(Lard) diet-induced non-alcoholic fatty liver disease in mice

Subscriber access provided by TUFTS UNIV

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

Journal of Agricultural and Food Chemistry

1

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

2

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

3

pectin

4 5

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

6

1

7

408100, China

8

2

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

9

3

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

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

10

China

11

*Corresponding authors:

12

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

13

district, Chongqing, China.

14

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

15

road, Xi'an, Shaanxi, China.

1

ACS Paragon Plus Environment


16

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

17

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

18

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

19

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

20

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

21

decreased oxidative stress and inflammation through a mechanism regulated by the

22

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

23

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

281

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

282

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

283

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

284

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

285

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

286

observed between palmitic acid levels and biochemical parameters, indicating that

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

14


Page 14 of 40

Page 15 of 40


296

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

297

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

298

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

299

positive correlation between antioxidant and acetic acid levels and between antioxidant

300

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

301

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

302

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

303

propionic acid played critical roles in preventing NAFLD by suppressing oxidative

304

stress and fatty acid synthesis.

305

Pectin Changes the Abundance of Specific Gut Bacteria. SCFAs are

306

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

307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

15



Page 16 of 40

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

contents

326

Porphyromonadaceae and Ruminococcaceae were significantly increased in the colonic

327

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

328

Lachnospiraceae and Lactobacillaceae were observed in the colonic digesta from

329

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

330

abundance of the genera Barnesiella, Anaerobacterium, Bacteroides, Parabacteroides

331

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

332

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

of

HF-fed

mice.

The

abundance

of

microbes

in

the

families

333

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

334

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

335

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

336

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

337

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

338

also had substantial influences on microorganisms associated with the physiology of

339

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

16


Page 17 of 40


340

Lactobacillus, Marvinbryantia and Allobaculum). The relative abundance of

341

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

342

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

343

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

344

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

345

Bacteroides, Parabacteroides, Olsenella, and Bifidobacterium were significantly

346

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

347

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

348

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

349

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

350

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

351

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

352

Bacteroides, Parabacteroides and Lactobacillus contributed to the mechanism

353

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

354

DISCUSSION

355

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

356

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

357

(HF) model that matches modern Western diets

358

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

359

increased serum transaminase levels, oxidative stress and abnormal proinflammatory

360

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

361

NAFLD as a reliable tool

23

23

. Long-term intake of HF has

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

17



Page 18 of 40

362

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

363

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

364

damage

365

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

366

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

367

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

368

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

369

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

370

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

371

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

372

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

373

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

374

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

375

gastrointestinal tract

376

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

377

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

378

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

379

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

380

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

381

de novo lipogenesis, namely ACC and FAS

382

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

383

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

18


Page 19 of 40


28

384

a previous study

385

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

386

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

387

inhibiting hepatic triglyceride accumulation induced by pectin consumption.

388

, 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

390

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

391

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

392

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

393

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

394

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

395

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

396

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

397

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

398

various antioxidants, including SOD and GSH-Px

399

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

400

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

401

p38 MAPK

402

responsible for protective responses and stress-dependent apoptosis reactions

403

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

404

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

405

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

19


34

.


Page 20 of 40

406

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

407

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

408

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

409

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

410

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

411

been shown to inhibit oxidative stress and proinflammatory cytokine production

412

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

413

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

414

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

415

NF-κB

416

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

417

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

418

be verified in further investigations, pectin consumption substantially increased the

419

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

420

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

421

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

422

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

423

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

424

that pectin effectively protected against HF-induced liver injury.

12, 38

36

.

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

425

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

426

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

427

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

20


Page 21 of 40


9, 12

428

and pectin, which are not digested by host enzymes

429

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

430

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

431

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

432

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

433

Henning and colleagues, the relative proportion of Parabacteroides significantly

434

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

435

mice

436

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

437

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

438

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

439

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

440

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

441

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

442

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

443

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

444

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

445

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

446

is associated with an increased abundance of Bifidobacterium

447

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

448

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

449

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


Page 22 of 40

450

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

451

manifestations of low-grade systemic inflammatory responses and improve liver

452

aminotransferase levels in patients with NAFLD

453

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

454

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

455

thereby reducing the risk of NAFLD progression

456

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

457

response

458

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

459

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

460

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

461

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

462

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

463

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

464

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

465

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

466

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

467

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

468

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

469

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

470

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

471

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

22


Page 23 of 40


472

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

473

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

474

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

475

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

476

health.

477 478

ASSOCIATED CONTENT

479

Supporting Information

480

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

481

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

482

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

483

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

484

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

485

abundance of Marvinbryantia in the mouse colon.

486

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

487

Histopathological observation.

488

ACKNOWLEDGEMENTS

489 490

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

491 492

REFERENCES

493

(1) Lopezbote, C. J.; Sanz, M.; Perez, A. P.; Flores, A. Effect of dietary lard on

23



494

performance, fatty acid composition and susceptibility to lipid peroxidation in

495

growing-finishing female and entire male pigs. Can. J. Anim. Sci. 1997, 77, 301-306.

496

(2) Leamy, A. K.; Egnatchik, R. A.; Young, J. D. Molecular mechanisms and the role of

497

saturated fatty acids in the progression of non-alcoholic fatty liver disease. Prog.

498

Lipid Res. 2013, 52, 165-174.

499 500

(3) Fan, J. G.; Cao, H. X. Role of diet and nutritional management in non-alcoholic fatty liver disease. J. Gastroen. Hepatol. 2013, 28, 81-87.

501

(4) Rolo, A. P.; Teodoro, J. S.; Palmeira, C. M. Role of oxidative stress in the

502

pathogenesis of nonalcoholic steatohepatitis. Free Radical Bio. Med. 2012, 52, 59-69.

503

(5) Chomto, P.; Nunthanid, J. Physicochemical and powder characteristics of various

504

citrus pectins and their application for oral pharmaceutical tablets. Carbohyd. Polym.

505

2017, 174, 25-31.

506

(6) Kang, H. J.; Kwon, J. H.; Ahn, D. U.; Lee, J. W.; Lee, W. K.; Jo, C. Effect of citrus

507

pectin oligosaccharide prepared by irradiation on high cholesterol diet b6.kor-apoe

508

mice. Food Sci. Technol. 2009, 18, 884-888.

509

(7) Jiang, T.; Gao, X.; Wu, C.; Tian, F.; Lei, Q.; Bi, J.; Xie, B.; Wang, H.Y.; Chen, S.;

510

Wang, X. Apple-derived pectin modulates gut microbiota, improves gut barrier

511

function, and attenuates metabolic endotoxemia in rats with diet-induced obesity.

512

Nutrients 2016, 8, 126.

513

(8) Khasina, E. I.; Kolenchenko, E. A.; Sgrebneva, M. N.; Kovalev, V. V.;

514

Khotimchenko, Y. S. Antioxidant activities of a low etherified pectin from the

515

seagrass zostera marina. Russ. J. Mar. Biol. 2003, 29, 259-261.

24


Page 24 of 40

Page 25 of 40


516

(9) Tian, L.; Scholte, J.; Borewicz, K.; van den Bogert, B.; Smidt, H.; Scheurink, A. J.;

517

Gruppen, H.; Schols, H. A. Effects of pectin supplementation on the fermentation

518

patterns of different structural carbohydrates in rats. Mol. Nutr. Food Res. 2016, 60,

519

2256-2266.

520

(10) Zhao, Y.; Liu, J.; Hao, W.; Zhu, H.; Liang, N.; He, Z.; Ma, K. Y.; Chen, Z. Y.

521

Structure-specific effects of short-chain fatty acids on plasma cholesterol

522

concentration in male syrian hamsters. J. Agric. Food Chem., 2017, 65, 10984-10992.

523 524

(11) Tilg, H.; Cani, P. D.; Mayer, E. A. Gut microbiome and liver diseases. Gut 2016, 65, 2035-2044.

525

(12) Postler, T. S.; Ghosh, S. Understanding the holobiont: how microbial metabolites

526

affect human health and shape the immune system. Cell Metab. 2017, 26, 110-130.

527

(13) Kondo, M.; Hada, T.; Fukui, K., Iwasaki, A., Higashino, K., Yasukawa, K.

528

Enzyme-linked immunosorbent assay (ELISA) for Aleuria aurantia lectin-reactive

529

serum cholinesterase to differentiate liver cirrhosis and chronic hepatitis. Clin. Chim.

530

Acta 1995, 241, 1-9.

531

(14) Smith, J. L.; Bencivengo, M. M. A review of methods for detection of

532

staphylococcal enterotoxins and evaluation of an enzyme-linked immunosorbent

533

assay applied to foods. J. Food Safety 2010, 7, 83-100.

534

(15) Yuan, L.; Han, X.; Li, W.; Ren, D.; Yang, X. Isoorientin prevents hyperlipidemia

535

and liver injury by regulating lipid metabolism, antioxidant capability, and

536

inflammatory cytokine release in high-fructose-fed mice. J. Agric. Food Chem. 2016,

537

64, 2682-2689.

25



538

(16) Han, L. D.; Xia, J. F.; Liang, Q. L.; Wang, Y.; Wang, Y. M.; Hu, P.; Li, P.; Luo, G. A.

539

Plasma esterified and non-esterified fatty acids metabolic profiling using gas

540

chromatography-mass spectrometry and its application in the study of diabetic

541

mellitus and diabetic nephropathy. Anal. Chim. Acta 2011, 9, 85-91.

542

(17) Li, W.; Li, Z.; Han, X.; Huang, D.; Lu, Y.; Yang, X. Enhancing hepatic protective

543

effect of genistein by oral administration with stachyose in mice with chronic high

544

fructose diet consumption. Food Funct. 2016, 7, 2420-2430.

545

(18) Rohart, F.; Gautier, B.; Singh, A.; Cao, K. A. L. mixOmics: An R package for

546

‘omics feature selection and multiple data integration. PLoS Comput. Biol. 2017, 13,

547

e1005752.

548

(19) Ren, D.; Hu, Y.; Luo, Y.; Yang, X. Selenium-containing polysaccharides from

549

Ziyang green tea ameliorate high-fructose diet induced insulin resistance and hepatic

550

oxidative stress in mice. Food Funct. 2015, 6, 3342-3350.

551

(20) Jeong, Y. H.; Park, J. S.; Kim, D. H.; Kim, H. S. Lonchocarpine increases

552

nrf2/are-mediated antioxidant enzyme expression by modulating AMPK and MAPK

553

signaling in brain astrocytes. Biomol. Ther. 2016, 24, 581-588.

554

(21) Kim, M.; Yoo, G.; Randy, A.; Kim, H. S.; Nho, C. W. Chicoric acid attenuate a

555

nonalcoholic steatohepatitis by inhibiting key regulators of lipid metabolism, fibrosis,

556

oxidation, and inflammation in mice with methionine and choline deficiency. Mol.

557

Nutr. Food Res. 2017, 61, 1600632.

558

(22) Anhê, F. F.; Roy, D.; Pilon, G.; Dudonné, S.; Matamoros, S.; Varin, T. V.; Garofalo,

559

C.; Moine, Q.; Desjardins, Y.; Levy, E.; Marette, A. Apolyphenol-rich cranberry

26


Page 26 of 40

Page 27 of 40


560

extract protects from diet-induced obesity, insulin resistance and intestinal

561

inflammation in association with increased Akkermansia spp. population in the gut

562

microbiota of mice. Gut 2015, 64, 872-883.

563 564

(23) Lau, J. K. C.; Zhang, X.; Yu J. Animal models of non‐alcoholic fatty liver disease: current perspectives and recent advances. J. Pathol. 2017, 241: 36-44.

565

(24) Le Roy, T.; Llopis, M.; Lepage, P.; Bruneau, A.; Rabot, S.; Bevilacqua, C.; Martin,

566

P.; Philippe, C.; Walker, F.; Bado, A.; Perlemuter, G.; Cassard-Doulcier, A. M.;

567

Gérard, P. Intestinal microbiota determines development of non-alcoholic fatty

568

liver disease in mice. Gut 2013, 62, 1787-1794.

569 570 571 572

(25) Koo, S. H. Nonalcoholic fatty liver disease: molecular mechanisms for the hepatic steatosis. Clin Mol. Hepatol., 2013, 19, 210-215. (26) Zhang, W.; Xu, P.; Zhang, H. Pectin in cancer therapy: A review. Trends Food Sci. Technol. 2015, 44, 258-271.

573

(27) Lin, Y.; Vonk, R. J.; Slooff, M. J.; Kuipers, F.; Smit, M. J. Differences in

574

propionate-induced inhibition of cholesterol and triacylglycerol synthesis between

575

human and rat hepatocytes in primary culture. Brit. J. Nutr. 1995, 74, 197-207.

576

(28) Weitkunat, K.; Schumann, S.; Petzke, K. J.; Blaut, M.; Loh, G.; Klaus, S., Effects

577

of dietary inulin on bacterial growth, short-chain fatty acid production and hepatic

578

lipid metabolism in gnotobiotic mice. J. Nutr. Biochem. 2015, 26, 929 -937.

579

(29) Li, W.; Huang, D.; Gao, A.; Yang, X. Stachyose increases absorption and

580

hepatoprotective effect of tea polyphenols in high fructose-fed mice. Mol. Nutr. Food

581

Res. 2016, 60, 502-510.

582

(30) Coccia, E.; Varricchio, E.; Vito, P.; Turchini, G. M.; Francis, D. S.; Paolucci, M.

583

Fatty Acid-Specific Alterations in Leptin, PPAR alpha, and CPT-1 Gene Expression 27



584

in the Rainbow Trout. Lipids 2014, 49, 1033-1046.

585

(31) Zhang, L.; Wang, D.; Wen, M.; Du, L.; Xue, C.; Wang, J.; Xu, J. Rapid modulation

586

of lipid metabolism in C57BL/6J mice induced by eicosapentaenoic acid-enriched

587

phospholipid from Cucumaria frondosa. J. Funct. Foods 2017, 28, 28-35.

588

(32) Toyama, T.; Nakamura, H.; Harano, Y.; Yamauchi, N.; Morita, A.; Kirishima, T.;

589

Minami, M.; Itoh, Y.; Okanoue, T. PPARα ligands activate antioxidant enzymes and

590

suppress hepatic fibrosis in rats. Biochem. Bioph. Res. Co. 2004, 324, 697-704.

591

(33) Fang, Y.; Su, T.; Qiu, X.; Mao, P.; Xu, Y.; Hu, Z.; Zhang, Y.; Zheng, X.; Xie, P.; Liu,

592

Q. Protective effect of alphamangostin against oxidative stress induced-retinal cell

593

death. Sci. Rep. 2016, 6, 21018.

594

(34) Hu, Y.; Hou, Z.; Liu, D.; Yang, X. Tartary buckwheat flavonoids protect hepatic

595

cells against high glucose-induced oxidative stress and insulin resistance via MAPK

596

signaling pathways. Food Funct. 2016, 7, 1523-1536.

597

(35) Ren, D.; Lin, D.; Alim, A.; Zheng, Q.; Yang, X. Chemical characterization of a

598

novel polysaccharide ASKP-1 from Artemisia sphaerocephala Krasch seed and its

599

macrophage activation via MAPK, PI3k/Akt and NF-κB signaling pathway in

600

RAW264.7 cells. Food Funct. 2017, 8, 1299-1312.

601

(36) Andrade-Oliveira, V.; Amano, M. T.; Correa-Costa, M.; Castoldi, A.; Felizardo, R.

602

J.; de Almeida, D. C.; Bassi, E. J.; Moraes-Vieira, P. M.; Hiyane, M. I.; Rodas, A. C.;

603

Peron, J. P.; Aguiar, C. F.; Reis, M. A.; Ribeiro, W. R.; Valduga, C. J.; Curi, R.;

604

Vinolo, M. A.; Ferreira, C. M.; Câmara, N. O. Gut Bacteria Products Prevent AKI

605

Induced by Ischemia-Reperfusion. J. Am. Soc. Nephrol. 2015, 26, 1877-1888.

28


Page 28 of 40

Page 29 of 40


606

(37) Kobayashi, M.; Mikami, D.; Kimura, H.; Kamiyama, K.; Morikawa, Y.; Yokoi, S.;

607

Kasuno, K.; Takahashi, N.; Taniguchi, T.; Iwano, M. Short-chain fatty acids, GPR41

608

and GPR43 ligands, inhibit TNF-ainduced MCP-1 expression by modulating p38 and

609

JNK signaling pathways in human renal cortical epithelial cells. Biochem. Bioph. Res.

610

Co. 2017, 486, 499-505.

611

(38) Segain, J. P.; Raingeard, de la Blétière, D.; Bourreille, A.; Leray, V.; Gervois, N.;

612

Rosales, C.; Ferrier, L.; Bonnet, C.; Blottière, H. M.; Galmiche, J. P. Butyrate inhibits

613

inflammatory responses through NFkappaB inhibition: implications for Crohn’s

614

disease. Gut 2000, 47, 397-403.

615

(39) Duncan, S. H.; Holtrop, G.; Lobley, G. E.; Calder, A. G.; Stewart, C. S.; Flint, H. J.;

616

Contribution of acetate to butyrate formation by human faecal bacteria. Brit. J. Nutr.

617

2004, 91, 915-923.

618

(40) El Kaoutari, A.; Armougom, F.; Gordon, J. I.; Raoult, D.; Henrissat, B. The

619

abundance and variety of carbohydrate-active enzymes in the human gut microbiota.

620

Nat. Rev. Microbiol. 2013, 11, 497-504.

621

(41) Henning, S.M., Yang, J., Hsu, M., Lee, R.P., Grojean, E.M., Ly, A., Tseng, C.H.,

622

Heber, D., Li, Z., Decaffeinated green and black tea polyphenols decrease weight

623

gain and alter microbiome populations and function in diet-induced obese mice. Eur.

624

J. Nutr. 2017, 7, 1-11.

625

(42) Duparc, T.; Plovier, H.; Marrachell, V. G.; Hul, M. V.; Essaghir, A.; Ståhlman, M.;

626

Matamoros, S.; Geurts, L.; Pardo-Tendero, M. M.; Druart, C.; Delzenne, N. M.;

627

Demoulin, J. B.; Merwe, S. W.; Pelt, J.; Bäckhed, F.; Monleon, D.; Everard, A.; Cani,

29



Page 30 of 40

628

P. D. Hepatocyte MyD88 affects bile acids, gut microbiota and metabolome

629

contributing to regulate glucose and lipid metabolism. Gut 2017, 66, 620-632.

630

(43) Avila-Nava, A.; Noriega, L. G.; Tovar, A. R.; Granados, O.; Perez-Cruz, C.;

631

Pedraza-Chaverri, J.; Torres, N. Food combination based on a pre-hispanic Mexican

632

diet decreases metabolic and cognitive abnormalities and gut microbiota dysbiosis

633

caused by a sucrose-enriched high-fat diet in rats. Mol. Nutr. Food Res. 2017, 61,

634

1501023.

635

(44)

Tarantino,

G.;

Finelli,

C.

Systematic

review

on

intervention

with

636

prebiotics/probiotics in patients with obesity-related nonalcoholic fatty liver disease.

637

Future Microbiol. 2015, 10, 889-902.

638 639

(45) Gomes, A. C.; Bueno, A. A.; de Souza, R. G.; Mota, J. F. Gut microbiota, probiotics and diabetes. Nutr J. 2014, 13, 60.

640

(46) Perry, R. J.; Peng, L.; Barry, N. A.; Cline, G. W.; Zhang, D.; Cardone, R. L.;

641

Petersen, K. F.; Kibbey, R. G.; Goodman, A. L.; Shulman, G. I. Acetate mediates a

642

microbiome-brain-β-cell axis to promote metabolic syndrome. Nature 2016, 534,

643

213-217.

644

(47) Lozupone, C. A.; Stombaugh, J. I.; Gordon, J. I.; Jansson, J. K.; Knight, R.

645

Diversity, stability and resilience of the human gut microbiota. Nature 2012, 489,

646

220-230.

30


Page 31 of 40


647

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


Page 32 of 40

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

34


Page 34 of 40

Page 35 of 40


Figure 2

35



Figure 3

36


Page 36 of 40

Page 37 of 40


Figure 4

37



Figure 5

38


Page 38 of 40

Page 39 of 40


Figure 6

39



TOC

40


Page 40 of 40

(Lard) diet-induced non-alcoholic fatty liver disease in mice

Recommend Documents