Da-KGM). Diet compositions are shown in Table 1. All mice were allowed free

123"

access to water and food throughout the study. Animal bedding and feed were

124"

replaced every three days, food intake was recorded daily and mice were weighed

125"

weekly. After a 10-week experimental period, mice were anesthetized and blood was

126"

collected from the vein of the eye-orbit. Blood serum was separated by centrifugation 6" "



127"

at 2,000 × g for 10 min at 4 °C, and stored at −80 °C. The liver was excised and weighed

128"

and a sample of the upper portion was removed and fixed in 10% buffered formalin

129"

for paraffin sectioning. The remaining liver was divided into two parts. One part was

130"

preserved in liquid nitrogen for RT-PCR and liver lipid analysis, and the other was

131"

homogenized in acetate at a ratio of 1:9 w/v then centrifuged at 12,000 × g for 10 min

132"

at 4 °C to obtain supernatant for liver enzymatic activity measurements. Adipose

133"

tissue from around the abdomen and testicles were excised and weighed. The

134"

contents of the small intestine were scraped out, weighed and stored at −20 °C for

135"

assessment of their viscosity. Small intestine used for determination of GPR41 and

136"

GPR43 expression was washed in PBS and frozen until processed. Fecal samples

137"

were collected during the last experimental week for fecal lipid excretion and SCFA

138"

analyses.

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Liver enzyme activity and blood lipids. Blood lipids (TC, TG, HDL-C, LDL-C),

140"

liver lipids (TC, TG), liver damage indices (AST, ALT) and hepatic antioxidant status

141"

(MDA, SOD, GPx) were measured by Hitachi Automatic Biochemical Analyzer

142"

(Model 7600-010, Hitachi High Technologies Corp., Tokyo, Japan. , following the

143"

manufacturer's instructions for each kit.

144"

Liver and fecal lipids. Total lipids were extracted by Folch’s method with

145"

modifications.17 Briefly, liver and freeze-dried feces were homogenized in a mixture

146"

of chloroform and methanol (2:1 v/v) at a ratio of 1:9 w/v, then centrifuged at 12,000 ×

147"

g for 10 min at 4 °C to obtain a supernatant. This supernatant was dried under 7" "


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nitrogen and redissolved in acetate. TC, TG and TBA of liver and feces were assayed

149"

with commercial kits from Biosino Bio-Technology and Science Co., Ltd.

150"

Small intestinal contents viscosity. Small intestinal contents were centrifuged at

151"

40,000 × g for 30 min at 37 °C. The viscosity of the supernatant was measured using a

152"

cone/plate viscometer (Brookfield Engineering Laboratories Inc., Middleboro, MA,

153"

USA) at 37 °C. Viscosity vs. shear rate was plotted on a log-log scale and the viscosity

154"

estimated by extrapolating the line to a shear rate of 23.0 s. Viscosity was expressed as

155"

millipascal seconds (mPa·s).

156"

Fecal SCFA. Fecal acetate, propionate and butyrate were determined as

157"

follows. Feces were dissolved in water at a ratio of 1:10 (w/v) then digested with

158"

concentrated sulfuric acid, mixed well and centrifuged at 12,000 × g for 20 min at 4 °C.

159"

The supernatant was then passed through a 0.22 μm filter before injection into a

160"

high-performance liquid chromatography (HPLC) instrument (CTO-10AS HPLC,

161"

Shimadzu Corp. Japan) fitted with a chromatographic column (3.5 μm, 4.6 × 150 mm

162"

Waters SunFireWaters, Waters Corp. USA). The flow rate was 1.0 ml/min, the

163"

wavelength of ultraviolet detection was 215 nm, and the eluent was phosphate buffer

164"

(pH 2.8) and acetonitrile at a ratio of 85:15 (v/v).

165"

Histological staining. Liver morphology was observed by hematoxylin-eosin

166"

staining. Liver sections were fixed in 10% formalin and embedded in paraffin after

167"

dehydration by gradient concentrations of ethanol and xylene. Paraffin sections of

168"

liver were then cut into slices of 5 mm thickness and stained with hematoxylin-eosin. 8" "



169"

Liver morphology was observed with an electron microscope (Olympus Optical,

170"

Tokyo, Japan).

171"

RNA extraction and reverse transcriptase-polymerase chain reaction. Total

172"

hepatic RNA was isolated from liver using Trizol reagent. The concentration and

173"

purity of each RNA sample were measured using a Nanodrop ultramicro

174"

spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). cDNA was

175"

synthesized from 1 μg of qualified RNA using the iScript Synthesis kit (Transgen,

176"

Beijing, China). Gene expression of lipid metabolism regulators was assayed using

177"

the Bio-Rad C1000 Thermal Cycler Real-Time PCR System (Bio-Rad," California

178"

USA) with TransStart Tip Green qPCR SuperMix(Transgen, Beijing, China).. β-actin

179"

was used as an internal control, and primer sequences are shown in Table 2. PCR

180"

products were analyzed by electrophoresis on 2% agarose gels stained with ethidium

181"

bromide. Relative gene expression was calculated according to the 2−ΔΔCt method.

182"

Statistical analysis. All experimental results are expressed as the mean ±

183"

standard deviation. Statistical analyses were performed with SPSS v. 19.0 software

184"

(IBM, Armonk, NY). Data were analyzed using one-way analysis of variance and

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Duncan’s multiple range post hoc test. For all comparisons, differences were

186"

considered to be statistically significant at p < 0.05.

187" 188"

!

RESULTS

9" "


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Influence of KGM and Da-KGM on body weight, weight gain, fat weight, fat

190"

index and food intake. After 10 weeks, body weight, weight gain, fat weight and the

191"

fat index of the HFD group were significantly higher than those of the NCD group.

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KGM significantly decreased weight gain, fat weight and fat index compared with

193"

the HFD group; however, no differences were found between Da-KGM and HFD

194"

groups (Table 3). Food intake of the NCD group was significantly higher than that of

195"

the HFD group, whereas there were no significant differences among the HFD, KGM,

196"

and Da-KGM groups (Table 3).

197"

Influence of KGM and Da-KGM on serum lipids. As shown in Figure 2, feeding

198"

of the HFD for 10 weeks resulted in the development of hyperlipidemia, where

199"

serum TC, TG and LDL-C concentrations were significantly higher in the HFD

200"

group than the NCD group. Dietary supplementation with KGM for 10 weeks

201"

induced decreases in serum TC (46.1%), TG (62.3%), and LDL-C (37.5%) compared

202"

with the HFD group, whereas Da-KGM supplementation did not exert a regulatory

203"

effect on blood lipids (Figure 2).

204"

Influence of KGM and Da-KGM on diet-induced hepatic steatosis. In gross

205"

appearance, the livers of HFD mice were a pale color, whereas the livers of mice fed

206"

KGM had a normal appearance (Figure 3A). Consistent with these observations,

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histological analysis showed that the HFD group exhibited characteristics typical of

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steatosis with an accumulation of lipid droplets in the liver. Compared with the HFD

209"

group, KGM and Da-KGM alleviated fat accumulation in liver, though this was more 10" "



210"

pronounced in the KGM group (Figure 3B). In addition, the liver weight and liver

211"

index of the HFD group were significantly higher (p < 0.05) than those of the NCD

212"

group, while there were no significant differences among the HFD, KGM and Da-

213"

KGM groups (Figure 3C). Liver TC and TG concentrations of the HFD group were

214"

significantly higher (p < 0.05) than those of the NCD group, while treatment with

215"

KGM significantly lowered the concentrations of liver TC and TG compared with

216"

those of the HFD and Da-KGM groups (Figure 3D). Furthermore, there were

217"

significant differences (p < 0.05) in ALT and AST values between the HFD and NCD

218"

groups, while KGM significantly lowered ALT levels compared with the HFD and

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Da-KGM groups (Figure 3E).

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Influence of KGM and Da-KGM on small intestinal contents viscosity and

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fecal lipid excretion. As shown in Figure 4A, the small intestinal contents viscosity

222"

of the NCD, HFD and Da-KGM groups was similar (p > 0.05), while KGM treatment

223"

significantly increased this viscosity (p < 0.05). Fecal TC, TG and TBA of mice in the

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HFD group were significantly higher (p < 0.05) than those of the NCD group, and

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KGM treatment further enhanced excretion of these lipids compared with the Da-

226"

KGM and HFD groups (p < 0.05). (Figure 4B,4C,4D)

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Influence of KGM and Da-KGM on fecal SCFA concentrations. As shown in

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Figure 5, the concentrations of propionate and total SCFA were significantly lower

229"

in the HFD group compared with those of the NCD group. Treatment with KGM

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significantly increased the fecal concentrations of acetate, propionate, butyrate and 11" "


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total SCFA compared with the HFD group. There was also a significant difference

232"

between the KGM and Da-KGM groups in the concentration of acetate (KGM 72.6 ±

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10.0 μmol/g vs. Da-KGM 54.5 ± 7.26 μmol/g), propionate (KGM 18.4 ± 4.52 μmol/g vs.

234"

Da-KGM 11.3 ± 1.87 μmol/g), butyrate (KGM 12.5 ± 1.54 μmol/g vs. Da-KGM 7.67 ± 1.38

235"

μmol/g) and total SCFA (KGM 103 ± 14.8 μmol/g vs. Da-KGM 74.5 ± 8.49 μmol/g).

236"

Influence of KGM and Da-KGM on hepatic antioxidant status. The effects of

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KGM and Da-KGM on hepatic antioxidant status are shown in Figure 6. The GPx

238"

and SOD levels of the HFD group were significantly lower (p < 0.05) than those of

239"

the NCD group, while MDA levels were greater in the HFD group than the NCD

240"

group. KGM significantly increased SOD levels and decreased MDA levels

241"

compared with the HFD group, whereas Da-KGM exerted no positive effects on

242"

hepatic antioxidant status.

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Influence of KGM and Da-KGM on gene expression of intestinal GPR43 and

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GPR41 and factors relating to lipid metabolism in liver. As shown in Figure 7,

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GPR41 and GPR43 gene expression was markedly higher in KGM mice compared

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with NCD, HFD or Da-KGM mice. Hepatic gene expression of CYP7A1 and FXR of

247"

the HFD group was significantly upregulated and LDLR and SRBI were

248"

downregulated compared with the NCD group. Treatment with KGM increased the

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gene expression of CYP7A1, LDLR and SRBI compared with the HFD and Da-KGM

250"

groups (p < 0.05). FXR gene expression was upregulated in high-fat diet-fed groups,

12" "



251"

but there were no statistically significant differences among HFD, KGM and Da-

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KGM group .

253" 254"

!

DISSCUSSION

255"

This is the first study to show that unlike native KGM, Da-KGM - the main

256"

component of konjac gel foods - does not help to prevent hyperlipidemia or hepatic

257"

steatosis. To explore the mechanism of action for the different effects of KGM and

258"

Da-KGM, we measured food intake, fecal lipid excretion, fermentation profiles,

259"

antioxidant capacity and expression of genes regulating lipid metabolism in a mouse

260"

model of high-fat feeding with KGM or Da-KGM supplementation. Our results show

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that KGM enhances fecal lipid excretion and SCFA concentration, improves

262"

antioxidant status, regulates the expression of genes related to lipid synthesis and

263"

transport and alleviates hepatic steatosis, whereas Da-KGM has little minimal effect

264"

on these parameters.

265"

The present study shows that feeding mice a high-fat diet for 10 weeks leads to

266"

hyperlipidemia and nonalcoholic fatty liver disease, as indicated by the significant

267"

increase in serum TC, TG and LDL-C and increased fat accumulation in hepatocytes.

268"

Treatment of these mice with KGM alleviated these symptoms, in accordance with

269"

previous studies. 10 The changes in serum TC, TG and LDL of the Da-KGM group

270"

were significantly smaller than those of the KGM group (Figure 2). Furthermore,

271"

KGM greatly decreased fat weight and fat index while Da-KGM did not (Table 2). 13" "


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KGM was also more effective than Da-KGM at lowering the TG and TC content of

273"

the liver, decreasing ALT and AST values, inhibiting hepatic steatosis, and reducing

274"

hepatocellular fat deposit (Figure 3). These results affirm the role of KGM in the

275"

regulation of lipid metabolism, and indicate that Da-KGM is less effective in this role

276"

than KGM. Thus, the notion that konjac foods that mainly consist of Da-KGM have

277"

a lipid-lowering function, and thereby provide health benefits, needs to be

278"

reconsidered.

279"

KGM is a soluble dietary fiber characterized by a high viscosity, though

280"

deacetylation reduces this viscosity (see Supporting information). A number of

281"

previous studies have demonstrated that dietary fiber can markedly reduce food

282"

intake by enhancing satiation or satiety, as a function of the viscosity of the fiber.18,19

283"

Surprisingly, in this study, no difference was seen in the food intake of mice fed a

284"

high-fat diet supplemented with KGM compared with those fed a high-fat diet alone

285"

or one supplemented with Da-KGM. This eliminates food intake as a mechanism for

286"

the different lipid-lowering effects of KGM and Da-KGM. Another potential

287"

mechanism for the different lipid-lowering effects is an increase in fecal bile acid and

288"

fat excretion and/or an impairment in lipid absorption.20 In our study, KGM

289"

significantly enhanced fecal lipid excretion compared with the HFD group, but there

290"

was no difference between the Da-KGM and HFD groups. Viscosity has been

291"

reported to affect gastric lipolysis and reduce lipid emulsification, which are key

292"

processes of lipid absorption in vitro.21,22 In animal studies, high-viscosity fibers have 14" "



293"

been shown to decrease adiposity and reduce hepatic steatosis in rats fed a high-fat

294"

diet.23 Moreover, an in vivo study showed that high-viscosity fiber has better

295"

hypocholesterolemic effects than low-viscosity fiber.24 It has been reported that fiber

296"

viscosity has a positive impact on the viscosity of the small intestinal contents, which

297"

is considered to lead to reduced lipid absorption and lower blood lipid

298"

concentrations.25 Gallaher et al. found that KGM decreased liver cholesterol via

299"

viscosity-mediated interference of cholesterol absorption, by increasing the viscosity

300"

of the small intestinal contents.20 In the current study, KGM supplementation

301"

increased intestinal contents supernatant viscosity and led to greater fecal lipid

302"

excretion, indicating that the lipid-lowering effects of KGM may be partly mediated

303"

by a viscosity-associated reduction in lipid absorption. Conversely, Da-KGM, with

304"

its reduced viscosity following deacetylation, did not increase intestinal contents

305"

supernatant viscosity and fecal lipid outputs, resulting in a lack of ability to decrease

306"

blood lipid concentrations. Therefore, it is likely that viscosity partly mediates the

307"

anti-hyperlipidemia effects, and we suggest that the difference in viscosity between

308"

KGM and Da-KGM contributes to their different effects on blood TG and TC

309"

concentrations.

310"

In addition to enhancement of fecal lipid excretion, changes in colonic

311"

fermentation is another possible mechanism for the lipid-lowering effects of dietary

312"

fiber.26 SCFA are the principal products of colonic fermentation, and 90-95% of

313"

SCFA are acetate, propionate or butyrate. Many studies have found that consuming 15" "


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a high-fiber diet can improve cholesterol metabolism through the SCFA produced

315"

by fermentation.27

316"

of cholesterol and fatty acids, thereby reducing serum cholesterol levels and

317"

regulating blood lipids.29,30 Propionic acid plays a role in lipid metabolism by

318"

inhibiting hepatic lipid biosynthesis.31 Like propionic acid, acetate can also suppress

319"

fat deposition by upregulating the expression of fatty acid oxidation enzymes in the

320"

liver.32 Moreover, butyrate affects the absorptive and metabolic functions of

321"

enterocytes, thus slowing down intestinal fat transport.33 GPR41 and GPR43, two

322"

orphan G protein-coupled receptors, have been reported to be activated by SCFAs.34

323"

In the present study, KGM supplementation resulted in a higher intestinal SCFA

324"

content and upregulated the gene expression of GRR41 and GPR43 compared with

325"

high-fat feeding alone. Conversely, there was no such increase in intestinal SCFA

326"

content or GPR41 or GPR43 expression with Da-KGM supplementation. Qin also

327"

found that Da-KGM presented no obvious effect on the increase of SCFA in vitro

328"

anaerobic fermentation. 35 Given the altered blood lipid profiles, it seems likely that

329"

elevated levels of SCFA, at least in part, contributed to the alleviation of

330"

hyperlipidemia.

28

Previous research suggests that SCFA can inhibit the synthesis

331"

Apart from their role in regulating lipid metabolism, fermentation products

332"

from dietary fiber are considered to exert beneficial physiological functions on

333"

antioxidative capacity. Butyrate has been shown to prevent hydrogen peroxide-

334"

induced DNA damage and modulate the antioxidant defense of colonocytes.36,37 16" "



335"

Prior studies have suggested that KGM can modulate this antioxidant defense

336"

through the antioxidant capacity of its fermentation products.38 An in vitro study also

337"

found that free radicals are eliminated by the fermentation products of KGM.39,40 In

338"

the current study, high-fat feeding led to fat-mediated oxidative stress and increased

339"

levels of lipid peroxidation, as characterized by increased MDA concentrations and

340"

decreased SOD and GSH activities. KGM supplementation improved these indexes

341"

of fat-induced lipid peroxidation, whereas Da-KGM supplementation had little

342"

effect. Furthermore, antioxidant capacity was associated with intestinal SCFA

343"

content in both the KGM and Da-KGM groups. Accordingly, our results indicate a

344"

role of KGM fermentation in the increase in antioxidant capacity, which agrees with

345"

previous studies. Enhancing antioxidant capacity by increasing SCFA production

346"

has been proposed as one of the mechanisms behind the lipid-lowering effects of

347"

KGM. Taken together, we suggest that part of the reason for the decreased efficacy

348"

in maintaining blood lipid homeostasis of Da-KGM compared with KGM is that the

349"

fermentation characteristics of Da-KGM are changed as a result of deacetylation.

350"

Studies have shown that formation of SCFA in the colon is dependent on the

351"

physicochemical characteristics of the fiber, such as solubility, monomeric

352"

composition and type of linkages.41 However, intestinal fermentation is a

353"

complicated process, and it is difficult to identify the specific characteristics

354"

influencing the formation of SCFA. Further studies are needed to determine the

17" "


Page 18 of 41

Page 19 of 41


355"

contributions of these structural characteristics to the differences between KGM and

356"

Da-KGM on the formation of SCFA.

357"

To investigate the underlying molecular mechanisms of KGM and Da-KGM, we

358"

determined the relative levels of gene transcripts involved in lipid metabolism in the

359"

liver. We found that mice fed KGM showed significantly increased expression levels

360"

of CYP7A1 and LDLR compared with Da-KGM-fed and high-fat-fed animals. These

361"

transcription parameters are considered to play a key role in the regulation of

362"

hepatic lipid homeostasis. LDLR plays an important role in lipid metabolism,

363"

maintaining normal blood lipid levels by clearing LDL-C.42,43 CYP7A1 is generally

364"

believed to be the first and rate-determining enzyme for bile acid synthesis in the

365"

liver, and is mainly regulated through feedback inhibition by bile acids reabsorbed

366"

from the intestine.44 SRBI is a key enzyme involved in reverse cholesterol transport,

367"

which plays an important role in preventing hyperlipidemia.45 This study found that

368"

KGM enhances the excretion of bile acids, interrupting the normal feedback

369"

repression of hepatic bile acid synthesis and thus promoting the conversion of

370"

hepatic cholesterol to bile acids by CYP7A1 activation. Then, as a result of the

371"

decreased concentration of hepatic free cholesterol, LDL receptors are stimulated,

372"

promoting the binding of LDL by LDLR-mediated endocytosis. In addition, SRBI

373"

enhancing the synthesis of HDL by facilitates the efflux of cholesterol from cells. In

374"

contrast, Da-KGM showed no effect on fecal excretion of bile acids and CYP7A1

375"

expression. It is therefore likely that fecal bile acid excretion leads to differences in 18" "



376"

liver protein expression, though a direct influence of KGM on liver cholesterol

377"

metabolism cannot be excluded.

378"

In summary, the present study indicates that unlike KGM, Da-KGM does not

379"

affect weight gain, blood lipid levels or hepatic steatosis. Thus, the claim that konjac

380"

gel foods consisting mainly of Da-KGM have excellent pharmacological activity

381"

should be reconsidered. There are two underlying mechanisms for functional

382"

difference between KGM and Da-KGM. First, KGM causes a greater increase in the

383"

viscosity of intestinal contents, which enhances fecal lipid excretion and reduces

384"

lipid absorption. KGM, but not Da-KGM, also increases fecal bile acid excretion,

385"

stimulating the catabolism of cholesterol to bile acids in the liver by upregulating

386"

CYP7A1 expression, thus lowering blood lipid concentrations and preventing liver

387"

steatosis. Second, KGM and Da-KGM exert different effects on SCFA production in

388"

the large intestine, which has a potential role in lipid metabolism and antioxidation.

389"

Taken together, the lipid-lowering and hepatic steatosis prevention effects of KGM

390"

during high-fat feeding may be attributed to its inhibition of lipid absorption and the

391"

formation of SCFA. Da-KGM does not exert the same effects as KGM because of its

392"

deacetylation.

393" 394"

!

395"

Supporting Information

ASSOCIATED CONTENT

19" "


Page 20 of 41

Page 21 of 41


396"

FT-IR spectrum of KGM and Da-KGM(Figure S1) and viscosity measurement of

397"

KGM and Da-KGM(Figure S2) were available.

398"

"

399"

!! AUTHOR IMFORMATION

400"

Corresponding Author

401"

Tel.: +86 20 8528 3448; Fax: +86 20 8528 0270. E-mail: [email protected] (Y.-M. Sun)

402"

"

403"

Acknowledgments

404"

The authors would like to thank Tan Jianbin and Li Xiongcai for their technical

405"

assistance.

406" 407"

Funding

408"

This work was supported by the National Natural Science Foundation of China

409"

(30070533), Provincal Science and Technology Plan Projects in Guangdong Province

410"

(2014A050503059) and The National Key Research and Development Program of

411"

China (2016YFE0106000)."

412" 413"

Notes

414"

The authors declare no competing financial interest.

20" "



415"

!

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416"

Khanna, S.; Tester, R. F. Influence of purified konjac glucomannan on the

417"

gelatinisation and retrogradation properties of maize and potato starches. Food

418"

Hydrocolloid. 2006, 20, 567-576. (2)

419" 420"

glucomannan in the mannan II polymorphic form. Carbohyd. Res. 1992, 229, 41-55. (3)

421" 422"

Yui, T.; Ogawa. K.; Sarko, A. Molecular and crystal structure of konjac

Zhang, Y. Q.; Xie, B. J.; Gan X. Advance in the applications of konjac

glucomannan and its derivatives. Carbohyd. Polym. 2005, 60, 27-31.

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428"

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435"

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Williams, M. A.; Foster,T. J.; Martin D. R.; Norton, I. T.; Yoshimura. M.;

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447"

Li, J.; Ye, T.; Wu, X.; Chen, J.; Wang, S.; Lin, L.; Li, B. Preparation and

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449"

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450"

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451"

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452"

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453"

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456"

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457"

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458"

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on subjective appetite, energy intake and body weight: a systematic review of

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466" 467"

272S-275S.

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reduction by glucomannan and chitosan is mediated by changes in cholesterol

470"

absorption and bile acid and fat excretion in rats. J. Nutr. 2000, 130, 2753-2759.

471"

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Fillery-Travis, A. J. Effect of meal viscosity and nutrients on satiety, intragastric

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dilution, and emptying assessed by MRI. Am. J. Physiol. 2001, 280(6 Part 1), G1227-G1233. 23" "


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Pasquier, B.; Armand, M.; Castelain, C.; Guillon, F.; Borel, P.; Lafont, H.;

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soluble dietary fibres in acidic gastric medium in vitro. Biochem. J. 1996, 314, 269-275. (23)

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Yamamoto, Y.; Sogawa, I.; Nishina, A.; Saeki, S.; Ichikawa, N.; Iibata, S.

478"

Improved hypolipidemic effects of xanthan gum-galactomannan mixtures in rats.

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Biosci. Biotec. Bioch. 2000, 64, 2165-2171. (24)

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Vuksan, V.; Jenkins, A. L.; Rogovik, A. L.; Fairgrieve, C. D.; Jovanovski E;

481"

Leiter, L. A. Viscosity rather than quantity of dietary fibre predicts cholesterol-

482"

lowering effect in healthy individuals. Brit. J. Nutr. 2011, 106, 1349-1352. (25)

483"

Carr, T. P.; Gallaher, D. D.; Yang, C.; Hassel, C. A. Increased intestinal

484"

contents viscosity reduces cholesterol absorption efficiency in hamsters fed

485"

hydroxypropyl methylcellulose. J. Nutr. 1996, 126, 1463-1469. (26) Chiu, Y.; Stewart, M. Comparison of konjac glucomannan digestibility and

486" 487"

fermentability with other dietary fibers in vitro. J. Med. Food. 2012, 15, 120-125.

488"

(27) Jakobsdottir, G.; Nilsson, U.; Blanco, N.; Sterner, O.; Nyman, M. Effects of

489"

soluble and insoluble fractions from bilberries, black currants, and raspberries on

490"

short-chain fatty acid formation, anthocyanin excretion, and cholesterol in rats. J.

491"

Agric. Food Chem. 2014, 62, 4359-4368.

492"

(28) Dos Reis, S. A.; Da Conceicao, L. L.; Rosa, D. D.; Dos Santos Dias, M. M.;

493"

Gouveia Peluzio, M. D. C. Mechanisms used by inulin-type fructans to improve the

494"

lipid profile. Nutr. Hosp. 2015, 31, 528-534. 24" "



495"

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496"

the formation of butyrate, but increases succinate, inflammation, liver fat and

497"

cholesterol in rats, while dietary fibre counteracts these effects. Plos One. 2013, 8,

498"

e8047611. (30) Hijova, E.; Chmelarova, A. Short chain fatty acids and colonic health. Bratisl

499" 500"

Med. J. 2007, 108, 354-358.

501"

(31) Todesco, T.; Rao, A. V.; Bosello, O.; Jenkins, D. J. Propionate lowers blood

502"

glucose and alters lipid metabolism in healthy subjects. Am. J. Clin. Nutr. 1991, 54, 860-

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865. (32)

504"

Kondo, T.; Kishi, M.; Fushimi, T.; Kaga, T. Acetic acid upregulates the

505"

expression of genes for fatty acid oxidation enzymes in liver to suppress body fat

506"

accumulation. J. Agric. Food Chem. 2009, 57, 5982-5986.

507"

(33) Marcil, V.; Delvin, E.; Garofalo, C.; Levy, E. Butyrate impairs lipid transport

508"

by inhibiting microsomal triglyceride transfer protein in Caco-2 cells. J. Nutr. 2003,

509"

133, 2180-2183.

510"

(34) Brown, A. J.; Goldsworthy, S. M.; Barnes, A. A.; Eilert, M. M.; Tcheang, L.;

511"

Daniels, D.; Muir, A. I.; Wigglesworth, M. J.; Kinghorn, I.; Fraser, N. J.; Pike, N. B.;

512"

Strum, J. C.; Steplewski, K. M.; Murdock, P. R.; Holder, J. C.; Marshall, F. H.; Szekeres,

513"

P. G.; Wilson, S.; Ignar, D. M.; Foord, S. M.; Wise, A.; Dowell, S. J. The orphan G

514"

protein-coupled receptors GPR41 and GPR43 are activated by propionate and other

515"

short chain carboxylic acids. J. Biol. Chem. 2003, 278, 11312-11319. 25" "


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Qin, Q. J.; Deng, L.; X, X. Q.; Wang, X. Y.; Zhong, G. Evaluation of prebiotic

517"

functions of konjac glucomannan and its derivatives by fermentation in vitro. Food

518"

Science. 2015 36 217-220.

519"

(36) Rosignoli, P.; Fabiani, R.; De Bartolomeo, A.; Spinozzi, F.; Agea, E.; Pelli, M.

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A.; Morozzi, G. Protective activity of butyrate on hydrogen peroxide-induced DNA

521"

damage in isolated human colonocytes and HT29 tumour cells. Carcinogenesis. 2001,

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22, 1675-1680.

523"

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524"

induces enzymes involved in detoxification of carcinogens and in antioxidative

525"

defence in human colon cells. Brit. J. Nutr. 2010, 104, 1101-1111.

526"

(38) Wu, W.; Chen, H. Konjac glucomannan and inulin systematically modulate

527"

antioxidant defense in rats fed a high-fat fiber-free diet. J. Agri. Food Chem. 2011, 59,

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9194-9200.

529"

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530"

Amorphophallus konjac on cytotoxicity and DNA damage induced by fecal water in

531"

caco-2 cells. Planta Med. 2007, 73, 1384-1388.

532"

(40) Yeh, S. L. Inhibitory effects of a soluble dietary fiber from Amorphophallus

533"

konjac C. Koch on cytotoxicity and DNA damage induced by fecal water in Caco-2

534"

cells. Planta Med. 2007, 73, 1522.

26" "



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535"

Lambo-Fodje, A. M.; Oste, R.; Nyman, M. E. G. L. Short-chain fatty acid

536"

formation in the hindgut of rats fed native and fermented oat fibre concentrates. Brit.

537"

J. Nutr. 2006, 96, 47-55. (42) Jain, K. S.; Kathiravan, M. K.; Somani, R. S.; Shishoo, C. J. The biology and

538" 539"

chemistry of hyperlipidemia. Bioorgan. Med. Chem. 2007, 15, 4674-4699. (43) Tammela, T.; Enholm, B.; Alitalo, K.; Paavonen, K. The biology of vascular

540" 541"

endothelial growth factors. Cardiovasc. Res. 2005, 65, 550-563. (44)

542"

Pullingr, C. R.; Eng, C.; Salen, G.; Shefer, S.; Batta, A. K.; Erickson, S. K.;

543"

Verhagen, A.; Rivera, C. R.; Mulvihill, S. J.; Malloy, M. J.; Kane, J. P. Human

544"

cholesterol 7 alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic

545"

phenotype. J. Clin. Invest. 2002, 110, 109-117.

546"

(45) Van Eck, M.; Twisk, J.; Hoekstra, M.; Van Rij, B. ,; Van der Lans, C.; Bos, I.;

547"

Kruijt, J. K.; Kuipers, F.; Van Berkel, T. Differential effects of scavenger receptor BI

548"

deficiency on lipid metabolism in cells of the arterial wall and in the liver. J. Biol.

549"

Chem. 2003, 278, 23699-23705.

27" "


Page 28 of 41

Page 29 of 41


550"

FIGURE CAPTIONS

551"

Figure 1. The schematic diagram of the mechanism on Konjak gel forming.

552"

(A)chemical structures of KGM (B)chemical structures of Da-KGM (C) schematic

553"

structures of Konjac gel.

554"

Figure 2. Serum TC, TG, LDL-C and HDL-C levels in the C57BL/6 mice fed the high

555"

fat diet containing KGM or Da-KGM. Data are presented as mean ± SD (n = 16) and

556"

were statistically analyzed using one-way ANOVA and Duncan’s test. Different

557"

letters (a, b) represent significant differences among groups when p < 0.05. NCD,

558"

normal control diet; HFD, high fat diet; KGM, high fat diet+KGM(100g/kg diet);Da-

559"

KGM, high fat diet+Da-KGM(100g/kg diet).

560"

Figure 3. Liver gross appearance(A), Photomicrographs of liver sections (B), Liver

561"

weight and liver index(C), hepatic TG and TC levels(D) and hepatic ALT and AST

562"

(E) . The liver stained with hematoxylin and eosin staining (×200 magnification).

563"

Liver index was calculated as liver weight (g)/100 g body weight. Data are presented

564"

as mean ± SD (n = 16) and were statistically analyzed using one-way ANOVA and

565"

Duncan’s test. Different letters (a, b) represent significant differences among groups

566"

when p < 0.05. NCD, normal control diet; HFD, high fat diet; KGM, high fat

567"

diet+KGM (100g/kg diet); Da-KGM, high fat diet+Da-KGM (100g/kg diet).

568"

Figure 4. Small intestinal contents viscosity(A), fecal bile acid (B), fecal

569"

triacylglycerol (C)and fecal total cholesterol (D) levels in the C57BL/6 mice fed the 28" "



570"

high fat diet containing KGM or Da-KGM. Data are presented as mean ± SD(small

571"

intestinal contents viscosity, n=6; fecal lipid ,n=8). Data were statistically analyzed

572"

using one-way ANOVA and Duncan’s test. Different letters (a-c) represent

573"

significant differences among groups when p < 0.05. NCD, normal control diet; HFD,

574"

high fat diet; KGM, high fat diet+KGM(100g/kg diet);Da-KGM, high fat diet+Da-

575"

KGM(100g/kg diet).

576"

Figure 5. Fecal acetate (A), propionate (B), butyrate (C) and total SCFA (D) levels in

577"

the C57BL/6 mice fed the high fat diet containing KGM or Da-KGM. Data are

578"

presented as mean ± SD (n = 8). Data were statistically analyzed using one-way

579"

ANOVA and Duncan’s test. Different letters (a-c) represent significant differences

580"

among groups when p < 0.05. NCD, normal control diet; HFD, high fat diet;

581"

KGM,high fat diet+KGM (100g/kg diet);Da-KGM, high fat diet+Da-KGM (100g/kg

582"

diet).

583"

Figure 6. Hepatic SOD (A), GPx (B) and MDA (C) levels in the C57BL/6 mice fed the

584"

high fat diet containing KGM or Da-KGM. Data are presented as mean ± SD (n = 16).

585"

Data were statistically analyzed using one-way ANOVA and Duncan’s test. Different

586"

letters (a, b) represent significant differences among groups when p < 0.05. NCD,

587"

normal control diet; HFD, high fat diet; KGM, high fat diet+KGM (100g/kg diet);Da-

588"

KGM, high fat diet+Da-KGM (100g/kg diet).

29" "


Page 30 of 41

Page 31 of 41


589"

Figure 7. Relative gene expressions of GPR41 (A), GPR43 (B) in intestine and CYP7A1

590"

(C), LDLR (D), FXR (E) and SRB1 (F) in the liver. The value of the NC group was

591"

designated as 1 for each gene. Data are presented as mean ± SD (n=6). Data were

592"

statistically analyzed using one-way ANOVA and Duncan’s test. Different letters (a,

593"

b) represent significant differences among groups when p < 0.05. NCD, normal

594"

control diet; HFD, high fat diet; KGM, high fat diet+KGM (100g/kg diet);Da-KGM,

595"

high fat diet+Da-KGM (100g/kg diet).

30" "



Figure 1

31" "


Page 32 of 41

Page 33 of 41


Figure 2.

32" "



Figure 3.

33" "


Page 34 of 41

Page 35 of 41


Figure 4.

34" "



Figure 5.

35" "


Page 36 of 41

Page 37 of 41


Figure 6 .

36" "



Figure 7.

37" "


Page 38 of 41

Page 39 of 41


Table1 Ingredient(g/100g NCD

Composition of Experimental Diets HFD

KGM

Da-KGM

diet) Corn starch

51.5

38.0

34.6

34.6

Casein

20

20

20

20

Sucrose

10

10

10

10

corn oil

7

-

-

-

Lard

-

20

20

20

methionine

0.2

0.2

0.2

0.2

Cholesterol

-

0.5

0.5

0.5

Choline

0.2

0.2

0.2

0.2

Cellulose

6.65

6.65

-

Mineral mix

3.5

3.5

3.5

3.5

Vitamin mix

1

1

1

1

KGM

-

-

10

-

Da-KGM

-

-

-

10

The diets were modified from American institute of Nutrition-93M diet.

38" "



Table 2 Sequences of primers used in quantitative real-time reverse transcription polymerase chain reaction Genes

Forward primer

Reverse primer

β-actin

5’-GGGTCAGAAGGACTCCTATG-3’

5’-GTAACAATGCCATGTTACCT-3’

GPR41

5’-GGTCAGTGTAGTCTGTTGGTTC-3’

5’-TTCCAGGTAGCAGGTTCCATT-3’

GPR43

5’-GGTGGAGGCTGTGGTGTTCA-3’

5’-AGGCAGGATTGCGGATCAGTAG-3’

CYP7A1

5’-CTTGAGCCAGAGTCCAATGC-3’

5’-AAGCTCTGTGTCCTCCTGTC-3’

SR-B1

5’-AGTTGGTGAGATCCTGTGGG-3’

5’-TCTTGCTGAGTCCGTTCCAT-3’

LDLR

5’-GACCAGGCCCCTAACTTGTC-3’

5’-ACTACGATGGCTCTGGGTCT-3’

PPARα

5’- GACCTCAGGCAGATCGTCACAG -3’

5’- GTTGTCAGCGGGTGGGACTTTC -3’

FXR

5’- ATGCTCTGCTTACGGCGACAAC -3’

5’-ATGCTGTGGGTCTTCTGGATGGT -3’

39" "


Page 40 of 41

Page 41 of 41


Table 3 Body weight, weight gain , fat weight, fat index , food intake in NCD, HFDfed mice and supplemented with KGM and Da-KGM varialble

NCD

HFD

KGM

Da-KGM

Initial BW,g

15.5±1.58

15.5±1.83

15.4±1.53

15.5±1.84

Final BW ,g

29.1±1.56a

32.2±1.87b

30.7±1.43ab

31.1±1.87b

Weight gain(g)

13.6±1.02a

16.8±1.31b

15.3±1.23a

15.7±1.12ab

Food intake,g/day

3.45±0.42b

2.98±0.51a

2.88±0.54a

2.97±0.56a

Fat weight,g

0.41±0.05a

0.68±0.06b

0.47±0.06a

0.67±0.08b

Fat index, g/100g bw

1.44±0.23a

2.30±0.05b

1.62±0.04a

2.22±0.06b

BW, body weight. Fat index was calculated as fat weight (g)/100 g body weight. Values are expressed as mean±SD (n=16). Means with different superscript letters within a row are significantly different (p

Deacetylated Konjac Glucomannan Is Less Effective in Reducing

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