Chloride intracellular channel 5 modulates adipocyte accumulation in skeletal muscle by inhibiting preadipocyte differentiation

Intramuscular fat, the total lipid deposited within skeletal muscle, has been regarded as a potential factor responsible for meat quality in animal production and insulin resistance in humans. The objective of present study was to identify candidate genes which control intramuscular fat accumulation through using animal models. PIC pigs (lean‐type) and Rongchang pigs (obese‐type) were used. By scanning the mRNA samples of longissimus dorsi muscle with Affymetrix Gene‐Chip microarray technology, sus scrofa chloride intracellular channel 5 (CLIC5) was isolated, and its mRNA abundance and protein expression level were reversely related with the intramuscular fat content of pigs. Furthermore, over‐expression of CLIC5 dramatically increased the proliferation of 3T3‐L1 preadipocytes, while inhibited adipocytic differentiation accompanied by the down‐regulation of c/EBPα, LPL, and PPARγ protein. Our results suggest that CLIC5 might be a crucial regulator of adipose accumulation in skeletal muscle of pigs. J. Cell. Biochem. 110: 1013–1021, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of <Emphasis Type="Italic">methionine adenosyltransferase 2β</Emphasis> gene expression in skeletal muscle and subcutaneous adipose tissue from obese and lean pigs

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit (MAT II β) that regulates the activity of the MAT2A-encoded isoenzyme and intracellular S-adenosylmethionine levels. Our previous work identified MAT2β as a candidate gene for intramuscular fat (IMF) deposition in porcine skeletal muscle by microarray technology. Here, we cloned porcine MAT2β cDNA and compared its expression pattern in subcutaneous adipose tissue and skeletal muscle from obese (Rongchang Breed) and lean (Pig Improvement Company, PIC) pigs (n = 6). The porcine MAT2β cDNA was 1,800 bp long and encodes for 334 amino acids sharing high similarity with other species. MAT2β is expressed at a higher level in liver and duodenum, followed by the stomach, fat and longissinus dorsi muscle. As expected, both subcutaneous fat content and IMF content were higher in obese than in lean pigs (both P < 0.01). MAT2β mRNA abundance was lower in both subcutaneous adipose tissue and skeletal muscle in obese pigs compared with lean pigs (both P < 0.01). MAT II β protein content was lower in skeletal muscle in obese than in lean pigs (P < 0.05), whereas the opposite was observed in subcutaneous adipose tissue (P < 0.01). These data demonstrated an obesity-related expression variation of the MAT II β subunit in skeletal muscle and adipose tissue in pigs, and suggest a novel role for the MAT2β gene in regulation of IMF deposition in skeletal muscle.     

Differential expression of lipid metabolism-related genes and myosin heavy chain isoform genes in pig muscle tissue leading to different meat quality

The aim of this study was to investigate the variations in meat quality, lipid metabolism-related genes, myosin heavy chain (MyHC) isoform genes and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) gene mRNA expressions in longissimus dorsi muscle (LM) of two different pig breeds. Six Rongchang and six Landrace barrows were slaughtered at 161 days of age. Subsequently, meat quality traits and gene expression levels in LM were observed. Results showed that Rongchang pigs not only exhibited greater pH, CIE a*_{24 h} and intramuscular fat content but also exhibited lower body weight, carcass weight, dressing percentage, LM area and CIE b*_{24 h} compared with Landrace pigs (P<0.05). Meanwhile, the mRNA expression levels of the lipogenesis (peroxisome proliferator-activated receptor gamma, acetyl-CoA carboxylase and fatty acid synthase) and fatty acid uptake (lipoprotein lipase)-related genes were greater in the Rongchang (P<0.05), whereas the lipolysis (adipose triglyceride lipase and hormone sensitive lipase) and fatty acid oxidation (carnitine palmitoyltransferase-1B)-related genes were better expressed in the Landrace. Moreover, compared with the Landrace, the mRNA expression levels of MyHCI, MyHCIIa and MyHCIIx were greater, whereas the mRNA expression levels of MyHCIIb were lower in the Rongchang pigs (P<0.05). In addition, the mRNA expression levels of PGC-1α were greater in Rongchang pigs than in the Landrace (P<0.05), which can partly explain the differences in MyHC isoform gene expressions between Rongchang and Landrace pigs. Although the small number of samples does not allow to obtain a definitive conclusion, we can suggest that Rongchang pigs possess better meat quality, and the underlying molecular mechanisms responsible for the better meat quality in fatty pigs may be partly due to the higher mRNA expression levels of lipogenesis and fatty acid uptake-related genes, as well as the oxidative and intermediate muscle fibers, and due to the lower mRNA expression levels of lipolysis and fatty acid oxidation-related genes, as well as the glycolytic muscle fibers.     

Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A

Background  

An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types.     

The influence of the <Emphasis Type="Italic">PRKAG3</Emphasis> mutation on glycogen,enzyme activities and fibre types in different skeletal muscles of exercise trained pigs

Background  

AMP-activated protein kinase (AMPK) plays an important role in the regulation of glucose and lipid metabolism in skeletal muscle. Many pigs of Hampshire origin have a naturally occurring dominant mutation in the AMPK γ3 subunit. Pigs carrying this PRKAG3 (R225Q) mutation have, compared to non-carriers, higher muscle glycogen levels and increased oxidative capacity in m. longissimus dorsi, containing mainly type II glycolytic fibres. These metabolic changes resemble those seen when muscles adapt to an increased physical activity level. The aim was to stimulate AMPK by exercise training and study the influence of the PRKAG3 mutation on metabolic and fibre characteristics not only in m. longissimus dorsi, but also in other muscles with different functions.     

Molecular characterization,expression pattern and association analysis of the porcine <Emphasis Type="Italic">BTG2</Emphasis> gene

B-cell translocation gene 2 (BTG2), a member of the B-cell translocation gene family with anti-proliferative properties, have been characterized to be involved in cell growth, differentiation and survival. In this study, we cloned the full length sequences of cDNA and genomic DNA of BTG2 gene from the porcine skeletal muscle. Spatial expression analysis showed that the porcine BTG2 gene is expressed predominantly in muscle. Temporal expression analysis in longissimus dorsi muscle demonstrated that the expression of BTG2 gene has the highest expression at 60 days old in Large White while with a peak expression at 120 days old in Meishan. Temporal analysis also revealed that the expression of BTG2 gene is generally higher in Large White than in Meishan at all the developmental stages tested (65 days of conception and 3, 35, 60, 120, and 180 days of postnatal). A single nucleotide polymorphism (G417C) in the intron of BTG2 gene was then detected by PCR-RFLP in Large White × Meishan F2 resource population and association analysis suggested that this polymorphic site had significant association (P < 0.05) with the buttock fat thickness, fat percentage, lean muscle percentage, ratio of lean to fat and carcass length.     

Breed difference and regulation of the porcine adipose triglyceride lipase and hormone sensitive lipase by TNFα

Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are major novel triglyceride lipases in animals. The aim of this study was to determine if there are differences in the porcine ATGL ( pATGL ) and HSL genes between Jinhua pigs (a fatty breed) and Landrace pigs (a leaner breed). In addition, the effect of TNFα and pATGL-specific siRNA ( pATGL-siRNA ) on the expression of pATGL and HSL in porcine adipocytes was also examined. Compared with Landrace pigs, the body weight ( BW ) of Jinhua pigs was lower ( P <  0.01), while intramuscular fat content (in the longissimus dorsi muscle), as well as the back fat thickness and body fat content were higher ( P <  0.01). The expression of pATGL and HSL mRNA in Jinhua pigs was lower ( P <  0.01) in subcutaneous adipose tissue, and greater ( P <  0.01) in longissimus dorsi muscle compared with Landrace pigs. In vitro treatment of porcine adipocytes with TNFα decreased ( P <  0.01) the glycerol release and the gene expression of pATGL , HSL and PPARγ in porcine adipocytes. Furthermore, transfection with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL , while it had no effect on the expression of HSL . Treatment with 25 ng/ml TNFα in conjunction with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL and HSL in cultured porcine adipocytes. These results provide useful information to further the understanding of the function of pATGL and HSL in porcine lipid metabolism, which should be applicable to the regulation of fat deposition and improvement of meat quality.     

Molecular characterization and expression patterns of <Emphasis Type="Italic">Lbx1</Emphasis> in porcine skeletal muscle

Ladybird-like genes were recently identified in mammals. The first member characterized, Lbx1, is expressed in developing skeletal muscle and the nervous system. However, little is known about the porcine Lbx1 gene. In the present study, we cloned and characterized Lbx1 from porcine muscle. RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. Lbx1 gene expressed at higher levels in biceps femoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.−1559C>G. Association analysis in our experimental pig populations showed that the mutation of g.752A>G was significantly associated with loin muscle area (P < 0.05) and internal fat rate (P < 0.05). Our results suggest that the Lbx1 gene might be a candidate gene of carcass traits and provide useful information for further studies on its roles in porcine skeletal muscle.     

Effect of age at the beginning of the free-range fattening period on growth and carcass and fat quality in Iberian pigs

Abstract

This experiment was carried out to study the influence of age at the beginning of the free-range fattening period (traditional pigs, TP, age 12 months vs. young pigs, YP, age 8 months) on the performance of Iberian pigs. During 152 days prior to the fattening period, TP and YP pigs received 1.7 and 2.6 kg feed per day, respectively. During fattening, TP pigs had a higher average daily gain (p < 0.05) than YP pigs. The proportions of PUFA and n-3 fatty acids of the outer and inner layers of subcutaneous backfat were higher in TP than in YP pigs (p < 0.05), while the proportions of C16:0 and SFA in the inner layer of subcutaneous backfat were greater in YP than in TP pigs (p < 0.05). The ratio of n-6/n-3 in subcutaneous backfat was lower in TP than in YP pigs (p < 0.05). The percentage of intramuscular fat in longissimus dorsi muscle was higher in TP than in YP pigs (p < 0.05). The relationship between the percentage of intramuscular fat in longissimus dorsi muscle and average daily gain during the free-range fattening period adjusted to a quadratic function (p < 0.05). The concentration of α- and γ-tocopherol in subcutaneous backfat at slaughter was significantly higher in TP than in YP pigs (p < 0.05). It is concluded that Iberian pigs that have 8 months of age at the beginning of free-range feeding have adequate commercial quality.     

Molecular cloning,polymorphism and association analyses of a novel porcine mRNA differentially expressed in the Longissimus muscle tissues from Meishan and Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Meishan and Large White pigs. One novel mRNA that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the mRNA is not homologous to any of the known porcine genes. Sequence prediction analysis revealed that the this mRNA is not protein-coding mRNA. Polymorphism analyses revealed that there was a C-T mutation on the position of 669 bp and PCR -Dra I-RFLP analyses revealed that Chinese indigenous pig breeds and exotic pig breeds displayed obvious genotype and allele frequency differences at this locus. Association analyses revealed that this polymorphic locus was significantly associated with the drip loss rate, skin percentage, meat color value (m.Longissimus Dorsi, LD), loin eye width, loin eye area, water holding capacity, carcass length, caul fat weight, intramuscular fat (m.Longissimus Dorsi, LD), lean meat weight, lean meat percentage, backfat thickness at buttock (P < 0.05).     

Effects of cannabinoid receptor 1 (brain) on lipid accumulation by transcriptional control of CPT1A and CPT1B

CB1 (also known as CNR1), a main receptor for cannabinoids acting at PPARs, can enhance fat deposition. Carnitine palmitoyltransferase‐1 (CPT1), an enzyme responsible for the transport of long‐chain fatty acids for β‐oxidation, is closely related to fat deposition. Whether CB1 can regulate intramuscular adipocytes lipid accumulation through regulation of CPT1 is unclear. Based on the investigation of tissue‐ and breed‐specific CPT1A and CPT1B mRNA expression levels in Jinhua and Landrace pigs, we studied the effects of CB1 on lipid accumulation and CPT1B expression by treating porcine intramuscular adipocytes with CB1 antagonist Δ9‐THC and antagonist SR141716. Results showed that muscle CPT1 mRNA was expressed at higher levels in the longissimus dorsi and subcutaneous fat. Liver CPT1A mRNA expression levels were higher in the pancreas, duodenum and liver. Compared with Landrace pigs, CPT1A and CPT1B in the longissimus dorsi of Jinhua pigs were significantly higher and positively correlated with intramuscular fat content. However, for subcutaneous fat, CPT1 levels were significantly lower and negatively correlated with body fat percentage. Δ9‐THC significantly increased CB1 mRNA levels and lipid accumulation but decreased CPT1A and CPT1B mRNA levels. Conversely, SR141716 reduced CB1 mRNA levels but increased CPT1A and CPT1B mRNA levels, resulting in decreased lipid accumulation. The CPT1 antagonist etomoxir did not affect CB1 expression, suggesting that CB1 is likely upstream of CPT1A and CPT1B. Meanwhile, PPARA expression was greatly decreased when CPT1A and CPT1B were inhibited and enhanced when CPT1A and CPT1B were activated. Taken together, these data indicate that CB1 can affect intramuscular fat deposition by regulating both CPT1A and CPT1B mRNA expression, with the PPARA signal pathway likely playing a major role in this process.     

Single nucleotide polymorphism scanning and expression of the FRZB gene in pig populations

Secreted frizzled-related protein 3 (sFRP3), encoded by the gene FRZB, is a member of the sFRP family with important roles in inhibition of the Wnt signalling pathway through competitive binding of the Wnt receptor. Here, we investigated pig FRZB as a candidate gene for growth traits and identified three polymorphic sites, an insertion (A-532B) and two SNPs (G636A and C650T) in its 5′-UTR. The genotype distributions of G636A and C650T were significantly different among mini-type indigenous (Diannan Small-ear and Tibetan), normal indigenous (Laiwu and Huai), and introduced (Large Yorkshire and Landrace) breeds. In semi-quantitative PCR expression analysis, expression of FRZB mRNA was abundant in tissues of hypophysis, longissimus dorsi muscle, and adipose tissues, and low in the heart, hypothalamus, and brain. Quantitative determination of mRNA level and protein expression analysis were corresponding. The results demonstrated that FRZB gene expression in longissimus dorsi muscle and liver tissue was significantly higher in Diannan Small-ear and Tibetan pigs than in the Large Yorkshire breed (P < 0.05); however, in back fat tissue, the expression was significantly higher in Diannan Small-ear pig than in Tibetan or Large Yorkshire breeds (P < 0.05). Given the known growth and fat characteristics of the breeds, these results indicate that FRZB expression has a negative association with muscle growth and a positive association with fat deposition. In conclusion, FRZB may be a major candidate gene for growth traits in pigs.     

Functional and association studies of the cholesteryl ester transfer protein (CETP) gene in a Wannan Black pig model

Some polymorphisms of the human CETP gene are causally and significantly associated with serum lipids levels; however, the information regarding this gene in pigs is sparse. To evaluate the effects of CETP on blood lipid traits and fat deposition in pig, porcine CETP tissue expression patterns were observed by quantitative real‐time polymerase chain reaction (qPCR) first. High expression was detected in liver, spleen, gluteus medius (GM) muscle and backfat. A de novo polymorphism (AF333037:g.795C>T) in the intron 1 region of porcine CETP was identified. This polymorphism was further genotyped by direct sequencing of the PCR products of 390 Wannan Black pigs, a Chinese native breed population. Association analyses at 45 and 300 days of age revealed highly significant associations between CETP genotypes and serum lipid traits. Furthermore, this polymorphism was proved to be associated with differences in liver CETP mRNA levels: pigs at 300 days of age with the TT genotype had higher levels than did those with other genotypes (P = 0.021). Additionally, analysis at 300 days of age showed that GM CETP mRNA expression correlated positively with serum lipids levels as well as with carcass backfat thickness and intramuscular fat content in GM. These results indicate that CETP is involved in serum, adipose and muscle lipid metabolism in pigs. The mechanisms underlying such relationships and their functional implications are worthy of further research.