Cellular Physiology and Biochemistry

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Original Paper

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Glucose Utilization, Lipid Metabolism and BMP-Smad Signaling Pathway of Porcine Intramuscular Preadipocytes Compared with Subcutaneous Preadipocytes

Wang S.a,b,e · Zhou G.a,b,c,e · Shu G.a, b · Wang L.a, b · Zhu X.a, b · Gao P.a, b · Xi Q.a, b · Zhang Y.a, b · Yuan L.d · Jiang Q.a, b

Author affiliations

aCollege of Animal Sciences, ALLTECH-SCAU Animal Nutrition Control Research Alliance, South China Agricultural University, Guangzhou; bGuangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, Guangzhou; cInstitute of Life Sciences, Fuzhou University, No.2 Xueyuan Rd., University Town, Fuzhou, Fujian Province; dCollege of Life Science, Xiamen University, Xiamen, Fujian Province; eSongbo Wang and Guixuan Zhou contributed equally to this work

Corresponding Author

Qingyan Jiang

Department of Animal nutrition and Feed Science, College of Animal Science

South China Agricultural University, Guangzhou, 510642 (P. R. China)

Tel. +8620-85284930, Fax +8620-85284901, E-Mail qyjiang@scau.edu.cn

Keywords: Intramuscular preadipocytesSubcutaneous preadipocytesPorcineGlucose utilizationLipid metabolismBMP-Smad signaling pathway

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Cell Physiol Biochem 2013;31:981-996
https://doi.org/10.1159/000350116

Abstract

Background/Aims: We previously reported that porcine intramuscular (i.m.) preadipocytes were different from subcutaneous (s.c.) preadipocytes on cell differentiation and lipid accumulation, but the underlying mechanisms remained unknown. The paper aims to investigate the underlying mechanisms by comparing the differences between i.m. and s.c. preadipocytes in glucose utilization, lipid metabolism, and the role of BMP signaling pathway. Methods: Experiments were performed in porcine primary i.m. and s.c. preadipocytes in culture. The mRNA and protein expression patterns were determined respectively by Quantitative real-time PCR and Western blot. Cytosolic triglycerides were examined by triglyceride assay. Results: The i.m. preadipocytes consumed more glucose by expression of GLUT1 and s.c. preadipocytes mainly utilized exogenic fatty acids for lipid synthesis by expression of LPL and FAT. Meanwhile, the expression of genes related to lipogenesis and lipolysis in s.c. preadipocytes increased more quickly than those in i.m. preadipocytes. The expression patterns of the genes involved in BMP-Smad signaling pathway were consistent with those of the genes participated in adipocytes differentiation in both i.m. and s.c. preadipocytes. Exogenous BMP2 significantly increased, whereas Noggin and Compound C, remarkablely decreased the triglycerides content in i.m. preadipoytes, without affecting s.c. preadipocytes. BMP2 shRNA significantly reduced the mRNA levels of the downstream genes of BMP-Smad signaling pathway and PPARγ in both i.m. and s.c. preadipocytes. Conclusion: These findings suggested that the differentiation and lipid accumulation differences between i.m. and s.c. preadipocytes might be caused by the different manners of glucose utilization, lipid metabolism and the BMP-Smad signaling pathway. The special feature of i.m. adipocytes implied that these cells might be a potential target for treatment of diabetes.

© 2013 S. Karger AG, Basel

Introduction

Intramuscular adipose tissue (IMAT) or intramuscular fat (IMF) is a kind of fat depot located on epimysium, perimysium, and endomysium. In farm animal, the content of IMF affects the tenderness, juiciness and flavor of pork [1,2,3] and is considered to be of great value in improving meat quality. In human, however, IMF is reported to be relevant to insulin sensitivity in obesity and diabetes [4,5,6]. It is of great economic value and medical value to disclose the metabolic feature and the mechanism involved in the development of IMF.

Due to the regional differences, the development, lipid content, and lipid metabolism of intramuscular (i.m.) adipocytes differ from those of subcutaneous (s.c.) adipocytes. It was reported that intramuscular adipose grew slower than those of subcutaneous adipose, and had the lowest lipid content than other adipose [7,8]. Our previous study also indicated that compared with lipids accumulated in s.c. adipocytes at late stage of differentiation in vitro, less lipids were accumulated in porcine i.m. adipocytes and that the significant difference occurred on the 6th differentiation day [9]. In addition, studies on bovine adipocytes had shown that i.m. and s.c. adipocytes utilized different carbon precursors for fatty acid synthesis during lipogenesis, and had different response to dexamethasone, a typical lipogenic inducer [10,11]. All these findings suggested that the developmental patterns and metabolism features of i.m. adipose were different from other adipose, but the underlying mechanism of these processes still remained unknown.

It is well documented that adipocytes obtain fatty acids for triglycerides synthesis in the following two ways: one is to utilize acetyl-CoA obtained from glycolysis for fatty acids de novo synthesis; the other way is to uptake exogenous fatty acids by lipoprotein lipase (LPL) and fatty acid transporters (FAT) [12,13,14]. Several enzymes and proteins play pivotal roles in these two processes, including glucose transporters (GLUT), acetyl CoA carboxylase (ACC), fatty acid synthase (FASN), LPL, and FAT [14,15]. Our previous study revealed that the mRNA expression level of LPL was significantly higher in s.c. adipocytes on the 6th of differentiation day than that in i.m. adipocytes and that PDK4, a gene involved in glycolysis, was rich in i.m. adipocytes [9]. These findings suggested that these enzymes genes might account for the differences of lipid accumulation between i.m. and s.c. adipocytes. However, the expression patterns of these genes during i.m. and s.c. preadipocytes differentiation are not yet clear.

Recently, more and more studies indicated that bone morphogenetic proteins (BMPs) and BMP signaling pathway played the very important roles in adipogenesis in mesenchymal stem cells (MSCs) [16,17,18,19] and in cell lines of preadipocytes [20,21]. However, little evidence was found to support the effects of BMPs and BMP signaling pathway on the differentiation porcine preadipocytes. Interestingly, we proved that the mRNA levels of both BMP4 and BMP7 were specifically higher in i.m. adipocytes than those in s.c. adipocytes by microarray and quantitative real-time PCR in our previous study [9], suggesting that BMPs might participate in the differentiation regulation of i.m. adipocytes. Thus, the elucidation of the role of BMP signaling pathway in the differentiation of i.m. and s.c. adipocytes may enhance the understanding of the mechanisms that regulate the development of these two kinds of cells.

In the present study, i.m. and s.c. preadipocytes obtained from neonatal Landrace pigs were induced to differentiate into mature adipocytes, and glucose utilization and glycerol release were determined to explore the lipogenesis and lipolysis. Meanwhile, the mRNA and protein expression levels of genes related to lipogenesis, lipolysis, BMP-Smad signaling pathway and adipocyte differentiation were respectively detected by quantitative real-time PCR and western blot. Furthermore, the effects of BMPs and antagonists of BMP-Smad signaling pathway on the adipogenesis of i.m. and s.c. adipocytes were investigated. Finally, by using RNA interference strategy, the influences of BMP2 and BMP4 shRNA on the expression of the genes involved in BMP-Smad signaling pathway and adipocyte differentiation were explored. Our study helps to elucidate the difference between porcine i.m. and s.c. preadipocytes in glucose utilization and lipid metabolism, reveals the role of the BMP-Smad signaling pathway, and improves our understanding the underlying mechanisms involved in the difference between i.m. and s.c. adipocytes in pigs.

Materials and Methods

Cell culture

Postnatal Landrace pigs aged of 5 to 7 days were killed via intraperitoneal injection of pentobarbital sodium (50 mg/kg bodyweight) followed by exsanguinations. Intramuscular and subcutaneous preadipocytes were isolated from longissimus dorsi muscle (LM) and subcutaneous adipose tissue (SAT) of the pigs by collagenase digestion respectively, and purified by a percoll density gradient centrifugation method as described previously [9]. The procedure was conducted in accordance with “The Instructive Notions with Respect to Caring for Laboratory Animals” issued by the Ministry of Science and Technology of the People's Republic of China. The purified cells were then seeded with a density of 3.0×104 cells/well or 2.0×105 cells/well in 24-well or 6-well plates, respectively. After 3 or 4 days, when the cells reached confluence, medium was exchanged to the adipogenic cocktail as described previously [9], which was DMEM/F12 (DMEM/F12, 1:1, GIBCO, Grand Island, NY, USA) containing 5% newborn bovine serum (NBS, GIBCO, Grand Island, NY, USA), 100000 U/L of penicillin sodium, 100 mg/L of streptomycin sulfate, 50 nM insulin, 50 nM dexamethasone, 50 μM oleate and 0.5 mM octanoate (penicillin sodium and streptomycin sulfate were purchased from GIBCO, Grand Island, NY, USA, whereas bovine recombinant insulin, dexamethasone, oleate and octanoate were all purchased from Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in adipogenic cocktail for 6 or 7 days. On each day of differentiation, 8 wells of cells from 24-well plates were used for triglyceride assay and culture medium were collected for glucose and glycerol assay, 6 wells of cells from 6-well plates were used for quantitative real-time PCR and 4 wells of cells were used for western blot assay.

Glucose assay

Culture medium obtained from 24-well plates on each day of differentiation was diluted in Ca2+, Mg2+-free PBS (1:5, v/v), then 50 μL of the diluted sample was used for glucose assay by using a Glucose Kit (Biosino Bio-Technology and Science Inc., Beijing, China) based on GOD-POD method [22] according to the manufacturer's protocol. Adipogenic medium that prepared at the same time but wasn't used for cell culture (un-culture medium) was also distributed in wells and incubated at the same condition with medium that used for cell culture. The glucose contents of these un-culture and culture medium were detected, and glucose utilization of cells was determined by subtracting the glucose content of the culture medium from that of the un-culture medium[23,24,25]. 8 wells of cells were used at each time point, and data was finally normalized by the content of total protein of cells detected by using a commercial kit (Bioteke Corporation, Beijing, China) based on bicinchoninic acid method [26] according to the manufacturer's procedure. The experiment was repeated for three times.

Glycerol assay

Glycerol content in culture medium of adipocytes served as an index of lipolysis [27,28,29]. To inactivate endogenous lipase, the culture medium was heated at 70 °C for 10 min, and the glycerol content of the samples were detected using a commercial kit (Beijing SINOPCR Co., LTD, Beijing, China) based on GPO Trinder reaction according to the manufacturer's protocol. Data was obtained from 8 wells of cells at each time point and normalized by the content of total protein of cells.

Triglyceride Assay

To investigate the effects of BMPs and antagonists of BMP-Smad signaling pathway on the differentiation of intramuscular and subcutaneous preadipocytes, cells were seeded in 48-well plates with a density of 1.0×104 cells/well. When the cells reached confluence, adipogenic medium containing 0, 25, 50, and 100 ng/mL of BMP2, BMP4, and Noggin, as well as 0, 2.5, 5, and 10 μM of Compound C (recombinant human BMP2 and Noggin were purchased from Shanghai PrimeGene Bio-Tech Co.,Ltd, Shanghai, China; recombinant human BMP4 were purchased from ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel; Compound C was purchased from Sigma-Aldrich, St. Louis, MO, USA), were used to treat the cells for 4 days, respectively. Cytosolic triglycerides were determined by a Triglyceride Kit (Biosino Bio-Technology and Science Inc., Beijing, China, based on lipase glycerol kinase enzymatic method) as described previously [9], and 6 wells of cells were used for each concentration of different supplements.

Quantitative real-time PCR

On each day of differentiation, cells cultured in 6-well plates were harvested and total RNA of each well of cells was extracted using a TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, and the trace amount of genome DNA was digested using RNase-free DNase I (Takara Bio Inc, Shiga, Japan). 1 μg of total RNA was reverse-transcribed using random primer N10 (N=A, T, C or G) and murine Moloney leukemia virus reverse transcriptase (MMLV, Promega, Madison, WI, USA). Primers for genes related to glucose utilization and lipid metabolism (Table 1) were designed using Primer Premier 5 (PREMIER Biosoft, Canda). SYBR Green Real-time PCR Master Mix reagents (Toyobo Co., Ltd., Osaka, Japan) and both sense and antisense primers (200 nM for each gene) were used for real-time quantitative PCR analysis in a final volume of 20 μL. GAPDH (glyceraldehydes-3-phosphate dehydrogenase) was used as a housekeeping gene according to its stability confirmed previously [9]. The real-time PCR reactions were performed in Mx3005p instrument (Stratagene, La Jolla, CA, USA), and a melting-curve analysis was also performed for each gene to confirm the specific amplification product. The relative gene expression for each gene was calculated using the formula as described in previous report [30]: R0,T/R0,R= (1+ER)Ct,R/(1+ET)Ct,T.

Table 1

The primers sequences of target genes

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Western blot

After RNA extraction, the protein contained in the organic phase of the Trizol reagent was extracted according to the manufacturer's protocol and the concentration of protein was detected using a commercial kit (Bioteke Corporation, Beijing, China) based on bicinchoninic acid method. Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking by 5% non-fat milk at room temperature for 2 h, the membranes were incubated with different primary antibodies, including FASN (1:500), ATGL (1:500), SREBP1 (1:500), PDK4 (1:500), PLIN (1:500), and β-actin (1:1000), at 4 °C overnight (antibodies FASN, ATGL, SREBP1, PDK4, and PLIN were all purchased from Santa Cruz, whereas β-actin was purchased from Biosen, China). Then the membranes were washed for 5×5 min in TBST buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 0.05% Tween-20), and incubated with different HRP-labeled secondary antibodies at room temperature for 2 h. The blots were developed with enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology, Jiangsu, China). The optical densities of the bands were analyzed using UVP image software.

RNA interference

Both intramuscular and subcutaneous preadipocytes were seeded in 12-well plates 24 hours before transduction with a density of 5.0×104 cells/well. Then culture medium was replaced by fresh culture medium containing 5 μg/mL Polybrene (Santa Cruz Biotechnology,Inc., Santa Cruz, CA, USA) and three Lentiviral Particles, BMP2 shRNA(h) Lentiviral Particles, BMP4 shRNA(h) Lentiviral Particles, and Control shRNA Lentiviral Particles (these Lentiviral particles were all purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA ), respectively. Cells were incubated with the Lentiviral particles for 24 hours, and culture medium was replaced by fresh culture medium without Polybrene or virus. 2 days post-infection, cells were split 1:3 to new 12-well plates, and medium containing 4 μg/mL of Puromycin dihydrochloride (AMRESCO Inc., Solon, OH, USA) was used for selecting stable cells expressing shBMP2, shBMP4, and control shRNA when the cells reached confluence. Stable clones were then expanded and seeded in 6-well plates for gene expression analysis.

Statistical Analysis

The results of glucose assay represented for means of three independent experiments. 8 parallels at each time point were used in glycerol assay, whereas 6 wells of cells were used for each concentration of different supplements in triglyceride assay in one experiment. For quantitative real-time RT-PCR and western blot analysis, 6 or 4 wells of cells were used respectively in one experiment. Data was presented as means ± standard error of the mean (SEM). Statistical analysis was performed by means of Student's t test or one way analysis of variance (ANOVA) followed by Duncan's multiple range test (SAS Institute Inc., Cary, NC, USA) when appropriate and a confidence level of P<0.05 was considered statistically significant.

Results

The differences of lipid metabolism and glucose utilization between porcine i.m. and s.c. preadipocytes

To compare the differences of lipid metabolism and glucose utilization between porcine i.m. and s.c. preadipocytes, we determined the contents of cytosolic triglycerides (index of lipogenesis), glycerol release (index of lipolysis) and glucose utilization at various time points of differentiation. The results of glucose assay revealed that i.m. and s.c. preadipocytes consumed glucose in different manners. The glucose utilization decreased in i.m. preadipocytes and increased in s.c. preadipocytes in a time-dependent manner (Fig. 1 A). From day 0 to day 3 of differentiation, the glucose uptakes in i.m. preadipocytes were significantly higher than those in s.c. adipocytes (P<0.01, or P<0.05) (Fig. 1 A). In contrast to glucose consumption, glycerol release to culture medium by i.m. and s.c. preadipocytes changed in a similar manner: it increased gradually and peaked on day 5 of differentiation and then declined (Fig. 1 B). The concentrations of glycerol on day 5 (P<0.05) and day 7 (P<0.001) of differentiation in culture medium of i.m. preadipocytes were higher than those of s.c. preadipocytes (Fig. 1 B).

Fig. 1

Differences between i.m. and s.c. preadipocytes on glucose uptake and glycerol release during differentiation. Both i.m. and s.c. preadipocytes were seeded in 24-well plates and induced to undergo adipogenesis. Culture mediums on day 0, day 1, day 3, day 5, day 7 of differentiation were collected and the glucose utilization (A) and glycerol release (B) were detected by using commercial kits, respectively. For glucose assay, data were means ± SEM of three independent experiments (For each experiment, 8 wells of cells were used at each time point). In glycerol assay, 8 wells of cells were used at each time point in one experiment.* P<0.05, ** P<0.01, *** P<0.001.

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The expression patterns of the genes involved in glucose utilization and lipid metabolism in i.m. and s.c. preadipocytes

The expression pattern of GLUT1 in i.m. preadipocytes differed from that in s.c. preadipocytes. GLUT1 levels on day 0 (P<0.05) and day 5 (P<0.001) of differentiation in i.m. preadipocytes were significantly higher than those of s.c. preadipocytes (Fig. 2 A). Another glucose transporter, GLUT4, however, changed in a similar manner in these two kinds of cells and mRNA level on day 3 of differentiation (P<0.01) in s.c. preadipocytes was higher (Fig. 2 B). The mRNA level of pyruvate kinase, PKM2, in i.m. preadipocytes (non-differentiated cells) was significantly higher than that in s.c. preadipocytes (P<0.05) and higher mRNA level was found in s.c. preadipocytes on day 3 of differentiation (Fig. 2 C). The mRNA levels of PDK4 in these two kinds of cells increased gradually during differentiation and the mRNA levels in i.m. preadipocytes were significantly higher than those in s.c. preadipocytes on day 5 (P<0.001) and day 6 (P<0.01) of differentiation (Fig. 2 D). The PDK4 protein level in i.m. preadipocytes was also higher than that in s.c. preadipocytes on day 5 (P<0.01) (Fig. 3 A, E).

Fig. 2

The expression patterns of genes involved in glucose utilization and lipid metabolism of i.m. and s.c. preadipocytes during differentiation. The changes of mRNA levels of genes related to glucose utilization (GLUT1 and GLUT4, A, B) and glycolysis (PKM2 and PDK4, C, D), fatty acid transport (LPL and FAT, E, F) and fatty acid synthesis (ACC and FASN, G, H), and lipolysis (ATGL and HSL, I, J) were detected by quantitative real-time PCR, respectively (n = 6). The relative expression levels of target genes compared to housekeeping gene, GAPDH, were calculated. * P< 0.05, ** P< 0.01, *** P< 0.001.

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Fig. 3

The differences of protein levels of genes related with glucose utilization and lipid metabolism between i.m. and s.c. preadipocytes during differentiation. Immunoblots of FASN, ATGL, SREBP1, PDK4, and PLIN on day 3 and day 5 of differentiation of intramuscular and subcutaneous preadipocytes (A), the relative protein levels of FASN, ATGL, SREBP1, PDK4, and PLIN between i.m. and s.c. preadipocytes on day 3 and day 5 of differentiation, respectively (n = 4) (B∼D). The relative protein levels of PDK4, and PLIN between i.m. and s.c. preadipocytes day 5 of differentiation (n=4) (E). Data were normalized to the expression levels of β-actin. ** P< 0.01.

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The mRNA levels of LPL in s.c. preadipocytes were significantly higher than those in s.c. preadipocytes from day 2 to day 6 of differentiation (P<0.05) (Fig. 2 E). The change of FAT mRNA expression levels in s.c. preadipocytes was similar to that of LPL and the mRNA levels of FAT on day 3 (P<0.001) and day 5 (P<0.01) of differentiation were higher than those in i.m. preadipocytes (Fig. 2 F). The mRNA levels of ACC and FASN increased more quickly in s.c. preadipocytes than that in i.m. preadipocytes, and the levels in s.c. preadipocytes on day 3 of differentiation were significantly higher(P<0.001, or P<0.05) (Fig. 2 G, H). The level of FASN protein in s.c. preadipocytes was higher on day 3 but significantly lower on day 5 (P<0.01) of differentiation than those in i.m. preadipocytes (Fig. 3 A, B). Both ATGL and HSL in s.c. preadipocytes had higher mRNA levels than those in i.m. preadipocytes on day 3 of differentiation (P<0.001), whereas ATGL and HSL in s.c. preadipocytes on day 5 of differentiation had lower mRNA levels than those in i.m. preadipocytes. And significant differences were confirmed (P<0.05, or P<0.01) (Fig. 2 I, J). Just like mRNA, ATGL protein was higher on day 3 but lower on day 5 of differentiation in s.c. preadipocytes than those in i.m. preadipocytes (Fig. 3 A, C). Though no significant difference was confirmed, the levels of SREBP1 protein were both higher on day 3 and day5 of differentiation in s.c. preadipocytes than those in i.m. preadipocytes (Fig. 3 A, D). The Perilipin (PLIN) protein level in i.m. preadipocytes on day 5 of differentiation was higher than that in s.c. preadipocytes (Fig. 3 A, E).

The expression patterns of the genes involved in BMP-Smad signaling pathway were consistent with those of the genes related to preadipocytes differentiation

The mRNA levels of BMP2 in s.c. preadipocytes were significantly higher than those in i.m. preadipocytes on day 2 (P<0.05) and day 3 (P<0.01) of differentiation (Fig. 4 A), whereas the mRNA levels of BMP4 in i.m. preadipocytes were higher (P<0.05) than those in s.c. preadipocytes at the late stage of differentiation (Fig. 4 B). The mRNA levels of the genes in the downstream of BMP-Smad signaling pathway, such as BMPRIA, BMPRIB, BMPRII, Smad1, and Smad4 (Fig. 4 C∼G), changed in a similar manner in both i.m. and s.c. preadipocytes during differentiation. The highest mRNA levels of these genes were found in s.c. preadipocytes on day 3 of differentiation and significant differences were confirmed between i.m. and s.c. preadipocytes (P<0.05, or P<0.01). In contrast, in i.m. preadipocytes, the highest mRNA levels of these genes were observed on day 5 of differentiation. Similar patterns were found in genes related to preadipocytes differentiation such as PPARγ, C/EBPα, and FABP5 (Fig. 4 H∼J).

Fig. 4

The expression patterns of genes involved in BMP-Smad signaling pathway and preadipocytes differentiation in i.m. and s.c. preadipocytes during differentiation. The mRNA levels of different BMPs (BMP2, BMP4, A∼B), BMP receptors (BMPRIA, BMPRIB, and BMPRII, C∼E), Smads (Smad1 and Smad4, F∼G), as well as adipocyte differentiation markers (PPARγ, C/EBPα, and FABP5, H∼J) at different time-points of differentiation were detected by quantitative real-time PCR, respectively (n = 6). Data were normalized to the mRNA levels of GAPDH. * P< 0.05, ** P< 0.01.

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Effects of BMPs on the differentiation of i.m. and s.c. preadipocytes

In order to determine the effects of BMP2 and BMP4 on the differentiation of porcine i.m. and s.c. preadipocytes, we added various doses of BMP2 and BMP4 into the medium and measured the content of cytosolic triglycerides. As shown in Fig. 5 A and 5C, BMP2 other than BMP4 significantly (P<0.05) increased cytosolic triglycerides content in i.m. preadipocytes in a dose-dependent manner. In contrast, the accumulations of cytosolic triglycerides in s.c. preadipocytes was not influenced by BMP2 and BMP4 (Fig. 5 B, D)

Fig. 5

Effects of BMPs and BMP-Smad signaling pathway antagonists on differentiation of i.m. and s.c. preadipocytes. Intramuscular (A, C, E, and G) and subcutaneous (B, D, F, and H) preadipocytes were treated with adipogenic medium containing 0, 25, 50, and 100 ng/mL of BMP2 (A, B), BMP4 (C, D) and Noggin (E, F), and 0, 2.5, 5, 10 μM of Compound C (G, H) for four days (n = 6), respectively, and cytosolic triglycerides were determined. Data without a similar letter above means the differences between two groups were significant (P< 0.05).

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Inhibition of BMP signaling prevented the fat accumulation of porcine i.m. adipocytes

BMP signaling pathway antagonists, such as Noggin and Compound C, were applied to investigate the effects of inhibition of BMP signaling pathway on the differentiation of porcine i.m. and s.c. preadipocytes. Our findings revealed that BMP signaling pathway antagonists (Noggin and Compound C) significantly (P<0.05) decreased cytosolic triglycerides in i.m. adipocytes in a dose-dependent manner (Fig. 5 E, G). In contrast, Noggin and Compound C had little effects on the accumulation of cytosolic triglycerides in s.c. preadipocytes (Fig. 5 F, H).

BMP2 and BMP4 shRNA treatment decreased the expressions of the genes involved in BMP-Smad signaling pathway and preadipocytes differentiation

In addition to pharmacological inhibition of BMP signaling by Noggin and Compound C, we used the shRNA to decrease endogenous BMP2 and BMP4 mRNA expression and observed the effects on the expressions of the genes involved in BMP-Smad signaling pathway and preadipocytes differentiation. i.m. and s.c. preadipocytes stably expressed control, BMP2, and BMP4 shRNA were respectively obtained after Puromycin selection for 10 days. As shown in Fig. 6 A and B, BMP2 and BMP4 shRNA inhibited the mRNA levels of BMP2 and BMP4 in both i.m. and s.c. preadipocytes to some extent. However, they dramatically decreased the mRNA levels of BMPRIA (Fig. 7 A, B) and BMPRIB (Fig. 7 C, D) in both i.m. and s.c. preadipocytes (P<0.05, P<0.01, or P<0.001). In addition, BMP2 shRNA could significantly reduce the mRNA levels of Smad1 (Fig. 7 E, F) and Smad4 (Fig. 7 G, H) in both two kinds of cells (P<0.05). BMP4 shRNA significantly repressed the mRNA expression levels of Smad4 in s.c. preadipocytes (P<0.05) (Fig. 7 H), while it had no significantly inhibition effect on the expression of Smad4 in i.m. preadipocytes as well as on Smad1 expression in the two kinds of cells. Interestingly, the mRNA levels of PPARγ in i.m. and s.c. preadipocytes were both dramatically inhibited by BMP2 and BMP4 shRNA (P< 0.05, or P< 0.001) (Fig. 7 I, J).

Fig. 6

The establishment of BMP2 and BMP4 knock-down i.m. and s.c. preadipocytes using BMP2- and BMP4-shRNAs. The changes of mRNA levels of BMP2 (A) and BMP4 (B) in i.m. and s.c. preadipocytes after being transfected with BMP2 and BMP4 shRNA, respectively (n = 6). Cells transfected with control shRNA served as control.

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Fig. 7

The changes of mRNA levels of genes involved in BMP-Smad signaling pathway and preadipocyte differentiation in i.m. and s.c. preadipocytes after being transfected with BMP2 and BMP4 shRNA. The mRNA levels of BMPRIA, BMPRIB, Smad1, Smad4, and PPARγ in i.m. (A, C, E, G and I) and s.c. (B, D, F, H and J) preadipocytes that were stably transfected with BMP2 and BMP4 shRNA were detected, respectively (n = 6). Cells transfected with control shRNA served as control. * P< 0.05, ** P< 0.01, *** P< 0.001 (compared with control).

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Discussion

The present study provided some new information about the difference between porcine i.m. preadipocytes and s.c. preadipocytes on glucose utilization, lipid metabolism and BMP-Smad signaling pathway.

With this in vitro culture system, the obtained results revealed that the utilization of glucose at early differentiation stage in i.m. preadipocytes was significantly higher than that in s.c. preadipocytes. According to this finding, the critical genes of glycolysis, pyruvate kinase muscle isoform 2 (PKM2) were found to be highly expressed in i.m preadipocytes at early differentiation stage, suggesting that the glycolysis was active in i.m adipocytes. These results, together with early findings in bovine adipose tissue culture [10], indicated that i.m. adipocytes mainly utilized glucose for lipogenesis at the early stage of differentiation. In contrast, Gardan et al. found that in presence of insulin, glucose incorporation into lipids was lower (P<0.05) in i.m. adipocytes than in s.c. adipocytes [31]. The reason for the opposite results obtained by Gardan et al. and us might be interpreted in two aspects. Firstly, the cells was not the same. In Gardan's work, the adipocytes isolated from trapezius muscle are mature adipocytes, and the glucose uptake of these cells were conducted in an acute experiment. In our experiment, however, we isolated preadipocyte from longissimus dorsi muscle and subcutaneous adipose tissue to compare the differences between intramuscular and subcutaneous preadipocytes during the whole differentiation period. Secondly, the age of the pigs was different (80 days vs. 5-7 days), which might affect the utiliazation of glucose by i.m. preadipocytes.

Glucose is an important material for the biosynthesis of triglycerides in adipose [32] and GLUT4 is generally regarded as a key glucose transporter in adipocytes [33,34]. However, in the present study, we found that the expression level of GLUT4 in i.m adipocytes was not higher but even lower than that in s.c. adipocytes in the early stages of differentiation. On the contrary, the expression levels of GLUT1 in i.m. adipocytes at the beginning and the late phase of differentiation (day 5) were significantly higher than that in s.c. adipocytes. The previous study in bovine intramuscular preadipocytes also indicated that, the GLUT1 protein was significantly increased during differentiation, whereas the GLUT4 protein remained undetectable [35]. Combined with the results obtained in our paper, it was suggested that i.m. preadipocytes might utilize glucose through GLUT1 other than GLUT4.

It was reported that, in some condition where GLUT4 was repressed, such as Type II diabetes, GLUT1, which was widely expressed in multiple tissues and accounted for basic glucose transport [36,37], could compensate for glucose transport [38]. Moreover, special attention was paid to the relationship between adipocyte and muscle in diabetes [39,40,41] and recent reports showed that co-culture with adipocytes could modify glucose and lipid metabolism of skeletal muscle [42,43]. Therefore, the high level of GLUT1, large consumption of glucose, and special location might make i.m. adipocytes the potential target for treatment of diabetes.

In contrast to i.m. adipocytes, s.c. adipocytes were found to mainly used fatty acids for lipogenesis [10], and grew faster and accumulated more lipids than i.m. adipocytes [7,8]. Our previous study also revealed that s.c. adipocytes accumulated more triglyceride than i.m. adipocytes during differentiation [9]. In agreement with this finding, we observed that the mRNA levels of genes related to lipogenesis such as LPL, FAT, ACC, and FASN, were increased more quickly and had higher expression levels in s.c. preadipocytes than those in i.m. preadipocytes in early stages. In the late stages, the mRNA levels of these genes in s.c preadipocytes were indistinguishable from those in i.m preadipocytes except for mRNA level of LPL, which was persistently increased in s.c preadipocytes (Fig. 2 E∼H and Fig. 8). The higher mRNA level of LPL in s.c. preadipocytes agreed with the results obtained in some other previous studies [44,45]. Based on the high levels of LPL and FAT and the previous finding that s.c. adipose mainly utilized acetic acid for fatty acid synthesis [10], we speculated that porcine s.c. adipocytes, in contrary to i.m. adipocytes, predominantly utilized exogenous fatty acid for lipid synthesis, especially at the late stages of differentiation.

Fig. 8

Schematic representation of the difference between i.m. and s.c. preadipocytes on differentiation patterns. Both s.c. preadipocytes (SCPA) and i.m. preadipocytes (IMPA) are committed from mesenchymal stem cells (MSC) by BMPs. The differentiation program of SCPA is faster and stimulated mainly by BMP2, followed with PPARγ, C/EBPα, as well as other genes relative to adipogenesis, and the accumulation of lipids in s.c. adipocytes are mainly dependent on transporting exogenous fatty acids (FA). In IMPA, however, the differentiation program is slower and stimulated mainly by BMP2 and BMP4. In contrast to s.c. adipocytes, i.m adipocytes mainly used glucose (Glu) for lipogenesis, and the accumulation of cellular lipids is less than s.c. adipocytes at the late phase of differentiation. Genes above or below the line represent its higher expression in relevant cells during differentiation.

http://www.karger.com/WebMaterial/ShowPic/181495

In addition to lipogenesis, lipolysis also plays an important role in the accumulation of lipids in adipocytes. Interestingly, we found that the content of glycerol in culture medium obtained from i.m. adipocytes was significantly higher than that from s.c. adipocytes. In coincide with this finding, the expression levels of ATGL and HSL in i.m. preadipocytes on day 5 of differentiation were all significantly higher than those in s.c. preadipocytes. The higher level of lipolysis in i.m. preadipocytes at the late stages of differentiation might be responsible for the residual less lipids in these cells.

Our previous study demonstrated that the expression levels of BMP4 and BMP7 in i.m. adipocytes were both higher than those in s.c. adipocytes [9]. In addition, BMP-Smad signaling has been proved to play an important role in the development of adipocytes [46,47,48,49]. So we wondered whether this signaling pathway played different roles in the two populations of cells. By using real-time quantitative PCR, we found that the expression patterns of the genes related to BMP-Smad signaling pathway, such as BMP2, BMPRIA, BMPRIB, BMPRII, Smad1, and Smad4, as well as the genes related to adipocytes differentiation, including PPARγ, C/EBPα, and FABP5, were very similar during the differentiation of i.m. and s.c. preadipocytes. The mRNA levels of these genes were all increased quickly in s.c. preadipocytes and peaked on day 3 of differentiation. In s.c. preadipocytes, they were increased slowly and peaked on day 5 of differentiation (Fig. 4 and Fig. 8). These results strongly implied that BMP-Smad signaling pathway might participate in the differentiation regulation of both i.m. and s.c. preadipocytes.

Furthermore, we found that BMP2 could significantly increase the content of cytosolic triglycerides of i.m. preadipocytes (Fig. 5 A, E), and this proadipogenic effect was in consistent with those found in preadipocyte cell lines or MSCs [18,50,51]. Meanwhile, Noggin and Compound C, two antagonists of BMP-Smad signaling pathway [18,52], significantly decreased the lipid accumulation in i.m. preadipocytes (Fig. 5 E, G). In s.c. preadipocytes, however, these four agents had no significant effect on the accumulation of lipids. Due to the limit of the replicate, the variation of the results cannot be ignored. However, these findings suggested that the differentiation of i.m. preadipocytes was more dependent on BMP-Smad signaling pathway than s.c. preadipocytes. Moreover, by using RNA interference strategy, we found that BMP2 and BMP4 shRNA had slightly effects on the expression of BMP2 and BMP4 in i.m. and s.c. preadipocytes. However, BMP2 and BMP4 shRNA significantly reduced the mRNA levels of the genes in the downstream of BMP-Smad signaling pathway, and dramatically reduced the mRNA levels of PPARγ. And the reason for the knockdown results of BMP2 and BMP4 being not statistically different might be mainly due to the limit of the replicates of samples and the experimental error in real-time PCR assay. Another possibility, though scarce, is that the sequences detected by qRT-PCR are not the target sites of shRNA, and have not been completely and efficiently digested, since different sites of mRNA away from the target site of siRNA have been shown to be digested at different rates [53]. Together with the results above, we inferred that BMP-Smad signaling pathway regulated the differentiation of both i.m. and s.c. preadipocytes and that the differentiation of i.m. pradipocytes seemed to be more dependent on this signaling pathway than s.c. preadipocytes.

Our previous work [9] and the present study have revealed different features between i.m. and s.c. adipocytes during differentiation. These findings showed that glucose and fatty acids might be differently utilized as sources of triglyceride synthesis by the two cells, and that the differentiation program, triggered by a lot of genes such as BMPs, PPARγ, and C/EBPα, in s.c. adipocytes began more early than i.m. adipocytes when responding to the same adipogenesis reagent (Fig. 8). These features may account for the different developmental rates of these two populations of cells in vitro as well as in vivo. Although the proportion of i.m. adipocyte in animal as well as human body is much smaller than that other depots of adipocytes, it is well documented that i.m. adipose is strongly associated with insulin resistance or Type 2 diabetes [54,55,56]. It is proved that i.m. adipose can change the metabolism feature of muscle by fatty acids flux [57,58] and cause Type 2 diabetes. Our present study showed that ATGL and HSL, two genes involved in lipolysis, were highly expressed in mature i.m. adipocytes (Fig. 2, Fig. 3, and Fig. 8). Furthermore, the development of i.m. adipocytes seemed to be more dependent on BMP-Smad signaling pathway (Fig. 5). According to these findings, to manipulating some relevant genes in i.m. adipocytes may be a new way for treatment of Type 2 diabetes.

In conclusion, our present study demonstrated that i.m. and s.c. preadipocytes accumulated lipids in two distinct manners: i.m. preadipocytes mainly utilized glucose whereas s.c. preadipocytes predominantly used exogenous fatty acid for lipids synthesis. The expressions of GLUT1 and LPL in these two cells might account for this difference respectively. The special feature of i.m. adipocytes implied that these cells might be a potential target for treating diabetes. Furthermore, the expression differences of the genes related to lipid metabolism and terminal differentiation between i.m. and s.c. preadipocytes were defined by the upstream signals, in which the BMP-Smad signaling pathway played a critical role (Fig. 8).

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 30901058 and No. 30972157), the National Key Project (No. 2009CB941601), the Joint Funds of the National Natural Science Foundation of China (No. u0731004), Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20094404120012 and No. 20124404130001) and Natural Science Foundation of Guangdong Province; Contract grant number: (No. S2012020011048 and No. S2012010010176).

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Author Contacts

Qingyan Jiang

Department of Animal nutrition and Feed Science, College of Animal Science

South China Agricultural University, Guangzhou, 510642 (P. R. China)

Tel. +8620-85284930, Fax +8620-85284901, E-Mail qyjiang@scau.edu.cn

Article / Publication Details

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Abstract of Original Paper

Accepted: June 09, 2013
Published online: July 02, 2013
Issue release date: July 2013

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ISSN: 1015-8987 (Print)
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References

  1. 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 Science 1999;53:59-65.
    External Resources
  2. van Laack R, Stevens SG, Stalder KJ: The influence of ultimate ph and intramuscular fat content on pork tenderness and tenderization. J Anim Sci 2001;79:392-397.
    External Resources
  3. Huang YC, Li HJ, He ZF, Wang T, Qin G: Study on the flavor contribution of phospholipids and triglycerides to pork. Food Sci Biotechnol 2010;19:1267-1276.
    External Resources
  4. Lim S, Son KR, Song IC, Park HS, Jin CJ, Jang HC, Park KS, Kim YB, Lee HK: Fat in liver/muscle correlates more strongly with insulin sensitivity in rats than abdominal fat. Obesity (Silver Spring) 2009;17:188-195.
    External Resources
  5. Dodson MV, Mir PS, Hausman GJ, Guan LL, Du M, Jiang Z, Fernyhough ME, Bergen WG: Obesity, metabolic syndrome, and adipocytes. J Lipids 2011;2011:721686.
    External Resources
  6. Bjorndal B, Burri L, Staalesen V, Skorve J, Berge RK: Different adipose depots: Their role in the development of metabolic syndrome and mitochondrial response to hypolipidemic agents. J Obes 2011;2011:490650.
    External Resources
  7. Kouba M, Sellier P: A review of the factors influencing the development of intermuscular adipose tissue in the growing pig. Meat Sci 2011;88:213-220.
    External Resources
  8. Kouba M, Bonneau M: Compared development of intermuscular and subcutaneous fat in carcass and primal cuts of growing pigs from 30 to 140 kg body weight. Meat Science 2009;81:270-274.
    External Resources
  9. Zhou GX, Wang SB, Wang ZG, Zhu XT, Shu G, Liao WY, Yu KF, Gao P, Xi QY, Wang XQ, Zhang YL, Yuan L, Jiang QY: Global comparison of gene expression profiles between intramuscular and subcutaneous adipocytes of neonatal landrace pig using microarray. Meat Science 2010;86:440-450.
    External Resources
  10. Smith SB, Crouse JD: Relative contributions of acetate, lactate and glucose to lipogenesis in bovine intramuscular and subcutaneous adipose-tissue. J Nutr 1984;114:792-800.
    External Resources
  11. Ortiz-Colón G, Grant AC, Doumit ME, Buskirk DD: Bovine intramuscular, subcutaneous, and perirenal stromal-vascular cells express similar glucocorticoid receptor isoforms, but exhibit different adipogenic capacity. J Anim Sci 2009;87:1913-1920.
    External Resources
  12. Brasaemle DL: Thematic review series: Adipocyte biology. The perilipin family of structural lipid droplet proteins: Stabilization of lipid droplets and control of lipolysis. J Lipid Res 2007;48:2547-2559.
    External Resources
  13. Ducharme NA, Bickel PE: Lipid droplets in lipogenesis and lipolysis. Endocrinology 2008;149:942-949.
    External Resources
  14. Vazquez-Vela ME, Torres N, Tovar AR: White adipose tissue as endocrine organ and its role in obesity. Arch Med Res 2008;39:715-728.
    External Resources
  15. Kopecky J, Flachs P, Bardova K, Brauner P, Prazak T, Sponarova J: Modulation of lipid metabolism by energy status of adipocytes - implications for insulin sensitivity. Ann N Y Acad Sci 2002;967:88-101.
    External Resources
  16. Date T, Doiguchi Y, Nobuta M, Shindo H: Bone morphogenetic protein-2 induces differentiation of multipotent c3h10t1/2 cells into osteoblasts, chondrocytes, and adipocytes in vivo and in vitro. J Orthop Sci 2004;9:503-508.
    External Resources
  17. Hino J, Miyazawa T, Miyazato M, Kangawa K: Bone morphogenetic protein-3b (bmp-3b) is expressed in adipocytes and inhibits adipogenesis as a unique complex. Int J Obes 2012;36:725-734.
    External Resources
  18. Bowers RR, Kim JW, Otto TC, Lane MD: Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: Role of the bmp-4 gene. Proc Natl Acad Sci U S A 2006;103:13022-13027.
    External Resources
  19. Huang H, Song TJ, Li X, Hu L, He Q, Liu M, Lane MD, Tang QQ: Bmp signaling pathway is required for commitment of c3h10t1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci U S A 2009;106:12670-12675.
    External Resources
  20. Bowers RR, Lane MD: A role for bone morphogenetic protein-4 in adipocyte development. Cell Cycle 2007;6:385-389.
    External Resources
  21. Suenaga M, Matsui T, Funaba M: Bmp inhibition with dorsomorphin limits adipogenic potential of preadipocytes. J Vet Med Sci 2010;72:373-377.
    External Resources
  22. Trinder P: Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 1969;22:158-161.
    External Resources
  23. Garcia-Diaz DF, Campion J, Arellano AV, Milagro FI, Moreno-Aliaga MJ, Martinez JA: Fat intake leads to differential response of rat adipocytes to glucose, insulin and ascorbic acid. Exp Biol Med 2012;237:407-416.
    External Resources
  24. Gardner DK, Wale PL, Collins R, Lane M: Glucose consumption of single post-compaction human embryos is predictive of embryo sex and live birth outcome. Hum reprod 2011;26:1981-1986.
    External Resources
  25. Manna P, Jain SK: Vitamin d up-regulates glucose transporter 4 (glut4) translocation and glucose utilization mediated by cystathionine-gamma-lyase (cse) activation and h2s formation in 3t3l1 adipocytes. J Biol Chem 2012;287:42324-42332.
    External Resources
  26. Goldschmidt RC, Kimelberg HK: Protein analysis of mammalian cells in monolayer culture using the bicinchoninic assay. Anal Biochem 1989;177:41-45.
    External Resources
  27. Zu L, Jiang H, He J, Xu C, Pu S, Liu M, Xu G: Salicylate blocks lipolytic actions of tumor necrosis factor-alpha in primary rat adipocytes. Mol Pharmacol 2008;73:215-223.
    External Resources
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2013, 31, No. 6

July 2013

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