Pectin alleviates high fat (Lard) diet-induced non-alcoholic fatty liver

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Bioactive Constituents, Metabolites, and Functions

Pectin alleviates high fat (Lard) diet-induced nonalcoholic fatty liver disease in mice: possible role of shortchain fatty acids and gut microbiota regulated by pectin Wenfeng Li, Kun Zhang, and Hongyan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02979 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Journal of Agricultural and Food Chemistry

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Pectin alleviates high fat (Lard) diet-induced non-alcoholic fatty liver disease in

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mice: possible role of short-chain fatty acids and gut microbiota regulated by

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pectin

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Wenfeng Li1,*, Kun Zhang2, and Hongyan Yang3,*

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1

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408100, China

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2

School of Clinical Medicine, Jining Medical University, Jining 272067, China

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3

School of Aerospace Medicine, Fourth Military Medical University, Xi’an 710032,

School of Life Science and Biotechnology, Yangtze Normal University, Chongqing

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China

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*Corresponding authors:

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W.F. Li: [email protected]; +86 23 72792193 (Fax); No. 16, Juxian road, Fuling

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district, Chongqing, China.

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H.Y. Yang: [email protected]; +86 29 83246270 (Fax); No. 169, Changle-West

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road, Xi'an, Shaanxi, China.

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ACS Paragon Plus Environment


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ABSTRACT: Consumption of pectin contributes to changes in the gut microbiota and

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the metabolism of short-chain fatty acids (SCFAs). We aimed to investigate the effects

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of and mechanism by which pectin prevented nonalcoholic fatty liver disease (NAFLD)

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in mice that were fed a high-fat diet containing 30% lard (HF). HF-fed mice that orally

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ingested pectin for eight weeks exhibited improvements in lipid metabolism, and

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decreased oxidative stress and inflammation through a mechanism regulated by the

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mitogen-activated protein kinase pathway. Pectin dose-dependently generated rise in

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acetic acid (from 566.4±26.6 to 694.6±35.9 μmol/mL, p 0.01

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was removed from the candidate pool of FA markers (Figure 4G). As shown in Figure

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4H and I, pectin intake significantly decrease the palmitic acid level in HF-fed mice,

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whereas oleic acid levels were not obviously reduced in mice in the HF-4% pectin

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group. Finally, the sPLS analysis was performed to reveal the correlation between the

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FA candidates and biochemical parameters (Figure 4J). A strong correlation was

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observed between palmitic acid levels and biochemical parameters, indicating that

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palmitic acid was a marker for the effects of pectin on HF-induced NAFLD.

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Pectin Affected the Short Chain Fatty Acid Profile. Pectin consumption is

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associated with changes in levels of short chain fatty acids, including acetic acid,

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propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid 9. As shown

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in the PCA score plot, the major modulator of SCFA metabolism is pectin consumption

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(Figure 5A). HF feeding prominently affected the cecal isovaleric acid content, whereas

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the levels of other SCFAs and the total SCFA content were unaffected (Figure 5B-H).

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Higher total SCFA, acetic acid and propionic acid levels were observed in the

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pectin-treated mice than in the HF-fed mice (Figure 5B-D). Conversely, pectin

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administration reduced the cecal isobutyric acid, isovaleric acid and valeric acid

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contents in HF-fed mice (Figure 5E, G, and H). The cecal butyric acid content was not

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affected by pectin consumption (Figure 5F). The sPLS analysis revealed a strong

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positive correlation between antioxidant and acetic acid levels and between antioxidant

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and propionic acid levels (Figure 5I). In addition, negative correlations were observed

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between parameters of de novo lipogenesis or oxidative stress and acetic acid or

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propionic acid levels, respectively (Figure 5I). Based on these findings, acetic acid and

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propionic acid played critical roles in preventing NAFLD by suppressing oxidative

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stress and fatty acid synthesis.

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Pectin Changes the Abundance of Specific Gut Bacteria. SCFAs are

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produced by different microbes 9, 22. Because we revealed a distinct change in the SCFA

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profile upon pectin intake, we further assessed whether this finding might be associated

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with changes in the gut microbiota composition. A Venn diagram displaying the

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overlapping OUT data from the four groups was developed to better understand their

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mutual microbes (Figure 6A). Four hundred fifty-one OTUs were shared by all samples.

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Moreover, 519 OTUs identified in the pectin-treated mice were not detected in the

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HF-fed mice. The nonmetric multidimensional scaling analysis (NMDS) showed an

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obvious difference in the composition of the gut microbiome between mice in the

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control, HF, HF-4% pectin and HF-8% pectin groups (Figure 6B). The first dimension

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separated control and HF mice from HF-4% pectin and HF-8% pectin mice, irrespective

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of the high fat diet, suggesting that changes may be induced by pectin intake (Figure

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6B). Additionally, the second dimension separated the control and HF-8% pectin groups

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from the HF and HF-4% pectin groups (Figure 6B). The most prevalent phyla in all

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samples were Firmicutes and Bacteroidetes (Figure S3A). Higher levels of Firmicutes

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were detected in the gut digesta from pectin-fed mice than in control- and HF-fed mice.

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At the class level (Figure S3B), Bacteroidia and Bacilli were influenced by pectin

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ingestion. In the colonic digesta, the most abundant order in all groups was

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Bacteroidales (Figure S3C). Long-term ingestion of HF decreased the proportion of

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Bacteroidales; however, pectin supplementation increased its proportion in the colonic

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contents

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Porphyromonadaceae and Ruminococcaceae were significantly increased in the colonic

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digesta of HF-fed mice (Figure S3D). Lower abundances of microbes in the families

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Lachnospiraceae and Lactobacillaceae were observed in the colonic digesta from

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pectin-fed mice than in HF-fed mice. Pectin consumption tended to increase the relative

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abundance of the genera Barnesiella, Anaerobacterium, Bacteroides, Parabacteroides

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and Clostridium IV, and decrease the relative abundance of the genera Lactobacillus,

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Helicobacter, Clostridium XIVa and Alistipes in HF-fed mice (Figure 6C).

of

HF-fed

mice.

The

abundance

of

microbes

in

the

families

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We cross-selected the results of the linear discriminant analysis effect size (LEfSe,

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Figure 6D) and the results of the sPLS analysis of SCFA and gut microbe datasets

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(Figure 6E) to explore which microbes were associated with the pectin-induced changes

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in SCFA metabolism in HF-fed mice. Although 16 genera were related to the regulation

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of SCFA metabolism in the gut of mice co-treated with HF and pectin (Figure 6E), 6

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also had substantial influences on microorganisms associated with the physiology of

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each group (Figure 6D; i.e., Bifidobacterium, Olsenella, Bacteroides, Parabacteroides,

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Lactobacillus, Marvinbryantia and Allobaculum). The relative abundance of

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Lactobacillus was increased by approximately 19-fold in response to HF feeding,

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whereas the abundance of other genera was not significantly altered (Figures 6F and S3).

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Strikingly, the pectin treatment dose-dependently reduced the HF-induced increase in

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the relative abundance of Lactobacillus. Moreover, the relative abundances of

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Bacteroides, Parabacteroides, Olsenella, and Bifidobacterium were significantly

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elevated by approximately 43-fold, 1379-fold, 466-fold and 74-fold, respectively,

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following the consumption of high-dose pectin (Figure 6F), and these effects were also

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dose-dependent. The administration of 4% pectin, but not the high dose of pectin, also

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affected the relative abundance of Allobaculum. Furthermore, the relative abundance of

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Marvinbryantia was not affected by the administration of pectin to HF-fed mice (Figure

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S3). Thus, pectin-induced changes in the abundances of Bifidobacterium, Olsenella,

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Bacteroides, Parabacteroides and Lactobacillus contributed to the mechanism

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regulating the short chain fatty acid profile in the gut of HF-fed mice.

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DISCUSSION

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Nonalcoholic fatty liver disease has emerged as a critical health issue in humans 3.

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The association between NAFLD and obesity led to the development of a high fat diet

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(HF) model that matches modern Western diets

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consistently been shown to cause NAFLD in mice, as evidenced by hepatic steatosis,

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increased serum transaminase levels, oxidative stress and abnormal proinflammatory

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cytokine production, and thus, HF has been applied to establish animal models of

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NAFLD as a reliable tool

23

23

. Long-term intake of HF has

. In the current study, C57BL/6 mice fed a HF including

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30% lard for continuous 12 weeks exhibited NAFLD, which have the hallmark features

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observed in human patients with NAFLD, including obesity, hepatic steatosis and liver

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damage

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widely recognized to comprise a two-hit model 4. Triglyceride accumulation in

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hepatocytes is the first “hit” that occurs as a result of alterations in fatty acid uptake,

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synthesis, or oxidation, as well as defects in fatty acid transporters 4. Pectin ingestion

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markedly inhibited abnormal fat accumulation in the enterocoelia and liver of HF-fed

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mice. ChREBP was identified as a key regulator of lipogenic genes, including FAS and

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ACC, and shown to be associated with increased de novo lipogenesis in NAFLD

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Pectin significantly reduced hepatic ChREBP, FAS and ACC levels in the present study,

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and these decreases primarily affected palmitic acid metabolism in the liver of HF-fed

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mice. Thus, pectin likely ameliorates hepatic steatosis through pathways involving the

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down-regulation of key lipogenesis-related proteins. Pectin is not digested in the

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gastrointestinal tract

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substantial regulatory effect on fat metabolism in the bowel in vivo remains unclear. As

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shown in the study by Tian and co-workers, pectin supplementation modulates the

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composition of the microbiota and SCFA production 9. Among SCFAs, proionate was by

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far the most abundant of the intestinal SCFAs, and it was suggested to inhibit

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lipogenesis by acting on the transcription of several rate-limiting enzymes involved in

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de novo lipogenesis, namely ACC and FAS

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contents negatively correlated with hepatic ChREBP, FAS and ACC levels, which were

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positively correlated with the hepatic palmitic acid level. Consistent with findings from

23, 24

. NAFLD is a continuous spectrum of diseases

26

23

, and its pathogenesis is

25

.

. Accordingly, the mechanism by which pectin exerted a

11, 27

. The acetic acid and propionic acid

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a previous study

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propionic acid concentrations increased in the gut in the present study. Therefore,

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increased acetic acid and propionic acid contents in the gut have important roles in

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inhibiting hepatic triglyceride accumulation induced by pectin consumption.

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, the hepatic palmitic acid level decreased as acetic acid and

The “second hit” of NAFLD is postulated to be associated with lipid peroxidation 29

and oxidative stress

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plays a major role in fatty acid β-oxidation and the regulation of oxidative stress

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Pectin inhibited PPARα activation and the formation of oxidative markers (MDA) in the

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liver of HF-fed mice, suggesting that pectin prevented lipid peroxidation and oxidative

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stress. Nrf2 is a transcription factor with a central role in the protective molecular

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response to oxidative stress in most cells and is activated by ROS 33. In the current study,

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HF significantly increased the total Nrf2 level in the liver, suggesting an overproduction

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of ROS in HF-fed mice. The ROS-activated Nrf2 translocates to the nucleus and

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interacts with antioxidant response elements (AREs), which mediate the transcription of

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various antioxidants, including SOD and GSH-Px

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SOD activities in the liver in the present study, which correlates with Nrf2 activation.

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Importantly, Nrf2 is also modulated by some signaling kinases, such as ERK, JNK and

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p38 MAPK

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responsible for protective responses and stress-dependent apoptosis reactions

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Interestingly, pectin attenuated HF-induced JNK and ERK activation, implying that

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pectin might activate Nrf2 in a MAPK-independent manner and that these changes

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might contribute to the strengthening of antioxidant activity. Moreover, phosphorylated

33

. Palmitic acid activates the nuclear receptor PPAR-α

30

389

33

, which 31, 32

.

. Pectin increased the GSH-Px and

, which are key mediators of stress signals and seem to be mainly

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34

.


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MAPK activates NF-κB to regulate the production of proinflammatory cytokines such

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as TNF-α 35. HF consumption substantially increased hepatic NF-κB and TNF-α levels,

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which reflect liver inflammation. In contrast, the pectin treatment markedly reduced the

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levels of NF-κB and TNF-α in the liver of HF-fed mice, clearly indicating that pectin

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may effectively protect hepatocytes against the inflammatory effects of HF. SCFAs have

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been shown to inhibit oxidative stress and proinflammatory cytokine production

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According to Kobayashi and colleagues, short chain fatty acids, particularly propionate,

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decrease TNF-α-induced MCP-1 expression by suppressing the phosphorylation of p38

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and JNK 37. Moreover, butyrate directly inhibits the activation of the transcription factor

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NF-κB

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acid levels, propionic acid levels and MAPK or NF-κB activity, and, to a lesser extent,

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between butyric acid levels and MAPK or NF-κB activity. Although these findings must

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be verified in further investigations, pectin consumption substantially increased the

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bacterial generation of SCFAs in the gut, thus largely contributing to the prevention of

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oxidative stress and inflammation by regulating the MAPK pathway. Collectively,

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pectin inhibited the first and second “hits” of NAFLD was related to the regulation of

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the MAPK signaling pathway and SCFA metabolism in HF-fed mice. Additionally, the

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analyses of transaminase (AST and ALT) levels and histological observations confirmed

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that pectin effectively protected against HF-induced liver injury.

12, 38

36

.

. In our study, strong negative correlations were observed between acetic

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Consistent with the aforementioned discussion, SCFAs exerted a positive effect on

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preventing HF-induced NAFLD in pectin-treated mice. SCFAs are produced in the large

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intestine by the bacterial fermentation of plant-derived polysaccharides such as cellulose

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and pectin, which are not digested by host enzymes

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the acetate and propionate, whereas Firmicutes, which tend to utilize polysaccharides

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less efficiently, are believed to be the primary producers of butyrate 12. In present study,

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Bacteroides, a genus in the family Bacteroidetes, was related to increases in acetic acid

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and propionic acid levels in the gut, consistent with previous findings 39, 40. According to

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Henning and colleagues, the relative proportion of Parabacteroides significantly

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correlates with weight loss, fat accumulation and the intestinal formation of SCFA in

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mice

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Bacteroides, was significantly increased upon pectin feeding, thereby increasing the

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generation of acetic acid and propionic acid. Interestingly, this study is the first to reveal

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that the intestinal formation of acetic acid and propionic acid are associated with an

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increased abundance of Olsenella in pectin-fed mice. Although butyric acid levels were

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not affected significantly in this study, elevated levels of enteral butyric acid were

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slightly associated with the increase in the abundances of Firmicutes and Allobaculum

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in pectin-treated mice, which could improve host metabolism to resist HF-induced

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NAFLD 42. Conversely, pectin consumption decreased the abundance of Lactobacillus,

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suggesting that Lactobacillus was irrelevant to the prevention of NAFLD in pectin-fed

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mice. As shown in the study by Avila-Nava and co-workers, dietary fiber consumption

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is associated with an increased abundance of Bifidobacterium

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significantly increased the abundance of Bifidobacterium to a greater extent than the HF

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in the present study, indicating that pectin exerts prebiotic effects. Notably, most clinical

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investigations recommend the use of prebiotics/probiotics by patients with different

41

. Bacteroidetes provide most of

. The abundance of another genus (Parabacteroides), which also belongs to

21


43

. Pectin consumption


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stages of NAFLD as an adjunct to standard treatment regimens because they decrease

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manifestations of low-grade systemic inflammatory responses and improve liver

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aminotransferase levels in patients with NAFLD

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with a more balanced secretion of gastrointestinal hormones, namely, a low level of

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conversion of non-digestible carbohydrates (fibers) into short-chain fatty acids and

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thereby reducing the risk of NAFLD progression

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reports, these effects of probiotics increase insulin sensitivity and reduce autoimmune

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response

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flora of mice that were subjected to a high-fat diet, leads to an increase of

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glucose-stimulated insulin secretion, ghrelin secretion, hyperphagia and obesity

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seems contradictory that both HF as risk factor and pectin as a protective factor

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encouraged an increase in the acetic level. Lozupone and colleagues reported that the

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functional gene profiles are quite similar, despite the highly disparate compositions of

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gut microbiota in different individuals 47. Thus, the increase in the acetic level of HF-fed

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and pectin-treated mice might be due to different gut microorganisms, which displayed

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a converse health influence in vivo. Although the therapeutic use of prebiotics and

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probiotics for NAFLD is not supported by high-quality clinical studies

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the present study suggest that pectin is a potential prebiotic that might prevent

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HF-induced NAFLD by modulating the gut microbial composition and production of

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short-chain fatty acids in the gut.

44, 45

44

. These changes may be associated

44

. However, according to some

. For example, enhanced generation of acetic acid by modified intestinal

44

46

. It

, results from

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In conclusion, pectin prevents nonalcoholic fatty liver disease in mice fed a high

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fat diet enriched in lard. More importantly, mechanisms by which pectin regulates lipid

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metabolism, anti-oxidants, inflammation, the generation of short-chain fatty acids and

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the gut microbiota play important roles in protecting the liver. All these findings may

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support the hypothesis that non-digestible pectin displays hepatoprotective activity in

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vivo and provide a scientific basis for the application of pectin for beneficial effects on

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health.

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ASSOCIATED CONTENT

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Supporting Information

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Supplementary Figure 1: Fatty acid composition of lard. Supplementary Figure 2:

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The effects of pectin on body form (A), epididymal fat (B and C), liver weight (D and E)

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and levels of two key proteins (F and G) involved in lipogenesis. Supplementary Figure

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3: Bacterial community at the phylum (A), class (B), order (C) and family (D) levels in

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the colon of mice. Supplementary Figure 4: The effects of different diets on the relative

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abundance of Marvinbryantia in the mouse colon.

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Supplementary method: Materials. Short-chain fatty acid (SCFA) measurements.

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Histopathological observation.

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ACKNOWLEDGEMENTS

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This study was supported by the Plan for Supporting the Development of Youth Scientific Research Talent of Yangtze Normal University.

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Figure captions

648

Figure 1 Effect of pectin on body weight and lipid metabolism in mice fed a high fat

649

diet for consecutive 12 weeks. (A) Body weight evolution; (B) BMI = body weight (g) /

650

(body length (cm))2; (C) epididymal fat weight; (D) fat index = epididymal fat weight (g)

651

/ body weight (g); and (E) food intake. (F) Serum total glycerin; (G) hepatic total

652

glycerin; (H) serum total cholesterol; (I) hepatic total cholesterol; (J) serum low-density

653

lipoprotein cholesterol; (K) serum high-density lipoprotein cholesterol; (L) hepatic

654

non-esterified fatty acid; (M) hepatic acetyl-coenzyme A carboxylase; and (N) hepatic

655

fatty acid synthase levels. (O) Representative histological images of liver Oil Red O

656

staining (400×). Data in histograms are presented as the means ± SD. For ELISA results,

657

each point in a group represents a value from a sample, and its average value is

658

presented as a horizontal line. Mean values with different alphabetical letters indicate a

659

statistically significant difference between the groups, as determined by ANOVA and

660

Tukey’s post hoc test (p < 0.05).

661 662

Figure 2 Pectin alleviated oxidative stress in the liver of mice fed a high fat diet. (A)

663

Levels of peroxisome proliferator activated receptor-α, (B) malondialdehyde, and (C)

664

hydroxyl radical scavenging activity. (D) Activities of glutathione peroxidase and (E)

665

total activities of superoxide dismutase. (F) Levels of phosphorylated extracellular

666

signal-regulated

667

phosphorylated p38; (I-K) ERK, JNK and p38, respectively. (L-N) Ratios of

668

pERK/ERK, pJNK/JNK and p-p38/p38, respectively. (O) Level of nuclear factor,

kinase;

(G)

phosphorylated

c-Jun

31


N-terminal

kinase;

(H)


669

erythroid 2-like 2. Data in histograms are presented as the means ± SD. For ELISA

670

results, each point in a group represents a value from a sample, and its average value is

671

presented as a horizontal line. Mean values with different alphabetical letters indicate

672

statistically significant difference between groups, as determined by ANOVA and

673


674 675

Figure 3 Pectin prevented inflammation and injury in the liver of mice fed a high fat

676

diet. (A) Levels of nuclear factor-κB and (B) tumor necrosis factor-. (C) Glutamic

677

oxaloacetic transaminase and (D) glutamic pyruvic transaminase activities. (E)

678

Representative histological images of H&E staining in the liver, scale bar = 100 μm.

679

Data in histograms are presented as the means ± SD. For ELISA results, each point in a

680

group represents a value from a sample, and its average value is presented as a

681

horizontal line. Mean values with different alphabetical letters indicate statistically

682

significant differences between groups, as determined by ANOVA and Tukey’s post hoc

683

test (p < 0.05).

684 685

Figure 4 Pectin alleviated disorders in fatty acid metabolism in the liver of mice fed a

686

high fat diet. (A) PCA score plots for hepatic fatty acids, (B) hepatic saturated fatty

687

acids, (C) hepatic monounsaturated fatty acids, and (D) hepatic polyunsaturated fatty

688

acids. (E) PLS-DA loading plot for hepatic fatty acids. (F) OPLS-DA s-plot for hepatic

689

fatty acids. (G) Z-score and false positive rate analysis of the potential markers (H)

690

hepatic palmitic acid levels and (I) hepatic linoleic acid levels. (J) Heat map showing

32


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Page 33 of 40


691

the results of the correlation analysis. Mean values with different alphabetical letters

692

indicate statistically significant differences between groups, as determined by ANOVA

693

and Tukey’s post hoc test (p < 0.05).

694 695

Figure 5 Pectin altered the cecal short-chain fatty acid metabolism in mice fed a high

696

fat diet. (A) PCA score plots for short-chain fatty acids, (B) total contents of short-chain

697

fatty acids in the cecum, (C) acetic acid, (D) propionic acid, (E) isobutyric acid, (F)

698

butyric acid, (G) isovaleric acid, and (H) valeric acid. (I) Heat map showing the results

699

of the correlation analysis. Mean values with different alphabetical letters indicate

700

statistically significant differences between groups, as determined by ANOVA and

701


702 703

Figure 6 Pectin regulated the composition of the gut microbiota in mice fed a high fat

704

diet. (A) Venn diagram illustrating the overlap of the OTUs identified in the gut

705

microbiota of the four groups. (B) Nonmetric multidimensional scaling analysis of

706

OTUs. (C) Linear relationships between samples and species. (D) Taxonomic

707

cladogram obtained using linear discriminant analysis effect size of the 16s rRNA

708

sequences. (E) sPLS analysis of the relationships between short-chain fatty acids and

709

the gut microbiota. (F) Relative abundances of filtered gut bacteria analyzed by Tukey’s

710

multiple comparisons test and the false discovery rate. Mean values with different

711

alphabetical letters denote significant differences (p < 0.05).

33



Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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TOC

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Pectin alleviates high fat (Lard) diet-induced non-alcoholic fatty liver

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