Antiobesity Effect of Flaxseed Polysaccharide via Inducing Satiety due

Article Cite This: J. Agric. Food Chem. 2019, 67, 7040−7049

pubs.acs.org/JAFC

Antiobesity Effect of Flaxseed Polysaccharide via Inducing Satiety due to Leptin Resistance Removal and Promoting Lipid Metabolism through the AMP-Activated Protein Kinase (AMPK) Signaling Pathway Jianming Luo,‡,† Jiamei Qi,§,† Wenjun Wang,∥ Zhenhuan Luo,∥ Liu Liu,‡ Guangwen Zhang,‡ Qinghua Zhou,*,∥ Jiesheng Liu,*,§ and Xichun Peng*,‡ Downloaded via GUILFORD COLG on July 17, 2019 at 15:31:59 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

Department of Food Science and Engineering, Jinan University, Guangzhou, 510632 Guangdong, China College of Life Science and Technology, Jinan University, Guangzhou, 510632 Guangdong, China ∥ The Center for Precision Medicine of First Affiliated Hospital, Biomedical Translational Research Institute, School of Pharmacy, Jinan University, Guangzhou, 510632 Guangdong, China §

ABSTRACT: Obesity is a metabolic syndrome worldwide that causes many chronic diseases. Recently, we found an antiobesity effect of flaxseed polysaccharide (FP), but the mechanism remains to be elucidated. In this study, rats were first induced to develop obesity by being fed a high-fat diet. The obese rats were then fed a control diet, AIN-93M (group HFD), or a 10% FP diet (group FPD). The body weight, body fat, adipose tissue and liver sections, serous total triglycerides, levels of fasting blood glucose in serum, serous insulin, inflammatory cytokines in serum, and serous proteins within the leptin−neuropeptide Y (NPY) and AMP-activated protein kinase (AMPK) signaling pathway were determined and analyzed. FP intervention significantly reduced body weight and abdominal fat from 530 ± 16 g and 2.15% ± 0.30% in group HFD to 478 ± 10 g and 1.38% ± 0.48% in group FPD, respectively. This effect was achieved by removing leptin resistance possibly by inhibiting inflammation and recovering satiety through the significant downregulation of NPY and the upregulation of glucagon-like peptide 1. Adiponectin was then significantly upregulated probably via the gut−brain axis and further activated the AMPK signaling pathway to improve lipid metabolism including the improvement of lipolysis and fatty acid oxidation and the suppression of lipogenesis. This is the first report of the proposed antiobesity mechanism of FP, thereby providing a comprehensive understanding of nonstarch polysaccharides and obesity. KEYWORDS: obesity, flaxseed polysaccharide, leptin resistance, AMPK signaling pathway

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INTRODUCTION Obesity occurs in 15.1% of women and 11.1% of men aged ≥18 globally on the basis of estimation of the World Health Organization Global Health Observatory data in 2016,1 and 37% of adults and 17% of youth according to the Centers for Disease Control and Prevention from 2011−2014 in the US.2 It is now increasingly recognized in China, where the obese population has risen to 43.2 million in 2014, accounting for 16.3% of global obesity.3 Obesity is recognized as one of the most common metabolic syndromes and is associated with multiple chronic diseases, such as type 2 diabetes, ischemic heart disease, chronic kidney disease, and nonalcoholic fatty liver disease (NAFLD).4,5 Treatments for obesity mainly include drugs, surgery, exercise, and diet.6−8 The key targets for obesity prevention and treatment interventions should be exercise and diet,8 which are two health-associated physiological regulators capable of activating AMP-activated protein kinase (AMPK), a common regulatory mechanism for inhibiting both cholesterol and fatty acid synthesis.9,10 Therefore, applying dietary interventions, including nonstarch polysaccharides (NSPs) such as Konjac glucomannan, has attracted much attention.11 It was reported that NSPs would alter the expression of the upstream proteins of AMPK,12 but only a few studies have reported that NSPs © 2019 American Chemical Society

also affected fatty acid synthase (FAS) or acetyl-CoA carboxylase (ACC) to lead to weight loss. Consequently, whether NSPs regulate other downstream proteins within the AMPK signaling pathway is still unclear. Additionally, one of possible reasons for the antiobesity effect caused by NSPs was the production of gut hormones.13 Gut hormones such as glucagon-like peptide-1 (GLP-1) were reported to improve metabolic status by modulation of microbiota−gut−brain axis in high-fat-diet-fed rats.14 However, the effects of the gut hormone generated from the interactions of NSP consumption and gut microbiota on triggering the AMPK signaling pathway via the gut−brain axis still needs to be investigated. Moreover, a systemic review reported that only some NSPs would manifest an antiobesity effect,15 which had also been indicated by our previous research demonstrating that rats fed high soybean fiber gained more body weight (BW).16 Recently, we found that flaxseed gum (flaxseed polysaccharide, FP) regulated gut microbiota and reduced BW and body fat.17 In Received: Revised: Accepted: Published: 7040

April 17, 2019 June 2, 2019 June 3, 2019 June 3, 2019 DOI: 10.1021/acs.jafc.9b02434 J. Agric. Food Chem. 2019, 67, 7040−7049

Article

Journal of Agricultural and Food Chemistry

93M thereafter, group HFD referred rats fed D12492 during the model development and fed AIN-93M thereafter, and group FPD referred to those rats fed D12492 during the model development and fed an FP containing diet thereafter. The detailed recipes of the diets that we used in this study are listed in Table 1 according to our

this study, we sought to further elaborate the antiobesity mechanism of FP.

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MATERIALS AND METHODS

Reagents and Materials. Araformaldehyde (4%), ethanol, hydrochloric acid, hematoxylin solution, eosin solution, Oil Red O powder, propylene glycol, glycerine, tris buffered saline tween (TBST), bovine serum albumin (BSA), electro-chemiluminescence (ECL) reagent, nonfat milk, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel solution, poly(vinylidene fluoride) (PVDF) membrane, and bicinchoninic acid (BCA) assay kits were purchased from Qiyun Biotechnology Co., Ltd. (Guangzhou, Guangdong, China). Blood glucose detecting test paper (OneTouch SelectSimple) was purchased from Johnson & Johnson Medical Devices Company (Shanghai, China). Enzymelinked immunosorbent assay (ELISA) kits for leptin, primary antibodies against AMP-activated protein kinase-α2 (AMPKα2), and carnitine palmitoyltransferase-1 (CPT-1) were purchased from Abcam Inc. (Shanghai, China). ELISA kits for neuropeptide Y (NPY) were purchased from EMD Millipore Co. (Billerica, MA, USA). ELISA kits for tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were purchased from Neobioscience Biotechnology Co., Ltd. (Shenzhen, Guangdong, China). ELISA kits for hormone sensitive lipase (HSL) were purchased from Huamei Bioengineering Co., Ltd. (Wuhan, Hubei, China). ELISA kits for signal transducer and activator of transcription 3 (STAT3), protein kinase B (AKT), suppressor of cytokine signaling 3 (SOCS3), adiponectin, insulin, GLP-1, peptide YY (PYY), adipose triglyceride lipase (ATGL), and sterol regulatory element binding protein 1 (SREBP-1) were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, Hubei, China). Primary antibody against ACC, FAS, and βactin and horseradish peroxidase (HRP)-linked secondary antibody (goat anti-rabbit or goat anti-mouse) were purchased from Cell Signaling Technology (Danvers, MA, USA). The flaxseed polysaccharide (FP) was generously provided by Professor Yong Wang in Jinan University. The polysaccharide content of FP is more than 93%. It includes two fractions, FP-1 and FP-2, that have molecular weights of 2626 kDa and 1182 kDa, respectively, based on their analysis. FP-1 primarily consists of rhamnose (23.81%), arabinose (10.62%), fucose (3.99%), xylose (35.24%), mannose (0.2%), glucose (2.66%), and galactose (23.39%), while FP-2 primarily consists of rhamnose (11.27%), arabinose (4.03%), fucose (2.82%), xylose (7.12%), mannose (2.99%), glucose (4.41%), galactose (11.61%), and galacturonic acid (55.75%). The main glycosidic bonds of FP-1 are T-linked β-D-xylan, (1 → 4)-linked β-D-xylan, (1 → 2, 3, 4)-linked βD-xylan, (1 → 3)-linked α-arabinose, and T-linked α-arabinose. In addition, the main glycosidic bonds of FP-2 are (1 → 4)-linked α-DGal, (1 → 2, 4)-linked α-D-Gal, and (1 → 2)-linked L-Rhap, with Tlinked D-Glu residues.18 Animals, Diets, and Sample Preparation. In total, 18 male Wistar rats (specific pathogen free (SPF) grade, 4 weeks of age) were purchased from Guangdong Medical Laboratory Animal Center (Foshan, Guangdong, China); they were housed in a SPF grade temperature-controlled room (23 ± 2 °C) with 12 h light/12 h dark cycles. After a 10-day adaptation with a standard diet, D12450B, the rats were randomly divided into 2 cohorts, which were cohort Con (n = 6) and cohort Obesity (n = 12), to develop the obesity model. Rats in cohort Con were fed the standard diet, D12450B, and those in cohort Obesity were fed the high fat diet (HFD), D12492, until the average BW of rats in cohort Obesity was 20% higher than that in cohort Con (approximately 8 weeks). After the obesity model was established, the rats in cohort Con were defined as group Con (n = 6), and the rats in cohort Obesity were randomly divided into two groups, group HFD and group FPD (n = 6 for each). During the diet intervention, group Con and group HFD were fed the control diet, AIN-93M (3.77 kcal/g), and group FPD was fed a 10% FP diet (10% of cornstarch in AIN-93M was replaced by the same amount of FP, 3.37 kcal/g) for 54 days. Generally, group Con referred to those rats fed D12450B during the obesity model development and fed AIN-

Table 1. Detailed Recipes of Each Diet Used in the Current Research (%) ingredients flaxseed polysaccharide corn starch dextrin casein sucrose cellulose soybean oil lard mineral mix ain93 vitamin mix ain93 L-cystine cholinebitartrate

standard diet (D12450B)

high-fat diet (D12492)

control diet (AIN93M)

flaxseed polysaccharide diet

0.00

0.00

0.00

10.00

33.00 3.35 19.13 34.47 4.78 2.39 1.91 3.35

0.00 16.35 26.17 9.00 6.54 3.27 32.06 4.58

46.57 15.50 14.00 10.00 5.00 4.00 0.00 3.50

36.57 15.50 14.00 10.00 5.00 4.00 0.00 3.50

0.96

1.31

1.00

1.00

0.29 0.24

0.39 0.33

0.18 0.25

0.18 0.25

previous reports.16,17 On day 53, all rats were fasted overnight. Rats were anesthetized with sodium pentobarbital. Blood samples from the abdominal aorta were collected and then centrifuged at 12 000 rpm for 30 min. The serum samples were collected and stored at −80 °C until further use. As soon as the rats were sacrificed, abdominal fat, epididymal fat, and liver were collected and weighed individually. The abdominal and epididymal fat ratios were defined as the adipose tissue mass divided by the BW, and the total fat ratio was the sum of abdominal and epididymal fat ratios. Fasting Blood Glucose and Serous Biochemical Measurements. The fasting blood glucose of fasted rats was measured via blood glucose detecting test paper (OneTouch SelectSimple, Johnson & Johnson Medical Devices Companies, Shanghai, China). Briefly, medical-grade alcohol (70% ethanol) was used to sterilize the tails of rats. A blood drop from the caudal vein was obtained by acupuncture and allowed to flow onto the test paper. The test paper was then placed into a reader, and the level of blood glucose was recorded. The analysis of serous total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) was performed via UV−vis spectrophotometer using Konelab 20XTi (Thermo Fisher Scientific, CA, USA). Adipose Tissue and Liver Sections and Histological Analysis. Adipose tissue and livers were sectioned to a proper size and fixed with 4% paraformaldehyde followed by paraffin embedding and then hematoxylin−eosin (HE) staining. The thickness of the slides with HE staining was 3 μm. A part of the liver was first stored at −80 °C and then made into slides by frozen section. A 5% Oil Red O working solution was prepared by dissolving Oil Red O powder in propylene glycol, and the solution was used to stain the sectioned tissues according to the manufacturer’s instructions. Counter staining was conducted with hematoxylin, and the sections were then mounted in glycerine. The thickness of the slides with Oil Red O staining was 3 μm. The histological sections were analyzed using light microscopy (×100 magnification for HE staining and ×200 magnification for Oil Red O staining). ELISA Determinations for Cell Factors in Leptin−NPY Signal and AMPK Signaling Pathway in Serum. ELISA tests for multiple cell factors (proteins) in serum were performed according to the instructions of the manufacturer. In brief, a series of standard solutions and samples (100 μL) were added into appropriate wells. 7041

DOI: 10.1021/acs.jafc.9b02434 J. Agric. Food Chem. 2019, 67, 7040−7049

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Figure 1. FP induced body weight loss and recovered the insulin level. (A) Average body weight (ABW) of the obesity model group (n = 12) and the control group (n = 6). *p < 0.05 and **p < 0.01. (B) ABW of rats during the FP intervention after the development of the obesity model (n = 6 for each group). *p < 0.05 and **p < 0.01 when comparing the ABW of rats in group HFD with those in group FPD. (C) Feed consumption during the whole trial. (D, E) Fasting blood glucose (D) and insulin (E) levels in the three groups (n = 6). The bottom and top of the box represented the 25th and 75th percentiles, respectively. The band indicates the mean value. The range between both ends of the whiskers indicates the values covered from the 10th to the 90th percentiles of the data. *p < 0.05; **p < 0.01; and ns, p > 0.05. Group Con indicates rats fed D12450B during the obesity model development and fed AIN-93M thereafter. Group HFD indicates rats fed D12492 during the model development and fed AIN-93M thereafter. Group FPD indicates rats fed D12492 during the model development and fed an FP containing diet thereafter. The wells were covered and incubated for 2.5 h or 90 min at 37 °C with gentle shaking. Then, the solution was discarded, followed by washing 4 times. Next, 100 μL of antibody solution was added into each well and further incubated for 1 h. The solution was discarded and washed 3−4 times. The HRP-linked secondary antibody working solution (100 μL) was then added and incubated at 37 °C for 15, 45, or 30 min depending on the instructions of different kits. The stop solution (50 μL) was added to each well, and the absorbance at 450 or 450 and 590 nm was read immediately. The levels of each cell factor in different groups were calculated based on the formula generated from the standard curve. Western Blot for AMPK, ACC, CPT-1, and FAS in Serum. The protein concentrations of collected serum were quantified using a BCA assay kit. The concentrations of total protein were normalized based on the results of the BCA assay. The same volume of cell lysate was resolved on a SDS-PAGE gel (10%) followed by trans-blotting onto PVDF membranes. After trans-blotting, the membranes were blocked in TBST containing 5% nonfat milk. The blocked membranes were then incubated with properly diluted primary antibodies in TBST containing either 5% nonfat milk or 5% BSA (following the instructions provided) at 4 °C overnight with gentle shaking. The membranes were then washed three times with TBST for 15 min and

incubated in appropriately diluted HRP-linked secondary antibodies in TBST containing 5% nonfat milk at room temperature for 3 h. Membranes were washed with TBST an additional three times for 10 min and visualized using ECL reagent. The integrated density of each band on the membrane was measured with ImageJ (https://imagej. nih.gov/ij/download.html). Ethics. The animal test was approved by the Institutional Animal Care and Use Committee of Jinan University. All Institutional Animal Care and Use Committee of Jinan University guidelines for the care and use of animals were followed (Ethical Approval No. 2019031224). Statistical Analysis. Statistical analysis was performed with SPSS 20.0 software (IBM Corporation, Armonk, New York). The results were presented as the mean values with standard deviations. Student’s t tests (two tailed) were conducted to compare the data in different groups. Statistical significance was set at a P value less than 0.05.

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RESULTS FP Induced Body Weight Loss and Recovered Insulin Level. We established the rodent obesity model by feeding rats a high fat diet until their BW was 20% higher than that of 7042


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Figure 2. FP reduced body fat and inflammation. (A) Fat ratios (the weight of corresponding fat tissue over the body weight) among the three groups (n = 6). *p < 0.05 when compared with the data in group Con; #p < 0.05 when compared with the data in group HFD; ##p < 0.01 when compared with the data in group HFD. (B) Total triglyceride (TG) among the three groups (n = 6). The bottom and top of the box represents the 25th and 75th percentiles, respectively. The band indicates the mean value. The range between both ends of the whiskers indicates the values 7043


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Journal of Agricultural and Food Chemistry Figure 2. continued

covered from the 10th to 90th percentiles of the data. **p < 0.01; ***p < 0.001; and ns, p > 0.05. (C) HE staining adipose tissue sections among the three groups; scale bar = 50 μm. (D) Oil Red O staining of liver tissue sections among the three groups; scale bar = 25 μm. (E) HE staining of liver tissue sections among the three groups; scale bar = 50 μm. (F) Concentration of inflammatory cytokines in the serum of rats among the three groups (n = 6). *p < 0.05 when compared with the data in group Con; #p < 0.05 when compared with the data in group HFD; ##p < 0.01 when compared with the data in group HFD. Group Con indicates rats fed D12450B during the obesity model development and fed AIN-93M thereafter. Group HFD indicates rats fed D12492 during the model development and fed AIN-93M thereafter. Group FPD indicates rats fed D12492 during the model development and fed an FP containing diet thereafter.

Figure 3. FP removed leptin resistance to offer satiety. (A) Leptin level among the three groups (n = 6). (B, C, E) Levels of downstream factors, including (B) AKT, (C) STAT3, and (E) NPY in the leptin signal (n = 6). (D) SOCS3 level among the three groups (n = 6). (F, G) Levels of gut hormones, including (F) PYY and (G) GLP-1, which could offer satiety (n = 6). The bottom and top of the box represented the 25th and 75th percentiles, respectively. The band indicates the mean value. The range between both ends of the whiskers indicates the values covered from the 10th to the 90th percentiles of the data. *p < 0.05; **p < 0.01; ns, p > 0.05. Group Con indicates rats fed D12450B during the obesity model development and fed AIN-93M thereafter. Group HFD indicates rats fed D12492 during the model development and fed AIN-93M thereafter. Group FPD indicates rats fed D12492 during the model development and fed an FP containing diet thereafter.

the control group (Figure 1A).19 To investigate the BW lowering effect of FP, the FP diet was given to rats (group FPD), which caused a lower BW than that of group HFD beginning on day 12, which was significantly different and

became highly significant on day 42. At the end of the trial (day 54), the BW of rats in group FPD became very similar to those in group Con (Figure 1B), which indicated that the FP intervention induced a loss of BW. In addition, the feed 7044


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Figure 4. FP promoted lipid metabolism through the AMPK signaling pathway. (A) Western blot of AMPKα2, ACC, CPT-1, FAS, and β-actin (internal standard) among the three groups. (B, C, D, E) Integrated densities of (B) AMPKα2, (C) ACC, (D) CPT-1, and (E) FAS over β-actin among the three groups (n = 6). (F, G, H, I) Levels of (F) adiponectin, (G) SREBP-1, (H) ATGL, and (I) HSL among the three groups (n = 6). The bottom and top of the box represents the 25th and 75th percentiles, respectively. The band indicates the mean value. The range between both ends of the whiskers indicates the values covered from the 10th to the 90th percentiles of the data. *p < 0.05; **p < 0.01; ***p < 0.001; ns, p > 0.05. Group Con indicates rats fed D12450B during the obesity model development and fed AIN-93M thereafter. Group HFD indicates rats fed D12492 during the model development and fed AIN-93M thereafter. Group FPD indicates rats fed D12492 during the model development and fed an FP containing diet thereafter.

ratio as well as the total fat ratio of group FPD was significantly lower than that of group HFD (Figure 2A). Serum biochemical indices, including TC, LDL-C, HDL-C, and TG concentrations, were also tested, but only the TG of group FPD was lower than that of group HFD (Figure 2B). To further validate the body fat loss induced by FP, we examined the adipose tissue sections with HE staining and liver sections with Oil Red O staining. The results showed that group HFD presented the largest size of adipocytes and most number of red spots (lipid droplets), which were reduced in group FPD (Figure 2C,D). Obesity will induce liver injury,20 and hence, histological analysis of liver sections with HE staining was also applied. It

consumption of rats in the three groups was similar (Figure 1C). The levels of fasting blood glucose and insulin were then determined. Rats in group HFD had significantly higher blood glucose and lower insulin than those in both group FPD and group Con, while no significant differences in fasting blood glucose and insulin levels were presented between group Con and group FPD (Figure 1D,F). Thus, blood glucose and insulin levels were recovered by the FP intervention. FP Reduced Body Fat and Inflammation. The reduction of fat tissue induced by FP was further investigated. We found no significant difference in the average epididymal fat ratio among the 3 groups, but the average abdominal fat 7045


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Figure 5. Possible antiobesity mechanism of FP. Briefly, FP was consumed by the obese rats, interacted with the gut microbiota, and removed the leptin resistance (probably by inhibiting inflammation). As the leptin resistance was removed, the leptin signal recovered and upregulated STAT3 and AKT, resulting in suppression of NPY expression to offer satiety. Moreover, the FP intervention elevated the gut hormone GLP-1, which delivered the signal (possibly via the gut−brain axis). The brain received the signal and increased the level of adiponectin, which would further stimulate the upregulation of AMPKα. The downstream proteins, such as ATGL, SREBP-1, FAS, ACC, and CPT-1, were then regulated and finally led to the enhancement of lipolysis and fatty acid oxidation as well as the suppression of lipogenesis to promote lipid metabolism. Therefore, the antiobesity effect of FP functioned simultaneously through offering satiety via leptin resistance removal and promoting lipid metabolism via the AMPK signaling pathway. Note that circles and text in red indicate upregulation of proteins in group FPD compared with group HFD. Circles and text in green indicate downregulation of proteins in group FPD compared with group HFD. Circles and text in blue indicate no alteration of proteins in group FPD compared with group HFD. Circles and text in gray indicate that proteins were not determined in the test. The lines with a bar at the end indicate inhibition; the arrows indicate promotion. Group FPD indicates rats fed D12492 during the model development and fed an FP containing diet thereafter. Group HFD indicates rats fed D12492 during the model development and fed AIN-93M thereafter.

(Figure 4F). Furthermore, activated AMPK signaling enhanced fatty acid oxidation by inhibiting ACC and increasing CPT-1 expression (Figure 4A,C,D). In addition, elevated AMPK reduced the level of SREBP-1 and the expression of FAS to inhibit lipogenesis (Figure 4A,G,E). Moreover, upregulated AMPK also caused lipolysis enhancement by upregulating the ATGL but not HSL (Figure 4H,I). Therefore, the antiobesity effect of FP occurred through the promotion lipid metabolism by activating the AMPK signaling pathway in these three aspects. Taken together, FP intervention can effectively reduce the accumulation of abdominal fat that was induced first by the removal of leptin resistance (probably via inhibiting inflammation) followed by the recovery of leptin signaling, which led to the downregulation of NPY. Satiety was then maintained because of the downregulated NPY along with the upregulated gut hormone GLP-1. Adiponectin was further upregulated by elevated GLP-1 (possibly via the gut−brain axis), and the AMPK signaling pathway was activated to improve lipid metabolism, including the improvement of lipolysis and fatty acid oxidation and the suppression of lipogenesis (Figure 5).

was found that the nuclei of liver cells in group HFD were more obvious. The intercellular space in group HFD was also large and arranged loosely, but the nuclei of the liver cells in group FPD became less obvious, and the intercellular space was arranged more closely when compared with those in group HFD (Figure 2E). This result implied that inflammation was reduced by the FP intervention, which was further confirmed by the serum levels of inflammatory cytokines, including TNFα, IL-6, and IL-1β that rose in group HFD and significantly dropped in group FPD (Figure 2F). FP Removed Leptin Resistance to Offer Satiety. Subsequently, the mechanism of the antiobesity effect of FP was explored. The first possible mechanism was related to the leptin signal. The average leptin level of group HFD was higher than that of group Con and group FPD (p < 0.01) (Figure 3A), which indicated that leptin signal transduction was blocked. After the intervention of FP (group FPD), the levels of AKT and STAT3 were significantly upregulated when compared with those of group HFD (Figure 3B,C). Although the level of SOCS3 was not altered (Figure 3D), the NPY content was significantly downregulated in group FPD (Figure 3E). This result implied that the FP intervention recovered the leptin signal transduction and affected the satiety-related hormone NPY. Moreover, the level of the gut hormone GLP-1 of group FPD was significantly higher than that in group HFD, while no significant alteration in the PYY level was found (Figure 3F,G). FP Promoted Lipid Metabolism through the AMPK Signaling Pathway. The AMPK signaling pathway is a classic pathway associated with metabolic function, such as lipid metabolism;21 therefore, we examined several key factors (proteins) within this signaling pathway to investigate the mechanism of the body fat lowering effect of FP after it was consumed and provided satiety. The expression of AMPKα2, the central molecule in the signaling pathway, was significantly upregulated in group FPD compared to group HFD (Figure 4A,B). This may rely on the upregulation of adiponectin, an upstream protein of AMPK

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DISCUSSION

Insulin resistance commonly exists in patients with diabetes who often simultaneously suffer from obesity.22 In our experiments, no insulin resistance was found in group HFD, as the control diet (AIN-93M) but not the high fat diet (D12492) was supplied to them after the obesity model had been established. Instead, the low amount of insulin was not sufficient to maintain the blood glucose at a normal level, and a significantly higher fasting blood glucose was thus detected in group HFD. Conversely, rats fed an FP diet (group FPD) secreted as much insulin as the lean rats (rats fed D12450B during obesity model development and then AIN-93M for the rest of the trial), which indicates that FP was capable of recovering the insulin level and maintaining the normality of the blood glucose level. 7046


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the leptin accumulation caused by a high-fat diet, and most of them had a reported antiobesity effect.38−40 Among those NSPs, inulin and resistant starch were considered to remove the leptin resistance by reducing adipose tissue that secretes leptin, while chitosan removed leptin resistance by increasing LepRb level. In contrast to their results, we proposed FP removed leptin resistance by inhibiting inflammation and then pulling up the leptin signaling pathway. Additionally, we examined two other gut hormones, PYY and GLP-1, which were capable of offering satiety.41 Apparently, FP could not affect the level of PYY but could enhance the secretion of GLP-1 based on our results. As mentioned above, the gut hormone GLP-1 was able to affect the brain through the gut−brain axis, and it possibly triggered the AMPK signaling pathway. It is known that AMPK regulates lipid metabolism by mediating a series of proteins within the AMPK signaling pathway.21 Adiponectin, one of the upstream proteins of the AMPK signaling pathway, can stimulate AMPK expression.42 Our current research displayed the increased level of adiponectin and elevated AMPK expression, which was in accordance with previous reports. The AMPK signaling pathway of lipid metabolism involves three aspects including lipolysis, lipogenesis, and fatty acid oxidation.43 As indicated by our results, only ATGL (and not HSL) was upregulated by FP, though both of them were positively related to the improvement of lipolysis.44,45 In addition, FP-elevated APMK could inhibit the level of SREBP-1, which is a transcription factor that promotes the expression of multiple lipogenic enzymes,46 including FAS. Since SREBP-1 was downregulated, the activity of FAS was also suppressed, which resulted in the inhibition of lipogenesis. Moreover, fatty acids were transported into the mitochondria for β-oxidation by CPT-1, and AMPK increased its activity by inhibiting ACC.47 The similar results in this study regarding ACC and CPT-1 implied that fatty acid oxidation was enhanced by the FP intervention. Some NSPs reportedly affect lipid metabolism via AMPK regulation. For example, soluble dietary fiber can upregulate AMPK, but there is no further discussion on the expression of upstream or downstream proteins in the AMPK signaling pathway.48 Other NSPs, such as sugar cane fiber,49 guar gum,50 oligofructose,51 plantago ovate husk,52 and hydroxypropyl methylcellulose,53 can also regulate AMPK and its downstream proteins SREBP-1, ACC, FAS, and CPT-1, which are involved in lipogenesis and fatty acid oxidation. However, FP regulated not only AMPK signaling pathway-related proteins in lipogenesis and fatty acid oxidation but also ATGL, which resulted in improved lipolysis. It should be noted that only FP could remove leptin resistance and simultaneously activate the AMPK signaling pathway to enhance lipolysis and fatty acid oxidation and to suppress lipogenesis. This is also the first report describing the potential antiobesity mechanism of FP.

As mentioned in our previous reports, FP can lower BW by reducing body fat.17 Therefore, the current results agreed with our previous report. In contrast to other NSPs, such as Ganoderma lucidum polysaccharides, that can lower the epididymal fat ratio,23 FP mainly led to abdominal adipocyte loss. Excessive abdominal adipocytes in the body is called hypertrophy and is considered to be one of the characteristics of obesity.24 Obesity is strongly linked with NAFLD,20 which is not only a chronic disease causing liver injury but also an emerging driving force in chronic kidney disease.25 At the end of the study, it is very possible that rats in group HFD had suffered from NAFLD, judging by the multiple lipid droplets in the liver as well as the histological status, but FP reduced the lipid droplets back to the normal level and relieved the abnormal state in the liver, thus showing a great potential to reverse the progress of NAFLD. Tissue injuries, including those caused by NAFLD, can induce inflammation.26 Obesity is also characterized by a proinflammatory condition in which hypertrophied adipose tissue together with immune cells contribute to increase the level of pro-inflammatory cytokines.27 Cytokines, such as TNF-α, IL-6, and IL-1β, have already been recognized to be positively correlated to obesity.28,29 According to our current data, the consumption of FP alleviated the obesity-inducing liver inflammation (group HFD) probably by reducing the level of inflammatory cytokines, including TNF-α, IL-6, and IL-1β. This is the first report that investigated the anti-inflammatory ability of FP. Since obesity and inflammation are closely related, we believe the antiobesity effect of FP is the cause and the result of inhibited inflammation. Leptin resistance is a phenomenon associated with obesity that is similar to insulin resistance in patients with type 2 diabetes.30 It is characterized as the elevated circulating levels of leptin as well as the disability of exogenous leptin, which can offer satiety to reduce BW.31 As a high level of leptin and high BW and body fat were shown in group HFD, these rats were in a state of leptin resistance. The possible mechanism of leptin resistance in obesity is the suppression of leptin receptor B (LepRb) signaling.31 LepRb can receive inhibitory signals from some negative feedback loops, such as SOCS3,31 but no alteration in the SOCS3 level was found among the three groups, which indicates that leptin signaling was suppressed via other paths. The signaling transduction of LepRb relies on the initial activation of Janus kinase 2 (JAK2) and subsequent phosphorylation of tyrosine residues within LepRb and STAT3,32,33 and endoplasmic reticulum (ER) stress has emerged as a key factor in obesity-induced inflammation and subsequently led to leptin resistance through JAK2 inhibition.34 We believe that the leptin resistance caused by the high-fat diet in our study was induced by inflammation-related ER stress. Therefore, FP consumption inhibited inflammation, prevented JAK2 inhibition, and then further relieved the ER stress, which eventually led to the removal of leptin resistance. The function of leptin was restored by FP, the signal was then delivered to the downstream proteins (including AKT and STAT3), and leptin ultimately inhibited the expression of NPY, whose overexpression consequently reduced satiety and caused obesity.35 Leptin sensitivity can be increased by combining some NSPs with other compounds, for example, iso-malto-oligosaccharide combined with green tea extract and fermented red ginseng combined with levan.36,37 Only a few NSPs, including inulin, chitosan, and resistant starch, had the ability to solely reduce

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AUTHOR INFORMATION

Corresponding Authors

*Q.Zhou E-mail: [email protected]. Tel: +86-20-85222787. *J.Liu E-mail: [email protected]. Tel: +86-20-85220006. *X.Peng E-mail: [email protected]. Tel: +86-20-85226630. ORCID

Xichun Peng: 0000-0002-4804-5203 7047


Article

Journal of Agricultural and Food Chemistry Author Contributions

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†

J.Luo and J.Qi contributed equally to this research. Q.Zhou, J.Liu, and X.Peng designed the research; J.Luo, J.Qi, and W.Wang conducted the research; W.Wang, Z.Luo, L.Liu, and G.Zhang provided essential reagents or materials; J.Luo and J.Qi analyzed the data; J.Luo and X.Peng wrote and revised the paper. Funding

This work was supported by the National Natural Science Funds (No. 31801543, No. 81601299, and No. 81800833) and the funds of the China Postdoctoral Science Foundation (No. 2018M643368 and No. 2018M631045). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Ruixia Qiu, Bing Yu, and Yong Wang from the Department of Food Science and Engineering, Jinan University, for their contributions to the experiments of this study.

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Antiobesity Effect of Flaxseed Polysaccharide via Inducing Satiety due

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