Mulberry 1-Deoxynojirimycin Inhibits Adipogenesis by Repression of

Jun 15, 2015 - Moreover, 4 μM DNJ significantly inhibited adipogenesis, whereas 0.4 mM RSG increased lipogenesis of porcine intramuscular adipocytes...
0 downloads 13 Views 7MB Size
Download PDF
Article pubs.acs.org/JAFC

Mulberry 1‑Deoxynojirimycin Inhibits Adipogenesis by Repression of the ERK/PPARγ Signaling Pathway in Porcine Intramuscular Adipocytes Guo-qiang Wang,†,∥ Li Zhu,‡,∥ Mei-lin Ma,† Xiao-chang Chen,† Yun Gao,† Tai-yong Yu,† Gong-she Yang,† and Wei-jun Pang*,† †

Laboratory of Animal Fat Deposition & Muscle Development, College of Animal Science and Technology, and ‡College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi 712100, China ABSTRACT: Intramuscular fat (IMF), which is modulated by adipogenensis of intramuscular adipocytes, plays a key role in pork quality associated with marbling, juiceness, and flavor. However, the regulatory mechanism of 1-deoxynojirimycin (DNJ) on adipogenesis is still unknown. Here, we found that both DNJ (2.0, 3.0, 4.0, 5.0, and 6.0 μM) and rosiglitazone (RSG; 0.1, 0.2, 0.3, 0.4, and 0.5 mM) had no effect on cell viability. Moreover, 4 μM DNJ significantly inhibited adipogenesis, whereas 0.4 mM RSG increased lipogenesis of porcine intramuscular adipocytes. Interestingly, DNJ sharply inhibited phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2), but did not change phosphorylation of AKT (protein kinase B) in intramuscular adipocytes. We further found that the inhibitory adipogenesis of DNJ was attenuated by RSG via up-regulation of PPARγ. On the basis of the above findings, we suggest that DNJ inhibited adipogenesis through the ERK/PPARγ signaling pathway in porcine intramuscular adipocytes. KEYWORDS: DNJ, pork, intramuscular adipocyte, adipogenesis, ERK/PPARγ signaling pathway

■

INTRODUCTION Surplus or inadequate fat has made pork quality bad. Recently, many efforts have been made to verify natural sources for searching a physiological functional food or compounds to improve meat quality or cure disease.1 1-Deoxynojirimycin (DNJ) is a kind of natural alkaloid, and its chemical structure is similar to that of D-glucose except for the oxygen atom of the pyranose ring that is replaced by an NH group.2 DNJ extracted from mulberry leaf, silkworm, and Bacillus species has an obvious inhibition effect on intestinal α-glucosidases, which plays an important role in the last step of carbohydrate digestion.3−6 Some experiments proved that DNJ inhibited postprandial intestinal glucose absorption, reduced the levels of blood sugar, and regulated hepatic glucose metabolic enzymes to accelerate glucose utilization, thereby preventing diabetes mellitus.3,7−10 Others showed that DNJ decreased lipid accumulation along with changing of some fatty acid metabolism enzymes in rat liver and stimulated adiponectin and glucose transporter 4 (GLUT4) expressions in 3T3-L1 adipocytes.11,12 Besides the role of carbohydrate and lipid metabolism, DNJ also alleviated the risk factors of atherosclerosis through enhancing cholesterol efflux in 3T3-L1 adipocytes and attenuating high glucose-accelerated senescence in human umbilical vein endothelial cells (HUVECs).13,14 Antimetastatic and antiviral effects of DNJ have also been reported, showing that DNJ prevents growth and migration of cancer cell and reduces replication of viruses.15−17 However, the effect and regulatory mechanism of DNJ on adipogenesis of porcine intramuscular adipocytes has not yet been reported. Intramuscular fat (IMF) is a class of fat depot existing in the epimysium, perimysium, and endomysium.18 It is generally considered that IMF may have a positive influence on meat quality including tenderness, juiciness, and flavor.19,20 However, © XXXX American Chemical Society

negative effects of IMF on human health have been reported. IMF secretes inflammatory cytokines to reduce the force of muscular fibers and is associated with increased diabetes risk in stroke survivors whose movement and independence were impaired.21−23 Therefore, the content of IMF is an important economic trait for meat quality and a healthy biomarker for human disease. As an ideal biomedical model, pigs are close to tissue development and disease occurrence of humans.24−26 Compared with rodents, pigs are more similar to humans in fat deposition patterns, so intramuscular adipocytes extracted from porcine longissimus dorsi muscle (LM) were studied to explore IMF deposition for improvement of marbling in pork and therapy of muscle-related diseases. In this study, we investigated the IMF distribution in LMs of fat-type and lean-type pigs and assessed the cell viability of DNJ and the optimal concentration of DNJ on inhibitory lipogenesis of porcine intramuscular adipocytes. Moreover, the signaling pathway involved in the inhibitory lipogenesis of DNJ was explored. The results suggested that DNJ attenuated adipogenesis of intramuscular adipocytes through the ERK/PPARγ signaling pathway. Our findings will help us understand intramuscular fat deposition by modulation of DNJ.

■

MATERIALS AND METHODS

Animals. Bamei and Large White pigs were supplied by the Experimental Farm of Northwest A&F University (Yangling, Shaanxi Province, China). All pigs were handled in accordance with the guidelines of the Northwest A&F University Animal Care Committee.

Received: April 3, 2015 Revised: June 15, 2015 Accepted: June 15, 2015

A

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. Nucleotide Sequences of the Primers Used for Real-Time Quantitative PCR gene

accession no.

direction

primer sequence

product length, bp

PPARγ

DQ013884

forward reverse

5′-TTCCAGGGGTGTCAGTTTCG-3′ 5′-TTGGTCATTCAGGTCAAGGTTTA-3′

99

aP2

NM_001002817

forward reverse

5′-TAGTAGATGATAAGTTGGTGGTGGAATG-3′ 5′-CTCTCATAAATTCTGGTAGCCGTGA-3′

67

FAS

EF589048

forward reverse

5′-TTGTCCTGGGAAGAGTGTAAGC-3′ 5′-CCTGAGGTCCCGAGATGGT-3′

94

HSL

AY686758

forward reverse

5′-ACCCTCGGCTGTCAACTTCTT-3′ 5′-ACTTTCTCCTCCTTGGTGCTAATCT-3′

90

18S rRNA

AY265350

forward reverse

5′-CCCACGGAATCGAGAAAGAG-3′ 5′-TTGACGGAAGGGCACCA-3′

Reagents. DNJ was purchased from Sigma (D9305; Sigma, St. Louis, MO, USA), and DNJ stock solutions were made in deionized distilled water and later diluted in media or phosphate-buffered saline (PBS) prior to use in culture. Rosiglitazone (RGZ), a PPARγ agonist, was purchased from Cayman Chemical (CAS Registry No. 122320-73-4; Ann Arbor, MI, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA), BODIPY from Life Technologies (Carlsbad, CA, USA), and 4′,6-diamidino-2-phenylindole (DAPI) from Roche (Basel, Switzerland). Frozen Section and HE Staining. Baimei is an important Chinese indigenous breed, an obese type with 32% fat in the carcass, whereas Large White pig is widely distributed in most of the world and is a lean type with 15% fat in the carcass. Four fat-type breed Bamei pigs and four lean-type breed Large White pigs at day 180 of age were provided by the experimental farm of Northwest A&F University. All pigs were killed at a slaughterhouse under the guidelines of Northwest A&F University Animal Care Committee. Half an hour post-mortem, 4−6 g samples were taken from LM muscle between the sixth and seventh rib. LM were dissected and rinsed with PBS. Samples were frozen in liquid nitrogen. For frozen section, the samples were fixed with 4% paraformaldehyde and kept at room temperature. Fixed tissues were dehydrated in 30% sucrose (v/v) and sectioned (10 μm) through a sliding microtome (Leica, Solms, Germany). Sections were stained with HE using standard pathologic procedures.27 The slides were then dehydrated with 95 and 100% ethanol successively followed by xylene (2 × 5 min) and mounted with coverslips. The sections were observed and pictures taken through a microscope (Olympus, New York, NY, USA) at 100× magnification. Isolation and Culture of Porcine Intramuscular Adipocytes. About 20−30 min post-mortem, 4−6 g of LM per piglet was collected from four piglets (2 of them at 1 day and the others at 2 days of age) and finely minced in 50 mL beakers containing 5 mL of Dulbecco’s modified Eagle’s medium (DMEM)/Nutrient Mixture F-12 (DMEM/F12; Gibco) with 0.2 mL of kanamycin solution (30 mg/mL) per gram of tissue, respectively. Intramuscular adipocytes were isolated using a type II collagenase (Gibco) method according to previous publications.28,29 The cells were cultured using a previously published method.30,31 Briefly, the finely minced tissues were digested at 37 °C for 120 min in a shaking water bath. Digesta were passed through sterile 178 and 74 μm steel mesh filters to isolate digested cells. Cells were rinsed with serumfree DMEM/F12 medium and centrifugation twice at 1500g for 10 min. Cells were resuspended in growth medium (GM), plated at 6 × 105 per 60 mm culture dish, and cultured in a 5% CO2 incubator at 37 °C. Because adipocytes attach much earlier than myoblasts, the culture cells were rinsed three times with PBS to remove myoblasts, insoluble myofibrillar proteins, and other debris at 1 h after plating. Cells were cultured in GM until reaching 70−80% confluence, digested with 0.05% trypsin, collected by centrifugation 1000g for 5 min, resuspended in GM, and plated at a density of 2 × 104 cells/cm2 in 6-well plates. Two days after 100% confluence, the cells were treated with DMEM/F12 supplemented with 10% FBS and the hormone cocktail IBMX−DEX−insulin

122

Figure 1. Morphologic observation of LM between obese and lean pigs at 180 days of age: (A) fat-type pig (Bamei) and lean-type pig (Large white); (B) intramuscular adipocyte in LM of fat-type and lean-type pigs. HE, hematoxylin and eosin staining, blue arrow indicates IMF adipocytes; BODIPY staining, lipids indicate green and blue nuclei stained with DAPI. Scale bar = 100 μm.

Table 2. Carcass Weight, Fat Percent and IMF Content of Bamei and Large White Pigs at 180 Days of Age pig breed

carcass weight, kg

Bamei Large White ** indicates

37.48 ± 68.74 ± 1.36

1.09**

fat, % 31.03 ± 17.61 ± 0.27

0.41**

IMF content, % 4.42 ± 0.45** 2.37 ± 0.29

P < 0.01.

(MDI; 0.5 mM IBMX, 1 nM DEX, and 5 ng/mL insulin) for 2 days. The medium was changed to differentiation medium (DM), including DMEM/F12 supplemented with 10% FBS and 5 ng/mL insulin, for another 2 days. Cell Viability Analysis. Porcine intramuscular adipocyte viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 24-well culture plates at a density of 2 × 104 cells/well, incubated for 48 h, treated with DNJ at various concentrations (2.0, 3.0, 4.0, 5.0, and 6.0 μM) or RSG (0.1, 0.2, 0.3, 0.4, and 0.5 mM) for 48 h to evaluate any dosedependent effects of DNJ or RSG on cell growth and viability, cultured with 0.5 mg/mL MTT at 37 °C in a humidified atmosphere of 5% CO2 for another 4 h, and solubilized with isopropanol. The viable cell number was directly proportional to the production of formazan measured spectrophotometrically at 563 nm. B

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 3. DNJ regulates mRNA expression of key adipogenic and lipolytic genes during lipogenesis of intramuscular preadipocytes: realtime qPCR analysis of (A) PPARγ, (B) AP2, (C) FAS, and (D) HSL mRNA levels, respectively. Porcine intramuscular preadipocytes treated with 4 μM DNJ were induced differentiation and collected at days 0, 2, 4, 6, and 8. Gene mRNA expression was determined by qPCR, normalized to 18S rRNA, and expressed relative to expression in the control at day 0. Data indicate the mean ± SEM: (∗) p < 0.05 and (∗∗) p < 0.01.

Figure 2. Effects of various concentrations of DNJ on porcine intramuscular preadipocyte differentiation. (A) Effect of DNJ on viability of porcine intramuscular preadipocyte as assessed by the MTT assay. Cells were treated with different concentrations (2.0−6.0 μM) of DNJ for 48 h and then assayed using MTT. (B) Intracellular lipids stained with Oil Red O. Intramuscular preadipocytes were treated with DMSO as control and the indicated concentrations of DNJ at day 8. (C) TG content (μg/104 cells) determined at day 8 after treatment. (D, E) mRNA levels of PPARγ and aP2 at day 8 after treatment. Gene mRNA expression was determined by qPCR, normalized to 18S rRNA and expressed relative to expression in the control. Each column represents the mean of four independent experiments ± SEM: (∗) p < 0.05.

adipocytes were incubated with 0.4 mM RSG for 2 days. At the indicated time points, cells were stained by Oil Red O or BODIPY, a triacylglycerol content assay was performed, and total RNA and protein were extracted for analysis of gene expression. Oil Red O Staining. Oil Red O staining was conducted according to a previously published method.32 BODIPY Staining. BODIPY staining was performed according to a previously published method.32 The sections or cells were observed under fluorescence microscope (Nikon, Tokyo, Japan). Triacylglycerol (TG) Content Assay. TG content analysis was conducted according to a previously published method.32 Real-Time Quantitative PCR. Total RNA was extracted from cells according to the manufacturer’s instruction of TRIZOL reagent (Takara, Japan). The RNA concentration was determined, and reverse transcription was performed using the First-Strand cDNA Synthesis Kit

Treatment with DNJ and RSG. For the DNJ experiment, porcine intramuscular adipocytes incubated in induced media of differentiation were treated with 0, 2, 3, 4, 5, or 6 μM DNJ for 8 days. For both DNJ and RSG experiment, at day 8 after treatment with 4 μM DNJ, intramuscular C

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 4. DNJ represses adipogenesis by inhibiting phosphorylation of ERK1/2: (A, B, C) ERK signaling in intramuscular adipocytes under DNJ stimulation condition. On reaching 70−80% confluence, the intramuscular preadipocytes were treated with DMSO as control and 4 μM DNJ for 2 days; the protein levels of adipogenic and lipolytic markers, phosphorylated ERK1/2 (p-ERK1/2), and ERK1/2 proteins were detected by Western blotting at day 8 of differentiation induction. GAPDH was applied as internal reference. Each column represents the mean of four batches ± SEM: (∗) p < 0.05 and (∗∗) p < 0.01.

Figure 5. DNJ did not change the levels of AKT and p-AKT: (A, B, C) AKT signaling in intramuscular adipocytes under DNJ stimulation condition. On reaching 70−80% confluence, the intramuscular preadipocytes were treated with DMSO as control and 4 μM DNJ for 2 days; p-AKT and AKT proteins were detected by Western blotting at day 8 of differentiation induction. GAPDH was applied as internal reference. Each column represents the mean of four batches ± SEM. (Takara, Japan) for qRT-PCR (Bio-Rad iQ5 multicolor Real-Time PCR Detection system) analysis of the functional genes. Real-time PCR was performed by using a Prime Script RT Reagent Kit (Perfect Real Time, Takara, Japan) according to the manufacturer’s instructions. Primers are listed in Table 1. In this study, 18S rRNA (18S) was used as the reference gene. The 2-ΔΔCT method is used to analyze the relative changes in the expression of each gene mRNA. Western Blot Analysis. Cells were lysed in lysis buffer (Beyotime, China), supplemented with 1 mM PMSF. Protein concentration was determined with a BCA protein assay kit (Tiangen, China). The proteins of each sample were separated by 12% SDS-PAGE and electrotransferred to PVDF membrane (Millipore, Bedford, MA, USA) for immunoblot analysis. The following primary antibodies were used: PPARγ (abcam, ab1948, 1:400), FAS (Santa Cruz, sc-20140, 1:300), HSL (cell signaling, 4107, 1:1000), ERK1/2 (cell signaling, 9102, 1:1000), p-ERK1/2 (cell signaling, 9101, 1:1000), AKT (Santa Cruz,

sc-8312, 1:500), p-AKT (Santa Cruz, sc-7985-R, 1:500), and antiGAPDH (Santa Cruz, sc-166574, 1:500), which was used as internal reference. After incubation with the appropriate HRP−conjugate secondary antibody, proteins were detected using a ChemiDoc XRS imaging system and analysis software Quantity One (Bio-Rad). Statistical Analysis. All experimental data are obtained from four independent experiments. Values are expressed as means ± standard error of the mean (SEM). Statistics are calculated with SPSS statistics v19.0 software. Student’s t test is used for individual comparisons. Multiple comparisons are assessed by one-way ANOVA followed by Dunnett’s tests. Differences between groups are considered statistically significant if P < 0.05.

■

RESULTS Intramuscular Adipocyte in Fat-Type and Lean-Type Pig Longissimus Dorsi Muscles. Bamei pig is a fat-type breed, and Large White pig is a lean-type breed (Figure 1A). Fat percent D

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry and IMF content of Bamei pigs were significantly greater than that of Large White pigs at 180 days of age (Table 2). Using HE and BODIPY staining, the intramuscular adipocytes and IMF in LM of Bamei pigs were much greater than that of Large White pigs at 180 days of age (Figure 1B). Effect of Various DNJ Concentrations on Adipogenesis of Porcine Intramuscular Adipocytes. As shown in Figure 2A, DNJ (2, 3, 4, 5, and 6 mM) was not toxic, so these five concentrations were used to test the effect of DNJ on adipogenesis of porcine intramuscular adipocytes. To evaluate the effect of DNJ on lipogensis of porcine intramuscular adipocytes, the cells were treated with different concentrations of DNJ including 0, 2, 3, 4, 5, and 6 μM for 8 days. The results indicated that lipid accumulation was significantly weakened in the treatment with 4 and 5 μM DNJ by Oil Red O staining (Figure 2B) and TG content assay (p < 0.05; Figure 2C). Moreover, the mRNA levels of key lipogenic gene PPARγ (4 and 5 μM DNJ; p < 0.05; Figure 2D) and aP2 (4, 5, and 6 μM DNJ; p < 0.05; Figure 2E) were markedly down-regulated at day 8 after treatment. On the basis of the above results, the following experiments were performed with 4 μM DNJ. DNJ Inhibited Lipid Accumulation during Intramuscular Adipocyte Differentiation. To further examine the effects of 4 μM DNJ on key lipogenic and lipolytic genes during adipocyte differentiation, the time course of mRNA expressions of aP2, PPARγ, FAS, and HSL were analyzed by real-time qRT-PCR at days 0, 2, 4, 6, and 8. The results indicated that mRNA levels of PPARγ were decreased at days 4, 6, and 8 compared with controls (p < 0.05; Figure 3A), mRNA levels of aP2 were significantly reduced at days 6 (p < 0.05; Figure 3B) and 8 (p < 0.01; Figure 3B), and mRNA levels of FAS were down-regulated at days 6 (p < 0.05; Figure 3C) and 8 (p < 0.01; Figure 3C), whereas the mRNA levels of HSL were up-regulated at days 6 and 8 (p < 0.05; Figure 3D). ERK Signaling Was Repressed in Intramuscular Adipocytes under DNJ Stimulation. To study the effect of DNJ on proteins that play a key role or involve signal transduction during differentiation, the total proteins were extracted from intramuscular adipocytes treated with 4 μM DNJ at day 8 after induction of differentiation for Western blot analysis. The results showed that protein levels of vital adipogenic genes, including PPARγ (p < 0.01) and FAS (p < 0.05), were significantly reduced (Figure 4), whereas the protein level of HSL (p < 0.05), which is a key lipolytic marker, markedly increased in the treatment (Figure 4). Interestingly, compared with the control, levels of ERK1/2 had no striking difference, but levels of phosphorylated ERK1/2 (p-ERK1/2) were markedly inhibited in the DNJ treatment (p < 0.05; Figure 4). In addition, levels of AKT and phosphorylated AKT (p-Akt) showed no difference (Figure 5). Therefore, the ERK signaling pathway was implicated in adipogenesis of intramuscular adipocytes under DNJ stimulation condition. RGZ Improves Adipogenesis of Porcine Intramuscular Adipocytes. As shown in Figure 6A, RGZ (0.1, 0.2, 0.3, 0.4, and 0.5 mM) was not toxic, so these five concentrations were used to test the effect of RGZ on adipogenesis of porcine intramuscular adipocytes. TG contents in intramuscular adipocytes treated with RGZ (0.3, 0.4, and 0.5 mM) were significantly greater than the control (p < 0.01; Figure 6B). Moreover, compared with the control, the levels of PPARγ mRNA in cells treated with RGZ (0.3, 0.4, and 0.5 mM) were markedly up-regulated (p < 0.01; Figure 6C). Inhibitory Adipogenesis of DNJ Is Attenuated by RGZ via Up-regulation of PPARγ Expression. To further

Figure 6. RSG improved adipogenesis in intramuscular adipocytes: (A) effect of rosiglitazone (RSG) on viability of porcine intramuscular preadipocyte as assessed by the MTT assay (cells were treated with different concentrations (0.1−0.5 mM) of RSG for 48 h and then assayed using MTT); (B) TG content (μg/104 cells) analyzed at day 8 after treatment; (C) real-time qPCR analysis of PPARγ mRNA levels. 18S rRNA was applied as internal reference. PPARγ mRNA expression was determined by qPCR, normalized to 18S rRNA, and expressed relative to expression in the control. Data indicate the mean ± SEM: (∗) p < 0.05 and (∗∗) p < 0.01.

investigate whether the ERK/PPARγ signaling pathway is involved in regulating adipogenesis of DNJ, the intramuscular adipocytes treated with 4 μM DNJ (for 8 days) were stimulated with 0.4 mM RGZ for 2 days (Figure 7A), then the treated cells were stained with BODIPY and Oil Red O to indicate lipogenesis; protein samples were subjected to a protein array to study the relevant kinases. The results showed that the inhibitory lipogenesis of DNJ was significantly attenuated by RGZ (p < 0.05; Figure 7B,C) and recovered the protein levels of PPARγ and FAS (p < 0.05; Figure 7D,E). Interestingly, the levels of ERK1/2 and its phosphorylation were not changed (Figure 7E,F) in intramuscular adipocytes treated with 4 μM DNJ plus 0.4 mM RSG. On the basis of the above results, the inhibitory adipogenesis of DNJ was attenuated by RSG via upregulation of PPARγ. These findings indicate that DNJ inhibits adipogenesis through the ERK/PPARγ signaling pathway.

■

DISCUSSION China is the world’s largest producer and consumer of pork. However, pork quality becomes bad because of the unfit IMF content. Our previous study showed that IMF content in LM of Bamei pig was much greater than that of Large White pig at 180 days of age.33 In this study, intramuscular adipocyte and IMF content in fat-type and lean-type porcine LMs were directly E

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 7. Inhibitory adipogenesis of DNJ was attenuated by RSG via up-regulation of PPARγ expression in intramuscular adipocytes: (A) experimental procedure for DNJ plus RSG treatment of porcine intramuscular adipocytes; (B) BODIPY and Oil Red O staining of intramuscular adipocytes under 4 μM DNJ condition at day 2 after treatment with 0.4 mM RSG; (C) TG content (μg/104 cells) analysis; (D, E, F) ERK signaling in intramuscular adipocytes under DNJ plus RSG stimulation condition. Each column represents the mean of four batches ± SEM: (∗) p < 0.05 and (∗∗) p < 0.01.

(Sirtuim 1),32 Akt2 (protein kinase B 2),32 C/EBPβ (CCAAT enhancer-binding protein B),34 PU.1 (spleen focus forming virus proviral integration oncogene spi1),35 resveratrol,36 and camptothecin31 regulated lipogenesis of porcine subcutaneous adipocytes through differentiation or apopotosis. It was reported that intake of DNJ suppressed lipid accumulation in rat liver and diet-induced obesity through increases in adiponectin in mice.11,37 At present, the regulatory adipogenesis by DNJ in

shown using HE and BODIPY staining. We think that the suitable IMF content and distribution in Bamei pork are involved in good meat quality. Improvement of domestic animal meat quality is implicated in the regulatory mechanism of IMF deposition, which is modulated by adipogenensis of intramuscular adipocytes. Our previous studies found that some functional genes and natural extractions, including FoxO1 (Forkhead box O 1),30,33 Sirt1 F

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

A 2.5−3% IMF level is best for pork, but it is much closer to 4−7% in many Chinese indigenous pig breeds, such as the Neijiang pig and Hanjiang Black pig,43 so DNJ may decrease pork IMF content. In introduced pig breeds, including Large White and Landrace,43 their IMF contents are often below 2.5%, so it should be increased in these breeds. In summary, DNJ suppresses lipid accumulation during differentiation of porcine intramuscular adipocytes by the ERK/PPARγ signaling pathway. We need to point out that the effects have been shown here for adipocytes of 1- or 2-day-old piglets and our method of possibly decreasing IMF deposition is only likely to be of practical usefulness for some breeds of pig that are particularly prone to fat deposition. These findings not only help us understand the mechanism whereby DNJ affects adipogenesis but may also provide a novel method to control intramuscular adipogenesis via DNJ for improving pork quality.

porcine intramuscular adipocytes is still unknown. In this study, to explore the effect of DNJ on lipogenesis of intramuscular adipocytes, intramuscular adipocytes were isolated and cultured using the differential speed adherence method.28 In accordance with Lee’s results,12 we found that DNJ did not affect intramuscular adipocyte viability. Likewise, RGZ, an agonist of PPARγ, was not toxic. DNJ at 4 μM decreased lipid acceleration and TG content, whereas 0.4 mM RGZ showed the opposite effects. Moreover, mRNA levels of key adipogenic genes including PPARγ and aP2 were down-regulated in intramuscular adipocytes treated with 4 μM DNJ. On the basis of above findings, DNJ and RGZ may regulate IMF deposition by modulated lipogenesis of porcine intramuscular adipocytes. Interestingly, in middle and later intramuscular adipocyte differentiation, mRNA and protein levels of PPARγ, aP2, and FAS were markedly decreased, but the mRNA and protein levels of HSL were increased in the DNJ treatment. These results suggested that DNJ inhibited lipogenesis not only by downregulation of lipogenic genes but also by up-regulation of lipolytic genes. To investigate whether DNJ influences the activation of ERK1/2 during intramuscular adipocyte differentiation, we detected the levels of total and phosphorylated ERK1/2 in adipocytes treated with 4 μM DNJ at day 8. ERK1 and ERK 2 are members of the mitogen-activated protein kinase (MAPK) family, which play a pivotal role in intracellular signaling pathways of cellular proliferation and differentiation.38 The blurry functions of ERK1/2 in adipogenesis were reported. Some researchers proved that phosphorylated ERK1/2 inhibited 3T3-L1 adipocyte differentiation by decreasing the activation of PPARγ.39 In contrast, others reported that ERK1/2 activation has a positive association with adipogenesis in human mesenchymal stem cells and 3T3-L1 adipocytes.38,40 We think that the opposite effects of ERK1/2 on lipogenesis may be related to different periods during differentiation. Consistent with the latter, our findings indicated that decreasing levels of phosphorylated ERK1/2 (p- ERK1/2) inhibited adipogenesis during the later period of intramuscular adipocyte differentiation at day 8 after treatment with DNJ. Meanwhile, expression levels of PPARγ and aP2 have an apparently declining trend at days 6 and 8. Interestingly, the levels AKT and p-AKT were not changed under the DNJ treatment, indicating that Akt signaling was not involved in the lipogenesis of DNJ. Therefore, we think that DNJ may inhibit adipogenesis through the ERK/PPARγ signaling pathway intramuscular adipocytes. To further explore whether ERK/PPARγ signaling is implicated in the regulatory lipogenesis of DNJ, the experiment of treatment with both DNJ and RSG was performed. Although levels of phosphorylated ERK1/2 and PPARγ in the treatment with DNJ were down-regulated, the level of PPARγ was recovered in treatment with DNJ plus RSG. DNJ inhibits lipogenesis of porcine intramuscular adipocyte via directly repressing the level of phosphorylated ERK1/2, then indirectly down-regulating the level of PPARγ through ERK signaling. Therefore, we conclude that DNJ inhibits IMF deposition by the ERK/PPARγ signaling pathway. DNJ has been reported to be the main component of mulberry leaves, fruits, and silkworms.41,42 Unlike chemical synthesis drugs, DNJ has no undesirable or significant side effects in vivo or in vitro.3 Therefore, pork palatability and marbling may be changed by feeding a feedstuff containing a suitable concentration of DNJ, which will be further explored in the future.

■

AUTHOR INFORMATION

Corresponding Author

*(W.-J.P.) Mail: No. 22 Xinong Road, Yangling, Shaanxi Province 712100, China. Phone: 86-29-87091017. Fax: 86-2987092430. E-mail: [email protected]. Author Contributions ∥

Guo-qiang Wang and Li Zhu contributed equally to this work.

Funding

This work was supported by the National Key Basic Research Programs of China (2015CB943102 and 2012CB124705), the National Natural Science Foundation of China (U1201213), the National Swine Industry Technology System Foundation (CARS-36), and the Natural Science Foundation of Shaanxi Province (2015JM3096). Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED AKT, protein kinase B; aP2, adipocyte fatty acid-binding protein 4; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DNJ, 1-deoxynojirimycin; Erk, extracellular regulated protein kinase; FAS, fatty acid synthetase; FBS, fetal bovine serum; HE, hematoxylin and eosin; HSL, hormone-sensitive lipase; HUVECs, human umbilical vein endothelial cells; IMF, intramuscular fat; IBMX, isobutylmethylxanthine; LM, longissimus dorsi muscle; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffer solution; PPARγ, peroxisome proliferatoractivated receptor-γ; RSG, rosiglitazone

■

REFERENCES

(1) Kumar, S.; Narwal, S.; Kumar, V.; Prakash, O. α-Glucosidase inhibitors from plants: a natural approach to treat diabetes. Pharmacogn. Rev. 2011, 5, 19. (2) Nakagawa, K.; Kubota, H.; Kimura, T.; Yamashita, S.; Tsuzuki, T.; Oikawa, S.; Miyazawa, T. Occurrence of orally administered mulberry 1deoxynojirimycin in rat plasma. J. Agric. Food Chem. 2007, 55, 8928− 8933. (3) Kwon, H. J.; Chung, J. Y.; Kim, J. Y.; Kwon, O. Comparison of 1deoxynojirimycin and aqueous mulberry leaf extract with emphasis on postprandial hypoglycemic effects: in vivo and in vitro studies. J. Agric. Food Chem. 2011, 59, 3014−3019. (4) Kong, W.; Oh, S.; Ahn, Y.; Kim, K.; Kim, J.; Seo, S. Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models. J. Agric. Food Chem. 2008, 56, 2613−2619. G

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

oxidants to depress myofibrillar force in murine skeletal muscle. J. Appl. Physiol. 2008, 104, 694−699. (22) Ryan, A. S.; Buscemi, A.; Forrester, L.; Hafer-Macko, C. E.; Ivey, F. M. Atrophy and intramuscular fat in specific muscles of the thigh: associated weakness and hyperinsulinemia in stroke survivors. Neurorehabil. Neural Repair 2011, 25, 865−872. (23) Noble, J. J.; Charles-Edwards, G. D.; Keevil, S. F.; Lewis, A. P.; Gough, M.; Shortland, A. P. Intramuscular fat in ambulant young adults with bilateral spastic cerebral palsy. BMC Musculoskeletal Disord. 2014, 15, 236. (24) Casellas, J.; Vidal, O.; Pena, R. N.; Gallardo, D.; Manunza, A.; Quintanilla, R.; Amills, M. Genetics of serum and muscle lipids in pigs. Anim. Genet. 2013, 44, 609−619. (25) Bendixen, E.; Danielsen, M.; Larsen, K.; Bendixen, C. Advances in porcine genomics and proteomics − a toolbox for developing the pig as a model organism for molecular biomedical research. Briefings Funct. Genomics 2010, 9, 208−219. (26) Lunney, J. K. Advances in swine biomedical model genomics. Int. J. Biol. Sci. 2007, 3, 179−184. (27) Hochstim, C. J.; Choi, J. Y.; Lowe, D.; Masood, R.; Rice, D. H. Biofilm detection with hematoxylin-eosin staining. Arch. Otolaryngol., Head Neck Surg. 2010, 136, 453−456. (28) Hausman, G. J.; Poulos, S. P. A method to establish co-cultures of myotubes and preadipocytes from collagenase digested neonatal pig semitendinosus muscles. J. Anim. Sci. 2005, 83, 1010−1016. (29) Jiang, S.; Wei, H.; Song, T.; Yang, Y.; Peng, J.; Jiang, S. Transcriptome comparison between porcine subcutaneous and intramuscular stromal vascular cells during adipogenic differentiation. PLoS One 2013, 8, e77094. (30) Pang, W. J.; Yu, T. Y.; Bai, L.; Yang, Y. J.; Yang, G. S. Tissue expression of porcine FoxO1 and its negative regulation during primary preadipocyte differentiation. Mol. Biol. Rep. 2009, 36, 165−176. (31) Pang, W. J.; Xiong, Y.; Wang, Y.; Tong, Q.; Yang, G. S. Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes. Exp. Cell Res. 2013, 319, 670−683. (32) Pang, W. J.; Wang, Y.; Wei, N.; Xu, R. X.; Xiong, Y.; Wang, P.; Shen, Q. W.; Yang, G. S. Sirt1 inhibits Akt2-mediated porcine adipogenesis potentially by direct protein-protein interaction. PLoS One 2013, 8, e71576. (33) Pang, W. J.; Wei, N.; Wang, Y.; Xiong, Y.; Chen, F. F.; Wu, W. J.; Zhao, C. Z.; Sun, S. D.; Yang, G. S. Obese and lean porcine difference of FoxO1 and its regulation through C/EBPβ and PI3K/GSK3β signaling pathway. J. Anim. Sci. 2014, 92, 1968−1979. (34) Xiong, Y.; Pang, W. J.; Wei, N.; Wang, Y.; Ren, W. K.; Yang, G. S. Knockdown of both FoxO1 and C/EBPβ promotes adipogenesis in porcine preadipocytes through feedback regulation. Cell Biol. Int. 2013, 37, 905−916. (35) Wei, N.; Wang, Y.; Xu, R. X.; Wang, G. Q.; Xiong, Y.; Yu, T. Y.; Yang, G. S.; Pang, W. J. PU.1 antisense lncRNA against its mRNA translation promotes adipogenesis in porcine preadipocytes. Anim. Genet. 2015, 46, 133−140. (36) Pang, W. J.; Xiong, Y.; Zhang, Z.; Wei, N.; Chen, N.; Yang, G. S. Lentivirus-mediated Sirt1 shRNA and resveratrol independently induce porcine preadipocyte apoptosis by canonical apoptotic pathway. Mol. Biol. Rep. 2013, 40, 129−139. (37) Tsuduki, T.; Kikuchi, I.; Kimura, T.; Nakagawa, K.; Miyazawa, T. Intake of mulberry 1-deoxynojirimycin prevents diet-induced obesity through increases in adiponectin in mice. Food Chem. 2013, 139, 16−23. (38) Xu, B.; Ju, Y.; Song, G. Role of p38, ERK1/2, focal adhesion kinase, RhoA/ROCK and cytoskeleton in the adipogenesis of human mesenchymal stem cells. J. Biosci. Bioeng. 2014, 117, 624−631. (39) Ling, H. Y.; Wen, G. B.; Feng, S. D.; Tuo, Q. H.; Ou, H. S.; Yao, C. H.; Zhu, B. Y.; Gao, Z. P.; Zhang, L.; Liao, D. F. MicroRNA-375 promotes 3T3-L1 adipocyte differentiation through modulation of extracellular signal regulated kinase signalling. Clin. Exp. Pharmacol. Physiol. 2011, 38, 239−246. (40) Sale, E. M.; Atkinson, P. G.; Sale, G. J. Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin

(5) Oku, T.; Yamada, M.; Nakamura, M.; Sadamori, N.; Nakamura, S. Inhibitory effects of extractives from leaves of Morus alba on human and rat small intestinal disaccharidase activity. Br. J. Nutr. 2006, 95, 933− 938. (6) Onose, S.; Ikeda, R.; Nakagawa, K.; Kimura, T.; Yamagishi, K.; Higuchi, O.; Miyazawa, T. Production of the α-glycosidase inhibitor 1deoxynojirimycin from Bacillus species. Food Chem. 2013, 138, 516− 523. (7) Li, Y.; Ji, D.; Zhong, S.; Lin, T.; Lv, Z.; Hu, G.; Wang, X. 1Deoxynojirimycin inhibits glucose absorption and accelerates glucose metabolism in streptozotocin-induced diabetic mice. Sci. Rep. 2013, 3, 1377. (8) Vichasilp, C.; Nakagawa, K.; Sookwong, P.; Higuchi, O.; Kimura, F.; Miyazawa, T. A novel gelatin crosslinking method retards release of mulberry 1-deoxynojirimycin providing a prolonged hypoglycaemic effect. Food Chem. 2012, 134, 1823−1830. (9) Asai, A.; Nakagawa, K.; Higuchi, O.; Kimura, T.; Kojima, Y.; Kariya, J.; Miyazawa, T.; Oikawa, S. Effect of mulberry leaf extract with enriched 1-deoxynojirimycin content on postprandial glycemic control in subjects with impaired glucose metabolism. J. Diabetes Invest. 2011, 2, 318−323. (10) Kimura, T.; Nakagawa, K.; Kubota, H.; Kojima, Y.; Goto, Y.; Yamagishi, K.; Oita, S.; Oikawa, S.; Miyazawa, T. Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. J. Agric. Food Chem. 2007, 55, 5869−5874. (11) Tsuduki, T.; Nakamura, Y.; Honma, T.; Nakagawa, K.; Kimura, T.; Ikeda, I.; Miyazawa, T. Intake of 1-deoxynojirimycin suppresses lipid accumulation through activation of the β-oxidation system in rat liver. J. Agric. Food Chem. 2009, 57, 11024−11029. (12) Lee, S. M.; Shin, M. J.; Hwang, K. Y.; Kwon, O.; Chung, J. H. 1Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectin and GLUT4 expressions in 3T3-L1 adipocytes. J. Microbiol. Biotechnol. 2013, 23, 637−643. (13) Shuang, E.; Kijima, R.; Honma, T.; Yamamoto, K.; Hatakeyama, Y.; Kitano, Y.; Kimura, T.; Nakagawa, K.; Miyazawa, T.; Tsuduki, T. 1Deoxynojirimycin attenuates high glucose-accelerated senescence in human umbilical vein endothelial cells. Exp. Gerontol. 2014, 55, 63−69. (14) Do, H. J.; Lee, S.; Kim, Y. S.; Shin, M. Effect of 1-deoxynojirimycin on cholesterol efflux through ABCA1-LXRα pathway in 3T3-L1 adipocytes. J. Funct. Foods 2014, 7, 692−699. (15) Wang, R.; Yang, C.; Hu, M. 1-Deoxynojirimycin inhibits metastasis of B16F10 melanoma cells by attenuating the activity and expression of matrix metalloproteinases-2 and-9 and altering cell surface glycosylation. J. Agric. Food Chem. 2010, 58, 8988−8993. (16) Zhao, Y.; Liu, W.; Zhou, Y.; Zhang, X.; Murphy, P. V. N-(8-(3Ethynylphenoxy)octyl-1-deoxynojirimycin suppresses growth and migration of human lung cancer cells. Bioorg. Med. Chem. Lett. 2010, 20, 7540−7543. (17) Papandréou, M.; Barbouche, R.; Guieu, R.; Kieny, M. P.; Fenouillet, E. The α-glucosidase inhibitor 1-deoxynojirimycin blocks human immunodeficiency virus envelope glycoprotein-mediated membrane fusion at the CXCR4 binding step. Mol. Pharmacol. 2002, 61, 186−193. (18) Wang, S.; Zhou, G.; Shu, G.; Wang, L.; Zhu, X.; Gao, P.; Xi, Q.; Zhang, Y.; Yuan, L.; Jiang, Q. Glucose utilization, lipid metabolism and BMP-Smad signaling pathway of porcine intramuscular preadipocytes compared with subcutaneous preadipocytes. Cell. Physiol. Biochem. 2013, 31, 981−996. (19) Wood, J. D.; Enser, M.; Fisher, A. V.; Nute, G. R.; Richardson, R. I.; Sheard, P. R. Manipulating meat quality and composition. Proc. Nutr. Soc. 1999, 58, 363−370. (20) Fernandez, X.; Monin, G.; Talmant, A.; Mourot, J.; Lebret, B. Influence of intramuscular fat content on the quality of pig meat-1. Composition of the lipid fraction and sensory characteristics of m. longissimus lumborum. Meat Sci. 1999, 53, 59−65. (21) Hardin, B. J.; Campbell, K. S.; Smith, J. D.; Arbogast, S.; Smith, J.; Moylan, J. S.; Reid, M. B. TNF-α acts via TNFR1 and muscle-derived H

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J. 1995, 14, 674. (41) Asano, N.; Yamashita, T.; Yasuda, K.; Ikeda, K.; Kizu, H.; Kameda, Y.; Kato, A.; Nash, R. J.; Lee, H. S.; Ryu, K. S. Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). J. Agric. Food Chem. 2001, 49, 4208−4213. (42) Isabelle, M.; Lee, B. L.; Ong, C. N.; Liu, X.; Huang, D. Peroxyl radical scavenging capacity, polyphenolics, and lipophilic antioxidant profiles of mulberry fruits cultivated in southern China. J. Agric. Food Chem. 2008, 56, 9410−9416. (43) Pang, W. J.; Bai, L.; Yang, G. S. Relationship among H-FABP gene polymorphism, intramuscular fat content, and adipocyte lipid droplet content in main pig breeds with different genotypes in western China. Yichuan Xuebao 2006, 33, 515−524.

I

DOI: 10.1021/acs.jafc.5b01680 J. Agric. Food Chem. XXXX, XXX, XXX−XXX