Effects of Medium- and Long-Chain Triacylglycerols on Lipid

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Effects of Medium- and Long-chain Triacylglycerols on Lipid Metabolism and Gut Microbiota Composition in C57BL/6J Mice Shengmin Zhou, Yueqiang Wang, Jörg Jacoby, Yuanrong Jiang, Yaqiong Zhang, and Liangli (Lucy) Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01803 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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Journal of Agricultural and Food Chemistry

1

Effects of Medium- and Long-chain Triacylglycerols on Lipid

2

Metabolism and Gut Microbiota Composition in C57BL/6J Mice

3 4

Shengmin Zhou,† Yueqiang Wang,§ Jörg J. Jacoby,§ Yuanrong Jiang,§ Yaqiong Zhang,†

5

Liangli (Lucy) Yu*,#

6 7

†

8

Shanghai Jiao Tong University, Shanghai, 200240, China

9

§

Institute of Food and Nutraceutical Science, School of Agriculture and Biology,

Wilmar (Shanghai) Biotechnology Research & Development Center Co., Ltd,

10

Shanghai, 200137, China

11

#

12

MD, 20742, United States

Department of Nutrition and Food Science, University of Maryland, College Park,

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ABSTRACT

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Obesity is related to an increasing risk of chronic diseases. Medium- and long-chain

15

triacylglycerols (MLCT) have been recognized as a promising choice to reduce body

16

weight. In this study, three MLCT with different contents of medium-chain fatty acids

17

(MCFA) (10%-30%, w/w) were prepared, and their effects on lipid metabolism and

18

fecal gut microbiota composition of C57BL/6J mice were systematically investigated.

19

MLCT with 30% (w/w) MCFA showed the best performance in decreasing body

20

weight gain as well as optimizing serum lipid parameters and liver triacylglycerol

21

content. The expression levels of genes encoding enzymes for fatty acid degradation

22

increased markedly and expression levels of genes encoding enzymes for de novo

23

fatty acid biosynthesis decreased significantly in the liver of mice treated with MLCT

24

containing 30% (w/w) MCFA. Interestingly, the dietary intake of a high fat diet

25

containing MLCT did significantly decrease the ratio of Firmicutes to Bacteroidetes

26

and down-regulate the relative abundance of Proteobacteria that may attribute to the

27

weight loss. Furthermore, we found a notable increase in the total short-chain fatty

28

acid (SCFA) content in feces of mice on a MLCT containing diet. All these results

29

may be concomitantly responsible for the anti-obesity effect of MLCT with relatively

30

high contents of MCFA.

31 32

KEYWORDS

33

Medium- and long-chain triacylglycerols, weight loss, lipid metabolism, gut

34

microbiota, short-chain fatty acids 2 ACS Paragon Plus Environment

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INTRODUCTION

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Obesity, the consequence of excess body fat, is associated with an increasing risk of

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chronic diseases including type 2 diabetes (T2D), cardiovascular diseases,

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hypertension, and some forms of cancer.1-3 Because the rate of obesity has risen

39

dramatically in many parts of the world, new efforts have been undertaken to suppress

40

weight accumulation in humans.4-8 Among them, the influence of dietary fat

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composition in energy and weight balance has attracted much more attention.9-11 In

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this respect, medium-chain triacylglycerols (MCT), which are formed by three

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medium-chain fatty acids (MCFA) with 8-12 carbon atoms attached to glycerol, have

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been investigated for their potential in weight loss.12-14 MCFA are absorbed directly

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through the portal vein to the liver and are rapidly oxidized because the

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intramitochondrial transport of MCFA does not require carnitine palmitoyltransferase,

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a rate-limiting enzyme in the mitochondrial β-oxidation pathway.15-16 In contrast, the

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common dietary intake of long-chain fatty acids (LCFA) with carbon atoms exceeding

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12 have a slower metabolism rate, because they must be re-esterified in the small

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intestine and transported by chylomicrons via the lymphatic and vascular system

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before being oxidized to release energy.17 Although MCT have great potential in

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weight management, they are not useful as regular cooking oils, due to their inferior

53

physical properties18-20. Thus, medium- and long-chain triacylglycerols (MLCT)

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containing both MCFA and LCFA have been synthesized to increase the smoking

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point and lower foaming and tested for weight management.12, 21-22

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In recent years, the gut microbiota has been recognized to affect host metabolism 3 ACS Paragon Plus Environment


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through two possible pathways: 1) extract energy and nutrients from food intake; 2)

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influence the host gene expression involved in energy metabolism.23 The relationship

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between the gut microbiota and nutrients in the regular diet including carbohydrates,

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fibers, proteins, and fats have been widely investigated.24 Meanwhile, the

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corresponding effects of the microbiota on human diseases including metabolic,

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immunological, and neurological diseases have also been systematically studied.23, 25

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In this respect, Zhang et al. summarized that the prediabetic individuals showed

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significantly lower abundance of Verrucomicrobia in comparison with the normal

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subjects, suggesting that Verrucomicrobia could be regarded as an effective diagnostic

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biomarker for the progression of T2D.26 Bajaj et al. found that cognitive impairment

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and inflammation in hepatic encephalopathy patients were related to some specific

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bacterial

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Enterobacteriaceae.27 Furthermore, many studies have indicated that energy-related

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obesity is highly associated with an altered composition of gut microbiota.23,

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However, there are only a few studies published focusing on the relationship between

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specific dietary fats with weight losing potential and their effect on the gut

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microbiota,30-31 but none looking at the influence of MLCT on the gut microbiota.

families

including

Alcaligenaceae,

Porphyromonadaceae,

and

28-29

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In our previous study, the safety of MLCT containing 30% (w/w) MCFA was

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assessed in mice and rats, and no dose-related adverse effects were observed in the

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90-day feeding, embryotoxicity and teratogenicity studies.16 In the present study, the

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relationship between MLCT-based diet and fecal gut microbiota was researched for

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the first time to test for the existence of a concomitant effect on weight loss. MLCT 4 ACS Paragon Plus Environment

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with different contents of MCFA (10%-30%, w/w) were prepared through the

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interesterification of long-chain triacylglycerols (LCT) and MCT. The body weight

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change, serum chemistry, liver triacylglycerol, lipid metabolism in liver, gut

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microbiota, and short-chain fatty acids (SCFA) production in C57BL/6J mice were

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comprehensively examined. Our results, for the first time, give a rational explanation

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on the obesity prevention ability of MLCT, which can be attributed in part through the

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modification of the gut microbiota.

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MATERIALS AND METHODS

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Materials. MCT and rapeseed oil (RSO) were kindly provided by Wilmar

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Biotechnology Research & Development Center Co., Ltd (Shanghai, China). MLCT

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including various contents of MCFA (10%, 20%, and 30% (w/w), labeled as MLCT-L,

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MLCT-M, and MLCT-H, respectively) were prepared according to our previous

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research.32 The fatty acid and triacylglycerol compositions of RSO and MLCT

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(synthesized according to our previous research32) are reported in Table S1 and Table

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S2, respectively. All other chemicals were analytical grade.

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Animals. All animal experiment procedures were approved by the Medical Ethics

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Committee of Shanghai Jiao Tong University (License Number, syxk (Hu) 2015-024),

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and were in accordance with the National Institute of Health regulations for the care

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and use of animals in research.33 Diet-induced obese C57BL/6J mice were obtained

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from Shanghai Slac Laboratory Animal Co. Ltd (China), where they had been bred

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under Specific Pathogen Free (SPF)-conditions. The mice were housed individually in

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a room at a controlled temperature of 22 ± 2 ºC, 52-56% humidity, and 12 hour 5 ACS Paragon Plus Environment


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light-dark cycle. Feed and drinking water were available ad libitum. After a 7-day

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acclimatization period, the mice entered the study.

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Study design. 40 healthy male C57BL/6J mice (7 weeks old) were used. They

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were randomly divided into five 5 groups (n = 8), including a control group fed with a

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low-fat diet containing 4.3% (w/w) RSO (labelled as LFD), a model group fed with a

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high-fat diet containing 20.0% (w/w) RSO (labelled as HFD), and three treatment

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groups containing 20.0% (w/w) MLCT with the gradually increased MCFA contents

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from 10% (w/w) to 30% (w/w) of total oil (labelled as MLCT-L, MLCT-M, and

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MLCT-H) for 6 weeks, respectively. The compositions of the diets are listed in Table

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1. The diets were kindly manufactured by Opensource Animal Diets Co. Ltd (Chang

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Zhou, China) as pellets.

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Body weight and food consumption. The mice were weighed on the initial day of

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administration, once per week during the experimental period, and on the day of

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necropsy. Similarly, food consumption was recorded on the initial day of

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administration, and once per week during the experimental period.

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Serum biochemistry. At the end of the treatment, all mice were deprived of food

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for approximately 16 h. All mice were deeply anaesthetized with isofluorane, and

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exsanguinated by intracardiac punctation and euthanized with CO2. Then, blood

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samples were collected and isolated by centrifugation at 3500 rpm (1200 g), 4 °C for

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10 min. Serum total cholesterol (TC) and triacylglycerol (TG) were enzymatically

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assayed with enzymatic/colorimetric kits (LabAssayTM Triacylglycerol/Cholesterol

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Kit, Wako, Osaka, Japan). Serum high-density lipoprotein cholesterol (HDL-C) was 6 ACS Paragon Plus Environment

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measured by the chemical precipitation method, and low-density lipoprotein

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cholesterol (LDL-C) were measured by the polyvinyl sulfuric acid precipitation

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method using reagents from Roche diagnostics on a MODULAR P-800 automated

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biochemistry analyzer (Roche Diagnostics, Indianapolis, IN, USA).34

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Visceral fat analysis. After blood samples collection, samples of visceral fats (including perirenal and epididymal fats) were quickly removed and weighted.

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Liver triacylglycerol analysis. Livers were excised from the carcass and

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immediately frozen in liquid nitrogen, and stored at −80 °C prior to analysis. Then,

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0.15 g of liver was minced and transferred into a test tube with 6 mL of

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chloroform/methanol (2:1, v/v). The mixture was homogenized for 2 min and

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sonicated for 30 s. After extraction for 2 h with shaking, 2 mL of distilled deionized

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H2O was added, and the samples were centrifuged for 20 min at 3500 rpm (1200 g).

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The bottom layer was carefully aspirated into a new test tube, incubated overnight,

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filtered through a 0.22 µm filter, and dried under nitrogen. Then, liver lipids were

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resuspended

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TriglycerideTM Kit, Wako, Osaka, Japan) for analysis of triacylglycerol.34

and

assayed

with

a

colorimetric/enzymatic

kit

(LabAssay

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RNA extraction, complementary DNA synthesis and gene expression analysis.

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First, liver tissues frozen in liquid nitrogen were preserved in RNALater (Invitrogen

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Life Technologies, Carlsbad, CA), followed by cutting into 0.1 to 0.2g pieces and

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homogenized with a Bertin homogenizer (Bertin Technologies, Villeurbanne, France).

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Then, total RNA was isolated using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA)

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according to the manufacturer’s instructions. First-strand cDNA was subsequently 7 ACS Paragon Plus Environment


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synthesized by using an IScript reverse transcriptase kit (Bio-Rad). Synthesized

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cDNA was amplified for quantitative real time Polymerase Chain Reaction (PCR)

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analysis on an ABI 7900 HT (Applied Biosystems, Carlsbad, CA, USA) in

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combination with the SYBR Green mix (Bio-Rad, Hercules, CA), specific primers,

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and DEPC (diethyl pyrocarbonate) H2O. Forward and reverse primer sequences are

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listed in Table S3. The corresponding amplification condition was as follows: 50 °C

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for 2 min, 95 °C for 10 min, and 40 cycles of amplification at 95 °C for 15 s and

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60 °C for 1 min. Relative mRNA expression levels for each gene were calculated

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using the 2-∆∆CT method.35

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Fecal DNA extraction, PCR amplification, and sequencing. 16S rRNA gene

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sequencing was performed on fecal samples collected from the test mice on Day 0,

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Day 1, Day 3, Week 1, Week 3, and Week 6. 240 samples were stored at -80 °C before

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analysis. Bacterial genome DNA was extracted using InviMag Stool DNA Kit/Kfml

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(STRATEC Molecular, Berlin, Germany) according to the manufacturer’s instructions.

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The extracted DNA was subsequently automated on the KingFisher Flex Magnetic

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Particle Processors (Thermo Scientific Inc., Bonn, Germany). The concentration of

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extracted DNA was evaluated using a Nano-Drop 1000 spectrophotometer

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(Nano-Drop Technologies, Wilmington, DE). The extracted DNA samples were stored

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at -20°C for further analysis. Then, the V1-V3 regions of the bacterial 16S rRNA gene

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were amplified in a PCR system (ABI GeneAmp1 9700, Carlsbad, CA, USA) with the

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following procedure: Denaturing step at 95°C for 2 min and 27 cycles of 95°C for 30

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s, annealing at 55°C for 30s and extension at 72°C for 45 s, followed by final 8 ACS Paragon Plus Environment

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annealing extension step at 72 °C for 10 min using the primers 27F

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(5′-AGAGTTTGATCCTGGCTCAG-3′)

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(5′-TTACCGCGGCTGCTGGCAC-3′). The PCR reactions were performed in

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triplicate in a 20 µL mixture containing 2 µL of 2.5 mM dNTPs, 0.4 µL of each primer

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(5 µM), 0.4 µL of Fast Pfu polymerase (TransGen, China), and 10 ng of template

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DNA. PCR products were visualized on a 2% agarose gel to assess the quality, then

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purified using an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City,

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USA)

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QuantiFluorTM-ST (Promega, USA). Pyrosequencing was performed on a MiSeq

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platform (Illumina, San Diego, USA) at Majorbio Bio-pharm Technology Co., Ltd

177

(Shanghai, China).

according

to

the

and

manufacturer’s

instructions

533R

and

quantified

with

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Bioinformatic analysis. Sequencing data were analyzed using the MOTHUR

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software (http://www.mothur.org/wiki/Main_Page). All the sequencing reads were

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denoised, and low quality sequences, pyrosequencing errors and chimera were

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removed. Thereafter, high-quality reads were classified into operational taxonomy

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units (OTUs) at 97% similarity level. The rarefraction curves, alpha diversity (within

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sample) indices (Chao1, ACE, Shannon and Simpson) and Good’s coverage analysis

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were performed using the MOTHUR software. Principal component analysis (PCA)

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was performed to provide an overview of gut microbial dynamics. BLASTs of

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taxonomic classification down to the phylum and genus level were performed using

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MOTHUR

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(https://www.arb-silva.de/documentation/release-106/).

and

the

Bacterial

SILVA106


database


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Quantitative PCR Analysis of Microbial DNA. Quantitative PCR (qPCR) was

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performed on the fecal samples employed in 16S rRNA gene sequencing. To quantify

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Helicobacter spp., validated primers specific for Helicobacter spp. (QF2:

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5′-ACCAAGGCTATGACGGGTATC-3′;

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5′-CGGAGTTAGCCGGTGCTTATT-3′) were used. To quantify total microbial DNA,

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we used universal bacterial primers AQF1: 5′-TCCTACGGGAGGCAGCAGT-3′ and

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AQR1:

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performed in a 20 µL mixture containing 1 µL DNA template, 10 µL SYBR Green

197

qPCR Mix (Applied Biosystems Carlsbad, CA), 7 µL nuclease-free H2O, and 1 µL of

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each primer. The following RT-PCR protocol was conducted: 95 °C for 4 min,

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followed by 40 cycles of 95 °C for 15 s, 55 °C (QF2/QR2) or 60 °C (AQF1/ AQR1)

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for 1 min. A melting curve (95 °C for 15s, 60 °C for 1min, 95 °C for 15s, 60 °C for

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15s) was performed after amplification to distinguish between the targeted and

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non-targeted PCR products. For qPCR analyses, sample-specific relative abundance

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of Helicobacter spp. was determined as genome equivalents amplified by QF2/QR2

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divided by genome equivalents amplified by AQF1/ AQR1. Relative abundance per

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gram of feces was determined by adjusting the concentrations of Helicobacter spp.

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and microbial DNA.

QR2:

5′-GGACTACCAGGGTATCTAATCCTGTT-3′.

The

reactions

were

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Fecal SCFA content analysis. Fecal SCFA content analysis was performed

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according to our previous study.36 Briefly, 1 g of fresh feces and 10 mL Milli-Q

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deionised H2O was added into 50 mL polypropylene vials. The mixture was shaken

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for 1 min on a vortex mixer. Then, the extraction was performed for 10 min using an 10 ACS Paragon Plus Environment

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ultrasonic cleaning bath (SK7200HP, Kudos Ultrasonic instrument co., Ltd, Shanghai,

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China) operating at 50 kHz frequency, followed by centrifugation for 10 min at 10000

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rpm (7500 g). 1 mL of the supernatant was transferred to 10 mL volumetric flask, 100

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µL of 50 mmol H2SO4 was added, and made up to a volume of 10 mL with Milli-Q

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deionised H2O. The solution was filtered using a 0.45 µm polyvinylidene fluoride

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filter. Standard solutions containing 1.0, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0 mg/kg of

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acetic acid, propionic acid,and butyric acid (Sigma-Aldrich Co Ltd, Shanghai, China),

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respectively, were used for calibration (R2 > 0.999). The concentration of SCFA was

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measured using a Metrohm ion chromatograph (850 Professional IC, Metrohm,

220

Herisau, Swizerland) system, fitted with a Metrosep Organic Acids polymer-based

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cation exchanger column (250 mm × 7.8 mm i.d., 9 µm particle size) and connected to

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a conductivity detector (Model 819, Metrohm, Herisau, Switzerland). The column

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oven was kept at 45 °C. An isocratic elution was obtained with 0.5 mmol/L H2SO4 as

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mobile phase at a flow-rate of 0.5 mL/min. Samples were applied with a loop injector

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(20 µL loop). A 50 mM LiCl solution was used as suppressor regenerant. The

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chromatograms were analyzed by MagIC Net software (Metrohm, Herisau,

227

Switzerland).

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Statistical analysis. Statistics were analyzed using SPSS for Windows (version rel.

229

21.0, SPSS Inc., Chicago, IL). Statistical significance was declared at P < 0.05.

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Continuous variables for body weight gain, visceral fat weight, serum biochemistry,

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liver TG, bacterial richness, diversity indexes, and SCFA content were reported

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separately as mean ± standard deviation (SD), and were subjected to one-way analysis 11 ACS Paragon Plus Environment


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of variance (ANOVA) followed by Tukey’s test.

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RESULTS AND DISCUSSION

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Food consumption and body weight gain. As shown in Table S4, all five groups

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showed no significant differences in Week 1 and Week 6 for food consumption

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(17.99-21.56 g in Week 1 and 16.80-21.77 g in Week 6). However, decreased food

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consumptions could be observed in the three MLCT-treated groups from Week 2 to

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Week 5, but only the MLCT-M group showed significant difference in comparison

240

with the HFD group during this period. This may be attributed to the replacement of

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dietary LCFA for MCFA that can increase satiety and decrease food intake.37-38 As

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expected, there was significant difference (P < 0.05) in the body weight gain between

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the LFD group (3.63 ± 1.69 g) and the HFD group (6.50 ± 1.77 g) after 6 weeks

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(Figure 1), which can be attributed to the notable increase of test oil intake from

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0.8-1.0 g/week to 2.8-3.9 g/week calculated on the basis of oil ratio in diet and food

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consumption. In comparison, the body weight gains in the three MLCT-treated groups

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(4.88 ± 1.25 g, 3.88 ± 1.17 g, and 3.75 ± 1.76 g) were lower than in the HFD group,

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although they consumed the same amount of fats, and were even similar to the LFD

249

group for the MLCT-M and MLCT-H groups, which can be ascribed to the special

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metabolism of MCFA in vivo.39 In this respect, recent studies based on different

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contents of MCFA in fats on body weight gain are consistent with the above result.14,

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40-41

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Serum biochemistry. In general, the changes of serum parameters are closely

254

associated with body weight gains. The serum parameters including TG, TC, HDL-C 12 ACS Paragon Plus Environment

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and LDL-C were analyzed (Figure 2). The HFD group had significantly higher TG

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level (4.63 ± 0.47 mmol/L) than the LFD group (3.29 ± 0.34 mmol/L) (P < 0.05).

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With the gradually increased content of MCFA from 10% to 30% (w/w), serum TG

258

were lowered from 4.00 to 2.55 mmol/L, which were significantly different from that

259

of the HFD group in the MLCT-M and MLCT-H groups (P < 0.05). Similarly, the

260

levels of serum TC and LDL-C in the three MLCT-treated groups were reduced in a

261

similar trend. In addition, the serum HDL-C levels in the MLCT-M and MLCT-H

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groups were significantly higher than the corresponding levels in the HFD group as

263

well as the LFD group (P < 0.05). Taken together, the above observations indicate that

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MLCT can effectively improve lipid metabolism in C57BL/6J mice.

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Visceral fat and liver TG analyses. The detailed data of visceral fat weight

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including perirenal and epididymal fats, and liver TG content are depicted in Figure 3.

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Both perirenal fat and epididymal fat weights were reduced after a diet containing

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MLCT in comparison to the HFD group. This was significant in the group treated

269

with MLCT containing 30% (w/w) MCFA (MLCT-H) for both fats (P < 0.05), but

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only significant for epididymal fat in the MLCT-M group. In addition, three MLCT

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groups showed gradually decreased relative visceral fat weight relative to the HFD

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group that the MLCT-H group had significant difference in comparison with the HFD

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group (P < 0.05), further indicating the good potential of MLCT in suppressing fat

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accumulation. Furthermore, there was a significant difference in the TG content of the

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liver between the LFD group (10.42 µmol/g) and the HFD group (15.88 µmol/g) (P
0.05). Beside fatty acid

292

catabolism by β-oxidation, the TG content of the liver is also influenced by hepatic de

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novo fatty acid synthesis. Therefore, the gene expression for ACC-1, ME, FAS, and

294

SREBP-1c was examined. SREBP-1c, a member of the SREBP family, controls

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predominantly the synthesis of fatty acids.44 The enzyme ACC-1 catalyzes the key

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step of the de novo fatty acid biosynthesis pathway by converting acetyl-CoA to

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malonyl-CoA,45 ME catalyzes the decarboxylation of malate to pyruvate and CO2,46

298

and FAS is a multi-functional homodimeric protein, important for the anabolic 14 ACS Paragon Plus Environment

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conversion of dietary carbohydrates to fatty acids in liver.47 As shown in Figure 4, all

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four genes were up-regulated in the HFD compared to the LFD but down-regulated in

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all three MLCT-treated groups relative to the HFD group, which reached statistical

302

significance in the MLCT-M and MLCT-H groups (P < 0.05), indicating a lower

303

hepatic de novo fatty acid synthesis enzyme expression after intake of MCFA. It

304

seems counterintuitive that a HFD would increase the expression of enzymes involved

305

in de novo FA synthesis in comparison to a LFD, but several recent studies have

306

shown an up-regulation of enzymes involved in de novo FA synthesis as well.48-50 In

307

conclusion, the gene expression analyses suggest that the lowered liver TG content in

308

mice fed with MLCT can likely be attributed to enhanced β-oxidation and decreased

309

de novo fatty acid biosynthesis in these mice.

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Fecal microbiota community. In recent years, it became obvious that the gut

311

microbiota plays an important part in influencing the metabolism of its host. To

312

investigate the effect of MLCT with different contents of MCFA on the gut microbiota,

313

we performed 16S rRNA gene sequencing. As shown in Table S5, a total of 1,138,959

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reads with an average read length of 389 bp were obtained from 240 samples. Each

315

library contains 2450 to 4638 reads, with different phylogenetic OTUs ranging from

316

525 to 1041. There was no significant difference in the fecal microbial richness or

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alpha diversity among all five groups (Table S5). This result indicates that neither the

318

amount of total dietary fat intake nor different ratios of long-chain to medium-chain

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FA could change the gut microbiota species diversity at 97% identity.

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In a next step, a principal component analysis (PCA) of the microbiota 15 ACS Paragon Plus Environment


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communities derived from the mice on Day 0 and the end of the study (Week 6) was

322

performed to further study the variation of different groups. Figure 5 shows the score

323

plot from the PCA that groups the samples based on their overall similarity. It was

324

easily found that the majority of variations in the microbiota communities can be

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explained by PC1 (47.89%), and PC2 accounted for further 27.91%. As expected, at

326

the beginning of the study (Day 0), no significant difference could be observed. After

327

the subsequent intake of a low fat diet or different high fat diets with RSO or MLCT,

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there were significant differences between the microbiota communities of the LFD

329

group and the HFD group after the 6 weeks study. Furthermore, obvious overlaps

330

among the MLCT-M and MLCT-H groups and the LFD group appeared. However, the

331

MLCT-L group does not show an obvious clustering with LFD. These results

332

indicated that dietary intake of MLCT-M and MLCT-H could enhance the overall

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structure of the gut microbiota in mice fed with a high fat diet by shifting the

334

microbiota towards those of mice on the LFD diet.

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Fecal microbiota compositions. As shown in Figure 6a, a total of 6 phyla

336

including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes,

337

Verrucomicrobia were identified taxonomically, and the rest were unclassified

338

bacteria. Among them, two groups of beneficial bacteria are dominant in the fecal

339

microbiota, the Bacteroidetes and the Firmicutes, accounting together for 98.94%

340

(LFD), 96.00% (HFD), 98.61% (MLCT-L), 95.70% (MLCT-M), and 99.21%

341

(MLCT-H) of the total identified bacteria, respectively. As expected, the ratio of

342

Firmicutes to Bacteroidetes (F/B) in the HFD group was notably higher (1.46) than 16 ACS Paragon Plus Environment

Page 16 of 41

Page 17 of 41


343

the ratio in the LFD group (0.43) (Table S7). This is in good agreement with several

344

studies in rodents and humans showing that a high F/B ratio is associated with a high

345

fat diet, weight gain, and obesity.51-54 Interestingly, after intake of MLCT with

346

different contents of MCFA, the ratios of F/B decreased to 0.94 (MLCT-L), 0.34

347

(MLCT-M), and 0.56 (MLCT-H), indicating that the microbiota is not only influenced

348

by the amount but also by the chain-length of triacylglycerol’s fatty acyl moieties. In

349

addition, the relative abundance of Proteobacteria in the HFD group was 2.45%,

350

which was much higher (P < 0.05) than in the LFD group (0.19%). In general, an

351

increased prevalence of Proteobacteria is an important marker for an unstable

352

microbial community (dysbiosis) and a potential diagnostic criterion for diseases like

353

obesity.55 Fei and others identified the obesogenic potential of Proteobacteria in

354

germ-free mice.56 And Everard et al. found the similar result in physiological and

355

pathological conditions.57 Notably, the relative abundance of Proteobacteria in the

356

three MLCT-treated groups markedly decreased to 0.19 (MLCT-L), 0.74 (MLCT-M),

357

and 0.15 (MLCT-H). Thus, the intake of a high fat MLCT diet improved the ratio of

358

F/B and decreased the relative abundance of Proteobacteria in comparison to the

359

HFD group. Indeed, the microbiota compositions of the MLCT diets were closer

360

related to the LFD than the HFD, therefore likely playing a role in the decreased body

361

weight gain observed in these groups.

362

The fecal bacterial composition at the genus level was shown in Figure 6b. It can be

363

observed the higher abundance of the Bacteroidetes phylum in the MLCT diet groups

364

was mainly driven by higher levels of Bacteroides and RC9_gut_group in comparison 17 ACS Paragon Plus Environment


Page 18 of 41

365

to the HFD group. On the contrary, the lower abundance of the Firmicutes phylum in

366

three MLCT-treated groups can be mainly attributed to the decreased levels of

367

Allobaculum and Lachnospiraceae_uncultured (Figure 6b). In addition, it could be

368

observed a major decrease of Helicobacter belonging to Proteobacteria phylum in

369

treatment groups (Figure 6b). Taken together, all these observations were in line with

370

the results obtained at the phylum level.

371

During the fecal 16S rRNA gene sequencing sequence analysis, a dramatic bloom

372

of Helicobacter spp. that is a member of the phylum Proteobacteria can be observed

373

in the HFD group (Figure 6b). Thus, the quantitative PCR of fecal DNA was further

374

performed to validate this result. It can be seen that Helicobacter was uniquely

375

elevated in the HFD group starting at Week 3 (Figure 7), but no significant differences

376

were observed in the other four groups. Generally, Helicobacter tend to colonize the

377

stomach, intestines, liver, bile ducts, and heart.58-59 Enterohepatic Helicobacter

378

species including H. hepaticus, H. bilis, and H. cinaedi, have been detected in the

379

lower intestinal and biliary tract of animals, which may lead to chronic inflammatory

380

bowel and liver diseases in rodents, poultry, and primates, as well as gastroenteritis,

381

cholecystitis, and liver diseases in human beings.58, 60-61. Thus, it can be speculated

382

that the abnormal expansion of Helicobacter may be partially attributable to the

383

retroaction of gene expression in liver for the HFD group.

384

Fecal SCFA analysis. SCFA including acetic acid, propionic acid, and butyric acid

385

are end products from fermentation of mainly dietary fibers by the gut microbiota in

386

the intestine.

They are exhibiting multiple beneficial effects on 18 ACS Paragon Plus Environment

energy

Page 19 of 41


387

metabolism,62-63 such as modulating the balance between fatty acid synthesis and

388

oxidation,64 and activating the sympathetic nervous system by stimulating G-protein

389

coupled receptors 41 (GPR41) in sympathetic ganglia that resulted in an improvement

390

of energy expenditure.65 Therefore, variations of the above gut microbiota may lead to

391

different concentrations of SCFA, thus influencing lipid metabolism in the host. The

392

concentrations of SCFA including acetic acid, propionic acid, and butyric acid were

393

measured in the feces of mice and are shown in Table 2. A significant decrease of total

394

SCFA concentration was obtained from feces of mice on the HFD (21.42 µmol/g)

395

relative to the LFD (31.64 µmol/g). This was mainly driven by a reduction in acetic

396

acid, which was the dominant SCFA of fecal samples in all five groups. After intake

397

of MLCT, the total SCFA concentrations increased dose-dependently from 24.15

398

µmol/g in the MLCT-L group to 35.28 µmol/g in the MLCT-H group, which was due

399

to an increase in the acetic acid concentration. These results are most likely attributed

400

to the increased abundance of Bacteroidetes, because many members of this phylum

401

are known to produce acetic acid as fermentation end products.36, 66 However, the

402

concentration of SCFA in feces not only depends on the production by bacteria in the

403

colon, but also on the absorption and the volume of fecal matter and we cannot

404

exclude that these factors may have played a role in the increase of acetic acid

405

observes in the MLCT diets.

406

In summary, the present study shows that MLCT not only have a direct effect on

407

lipid metabolism in the liver by influencing gene expression and decreasing TG

408

content, but also have an effect on gut microbiome composition, which can also 19 ACS Paragon Plus Environment


409

influence lipid metabolism, e.g. by modulating SCFA production. Thus, both

410

mechanisms may be concomitantly responsible for the obesity prevention of MLCT

411

with relatively high contents of MCFA.

412

AUTHOR INFORMATION

413

Corresponding Author

414

*(Liangli (Lucy) Yu) Phone: +1 301 405 0761. Fax: +1 301 314 3313. E-mail address:

415

[email protected]

416

Funding

417

This research was supported by a special fund for Agro-Scientific Research in the

418

Public Interest (Grant No. 201203069), National High Technology Research and

419

Development Program of China (Grant Nos. 2013AA102202; 2013AA102207), and a

420

grant from Wilmar (Shanghai) Biotechnology Research & Development Center Co.,

421

Ltd.

422

Notes

423

The authors declare no further conflict of interest.

424

ACKNOWLEDGMENTS

425

The authors thank Dr. Fang Liu for guidance with bioinformatics analysis and

426

Songyou Hu for assistance with the animal experiments.


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Tables

633

Table 1. Composition of experimental diets. Ingredients (g/100 g)

LFD

HFD, MLCT-L, MLCT-M, and MLCT-H

Casein, 80 mesh

17.23

14.40

L-Cystine

0.26

0.22

Corn starch

30.19

25.23

Maltodextrin 10

3.02

2.52

Sucrose

35.79

29.92

Cellulose, BW200

4.31

3.60

Test oilsa

4.3

20.0

Vitamin mix V10001

0.86

0.72

Mineral mix S10026

0.86

0.72

Dicalcium phosphate

1.1

0.94

Calcium carbonate

0.47

0.40

Potassium citrate, 1 H2O

1.4

1.2

Choline bitartrate

0.17

0.14

Protein

17.5

14.6

Carbohydrate

69.0

57.7

Fat

4.3

20.0

Total kcal/100g

388.1

472.0

634

a

635

MLCT-H are MLCT with different contents of MCFA (10%, 20% and 30%, w/w,

Test oils for LFD and HFD are rapeseed oil. Test oils for MLCT-L, MLCT-M, and



636

respectively).


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637

Table 2. Fecal content of SCFA in rats (µmol/g). LFD

HFD

MLCT-L

MLCT-M

MLCT-H

Acetic acid

30.91a ± 2.53

19.22b ± 2.28

22.15b ± 2.35

22.39b ± 2.26

33.06a ± 2.16

Propionic acid

0.27a ± 0.01

0.22b ± 0.01

0.11c ± 0.00

0.16d ± 0.01

0.18d ± 0.01

Butyric acid

0.46a ± 0.02

0.47b ± 0.01

0.31b ± 0.05

0.25b ± 0.04

0.27b ± 0.07

Total SCFA

31.64a ± 2.55

21.42b ± 2.31

24.15b ± 3.02

24.61b ± 2.91

35.28a ± 2.16

638

LFD stands for the control group fed with a low-fat diet containing 4.3% (w/w)

639

rapeseed oil; HFD stands for the model group fed with a high-fat diet containing 20.0%

640

(w/w) rapeseed oil. MLCT-L, MLCT-M, and MLCT-H stand for three treatment

641

groups containing 20.0% (w/w) MLCT with the gradually increased MCFA contents

642

from 10% (w/w) to 30% (w/w) of total oil, respectively. Data are expressed as mean ±

643

SD (n = 8). Values with different letters in the same row indicate significant

644

differences (P < 0.05).



645

Figures

646 647

Figure 1. Body weight gain of C57BL/6J mice after 6-weeks treatment. LFD stands

648

for the control group fed with a low-fat diet containing 4.3% (w/w) rapeseed oil. HFD

649

stands for the model group fed with a high-fat diet containing 20.0% (w/w) rapeseed

650

oil. MLCT-L; MLCT-M, and MLCT-H stand for three treatment groups containing

651

20.0% (w/w) MLCT with the gradually increased MCFA contents from 10% (w/w) to

652

30% (w/w) of total oil, respectively. Data are expressed as mean ± SD (n = 8).

653

Different letters indicate significantly different values (P < 0.05), whereas groups with

654

the same letter have no significant differences between each other.


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655

656 657

Figure 2. Serum TG (a), serum TC (b), serum HDL-C (c), and serum LDL-C (d) of

658

C57BL/6J mice after 6-weeks treatment. TC stands for total cholesterol; TG stands for

659

triacylglycerol; HDL-C stands for high-density lipoprotein cholesterol; LDL-C stands

660

for low-density lipoprotein cholesterol. Data are expressed as mean ± SD (n = 8).

661

Different letters indicate significantly different values (P < 0.05), whereas groups with

662

the same letter have no significant differences between each other.



663

664 665

Figure 3. Perirenal fat weight (a), epididymal fat weight (b), relative visceral fat

666

weight (perirenal and epididymal fat weights/body weight) (c), and liver TG (d) of

667

C57BL/6J mice after 6-weeks treatment. Relative visceral fat weight = Visceral fat

668

weight/Body weight. Data are expressed as mean ± SD (n = 8). Different letters

669

indicate significantly different values (P < 0.05), whereas groups with the same letter

670

have no significant differences between each other.


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671 672

β-oxidation

de novo fatty acids synthesis

673

Figure 4. mRNA expressions in the liver after 6-weeks treatment. LCAD stands for

674

long-chain acyl-CoA dehydrogenase; MCAD stands for medium-chain acyl-CoA

675

dehydrogenase; ME stands for malic enzyme; PPARα stands for peroxisome

676

proliferator activated receptor-alpha; FAS stands for fatty acid synthase; ACC1 stands

677

for acetyl-CoA carboxylase 1; SREBP-1c stands for sterol regulatory element binding

678

protein-1c. Data are expressed as mean ± SD (n = 8). Different letters indicate

679

significantly different values (P < 0.05), whereas groups with the same letter have no

680

significant differences between each other.



681 682

`

683

Figure 5. Principal component analysis (PCA) of microbiota communities before and

684

after 6-weeks treatment (n = 8).


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685

686 687

Figure 6. (a) Fecal bacterial composition at the phylum level after 6-weeks feeding (n

688

= 8); (b) Fecal bacterial composition at the genus level that had significant differences

689

between HFD and the other four groups after 6-weeks feeding (n = 8).



690 691

Figure 7. Variation of Helicobacter abundance during 6-weeks study determined by

692

qPCR (n = 8).

693


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