Myunggi Baik

Transcriptome changes favoring intramuscular fat deposition in the longissimus muscle following castration of bulls

Castration increases intramuscular fat (IMF) deposition, improving beef quality in cattle. The present study was performed to determine the global transcriptome changes following castration of bulls and to identify genes associated with IMF deposition in the longissimus dorsi (LM) of Korean cattle. A customized bovine CombiMatrix oligonucleotide microarray was constructed, and transcriptome changes following castration were determined by microarray hybridization. Transcriptome comparison between bulls and steers indicated that 428 of 8,407 genes were differentially expressed in the LM by greater than two fold (P < 0.05). Gene expression profiling indicated alterations in several pathways, including adipogenesis, fatty acid oxidation, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OP), following castration. Castration upregulated transcription of adipogenic perilipin 2 (PLIN2) and visfatin, lipogenic fatty acid synthase, fatty acid esterification 1-acylglycerol-3-phosphate O-acyltransferase 5, and many fatty acid oxidation-related genes. Many TCA cycle and OP genes were also transcriptionally upregulated. Correlation analysis indicated that the IMF content in the LM was highly correlated with mRNA levels of PLIN2 (r = 0.70, P < 0.001), adenosine triphosphatase (ATPase), H(+)-transporting, lysosomal 42 kDa, V1 subunit C1 (ATP6V1C1: r = 0.66, P < 0.001), and cytochrome c oxidase assembly homolog 11 (COX11: r = 0.72, P < 0.001) genes in a pooled animal group of steers plus bulls, and significant correlations in the steer-alone group were maintained in the 3 genes, PLIN2 (r = 0.47, P < 0.05), ATP6V1C1 (r = 0.50, P < 0.05), and COX11 (r = 0.60, P < 0.01). In conclusion, our study provided evidence that castration shifts transcription of lipid metabolism genes, favoring IMF deposition by increasing adipogenesis, lipogenesis, and triglyceride synthesis. This study also indicated that castration increases transcription of genes involved in fatty acid oxidation and subsequent energy production (TCA and OP genes). Our microarray analysis provided novel information that castration alters the transcriptome associated with lipid/energy metabolism, favoring IMF deposition in the LM.

Wnt/β-catenin signaling and adipogenic genes are associated with intramuscular fat content in the longissimus dorsi muscle of Korean cattle

Intramuscular fat (IMF) is an important trait that influences beef quality. In two studies, we examined the possible involvement of the Wnt/β-catenin signaling pathway in IMF deposition in Korean cattle. In study 1, using a group of bulls and steers, we found that castration, a non-genetic factor, decreased (Pxa0<xa00.01) the expression of both the WNT10B and CTNNB1 genes, whereas it increased the expression of the Wnt antagonist secreted frizzled-related proteins 4 (SFRP4, Pxa0<xa00.001) and the adipogenic CCAAT/enhancer binding protein (C/EPB), alpha (CEBPA, Pxa0<xa00.001) and peroxisome proliferator-activated receptor gamma (PPARG, Pxa0<xa00.05) genes in longissimus dorsi muscle (LM) tissue. The WNT10B and CTNNB1 mRNA levels showed strong (Pxa0<xa00.001) negative correlations (rxa0=xa0-0.68 and rxa0=xa0-0.73 respectively) with the IMF content, whereas the SFRP4, CEBPA and PPARG mRNA levels showed strong (Pxa0<xa00.01) positive correlations (rxa0=xa00.70, 0.70 and 0.64 respectively) with the IMF content. Large variation still exists in the IMF content of steers, implying that genetic factors affect IMF deposition. Using a different group of steers, a correlation analysis in study 2 also showed that the expression of the WNT10B and CTNNB1 genes, and SFRP4 and adipogenic genes was negatively and positively associated with the IMF content respectively. Our findings suggest that downregulation of the Wnt/β-catenin signaling pathway genes, but upregulation of Wnt antagonist SFRP4 and adipogenic gene expression following castration, contributes to increased IMF deposition in the LM. Our results demonstrate that both non-genetic factors (castration) and genetic variation within the steer group affect the gene expression pattern of the Wnt/β-catenin signaling pathway.

Association of DNA Methylation Levels with Tissue-specific Expression of Adipogenic and Lipogenic Genes in Longissimus dorsi Muscle of Korean Cattle

Epigenetic factors, such as DNA methylation status, may regulate adipogenesis and lipogenesis, thus affecting intramuscular fat (IMF) deposition in longissimus dorsi muscle (LM) of beef cattle. In Korean cattle steers, the LM consists mainly of muscle tissue. However, the LM tissue also contains IMF. We compared the gene expression levels between the IMF and muscle portions of the LM after tissue separation. Real-time polymerase chain reaction analysis showed that the mRNA levels of both adipogenic peroxisome proliferator-activated receptor gamma isoform 1 (PPARG1) and lipogenic fatty acid binding protein 4 (FABP4) were higher (p<0.01) in the IMF than in the muscle portion of the LM. We determined DNA methylation levels of regulatory regions of the PPARG1 and FABP4 genes by pyrosequencing of genomic DNA. DNA methylation levels of two of three CpG sites in the PPARG1 gene promoter region were lower (p<0.05) in the IMF than in the muscle portion of the LM. DNA methylation levels of all five CpG sites from the FABP4 gene promoter region were also lower (p<0.001) in the IMF than in the muscle portion. Thus, mRNA levels of both PPARG1 and FABP4 genes were inversely correlated with DNA methylation levels in regulatory regions of CpG sites of the corresponding gene. Our findings suggest that DNA methylation status regulates tissue-specific expression of adipogenic and lipogenic genes in the IMF and muscle portions of LM tissue in Korean cattle.

The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages.

1. The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ([3H]-lysine, [14C]-cycloleucine) in rat mammary epithelial cell cultures. The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages. 2. Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake. But increase of these activities was different at each physiological stage. 3. The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation. And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

Differential Expressions of G0/G1 Switch Gene 2 and Comparative Gene Identification-58 are Associated with Fat Content in Bovine Muscle

Regulation of lipolysis in muscle is a potential mechanism affecting marbling in beef carcasses and fat accumulation in muscles of humans, which is a known risk factor for type 2 diabetes. Adipose triglyceride lipase-mediated lipolysis is inhibited by G0/G1 switch gene 2 (G0S2) and co-activated by comparative gene identification-58 (CGI-58). In this study, bovine G0S2 and CGI-58 were sequenced, and expressions of these genes were compared among various tissues and in muscles between bulls and steers with different degrees of marbling. The protein coding sequences of bovine G0S2 and CGI-58 revealed breed-specific SNPs, causing two amino acid variations for each protein. Bovine CGI-58 mRNA showed two isoforms from alternative splicing. The G0S2 gene was preferentially expressed in fat and, to a lesser degree, in the liver; whereas, CGI-58 was highly expressed in the muscle and fat (Pxa0<xa00.05), suggesting their association with lipid metabolism in those tissues. The longissimus dorsi muscle (LM) of steers showed higher FABP4, G0S2 and CGI-58 mRNA expression levels than the LM of bulls, implying the roles of those genes more in marbling of steers than in that of bulls. The G0S2 expression was markedly higher in the intramuscular fat (IMF) (Pxa0<xa00.001); whereas, the CGI-58 expression was significantly higher in the pure muscle portion of the LM of steers (Pxa0<xa00.01), suggesting that G0S2 and CGI-58 may regulate IMF and intramyocellular triglycerides, respectively. Taken together, our data suggest that G0S2 and CGI-58 are associated with fat content in bovine species.

Heat-shock protein beta 1 regulates androgen-mediated bovine myogenesis

To elucidate the functional significance of heat-shock protein beta 1 (HSPB1) in androgen-mediated myogenesis of bovine cells, we conducted ‘loss and gain of function of HSPB1’ assays by siRNA inhibition and gene overexpression. siRNA inhibition of HSPB1 expression reduced the expression of desmin (a myogenic marker) and repressed the formation of myotubes in cells induced for myogenic differentiation. In contrast, overexpression of HSPB1 enhanced the expression of desmin and accelerated formation of myotubes. The loss and gain of HSPB1 function was closely associated with the expression level of androgen receptor (AR). Our findings suggest that HSPB1 mediates androgen signaling by binding directly to AR and then enhancing androgen-mediated myogenesis in myogenic cells.

Comparison of Carcass and Sensory Traits and Free Amino Acid Contents among Quality Grades in Loin and Rump of Korean Cattle Steer.

This study was performed to compare carcass traits, sensory characteristics, physiochemical composition, and contents of nucleotides, collagen, and free amino acids among quality grades (QG) and to understand the association between QG and above parameters in loin and rump of Korean cattle steer. Loin and rump samples were obtained from 48 Korean cattle steers with each of four QG (QG 1++, 1+, 1, and 2; average 32 months of age). Carcass weight and marbling score (MS) were highest in QG 1++, whereas texture score measured by a meat grader was highest in QG 2. A correlation analysis revealed that MS (r = 0.98; p<0.01) and fat content (r = 0.73; p<0.01) had strong positive correlations with QG and that texture had a strong negative correlation (r = −0.78) with QG. Fat content in loin was highest but protein and moisture contents were lowest in QG 1++. Our results confirmed that a major determinant of QG is the MS; thus, intramuscular fat content. The International Commission on Illumination L*, a*, and b* values in loin were highest in QG 1++. Numeric values of shear force in loin were lowest in QG 1++, whereas those of tenderness, juiciness, and overall acceptability tended to be highest in QG 1++ without statistical significance. QG was strongly correlated with juiciness (r = 0.81; p<0.01) and overall acceptability (r = 0.87; p<0.001). All sensory characteristics were higher (p<0.05) in loin than those in rump. Adenosine-5′-monophosphate (AMP) and inosine-5′-monophosphate (IMP) contents in both loin and rump did not differ among QGs. No nucleotide (AMP, IMP, inosine, hypoxanthine) was correlated with any of the sensory traits. Total, soluble, and insoluble collagen contents in loin were higher in QG 1++ than those in QG 1. All three collagens had lower content in loin than that in rump. All three collagens were positively correlated with tenderness, juiciness, and overall acceptability. Glutamic acid content did not significantly differ among the four QGs in either loin or rump. In conclusion, it is confirmed that QG is associated with sensory traits but nucleotide contents in beef may not be a major factor determining meat palatability in the present study.

Liver-specific deletion of the signal transducer and activator of transcription 5 gene aggravates fatty liver in response to a high-fat diet in mice

Growth hormone (GH) signal is mediated by signal transducer and activator of transcription 5 (STAT5), which controls hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) is clinically associated with a deficiency in GH. This study was performed to understand the role of local STAT5 signaling on hepatic lipid and glucose metabolism utilizing liver-specific STAT5 gene deletion (STAT5 LKO) mice under both normal diet and high-fat diet (HFD) feeding conditions. STAT5 LKO induced hepatic steatosis under HFD feeding, while this change was not observed in mice on normal diet. STAT5 LKO caused hyperglycemia, hyperinsulinemia, hyperleptinemia and elevated free fatty acid and cholesterol concentrations under HFD feeding but induced only hyperglycemia on normal diet. At the molecular level, STAT5 LKO up-regulated the expression of genes involved in lipid uptake (CD36), very low-density lipoprotein receptor (VLDLR), lipogenic stearoyl-CoA desaturase and adipogenic peroxisome proliferator-activated receptor gamma, in both diet groups. In response to HFD feeding, further increases in CD36 and VLDLR expression were found in STAT5 LKO mice. In conclusion, our study suggests that low STAT5 signaling on normal diet predisposes STAT5 LKO mice to early development of fatty liver by hyperglycemia and activation of lipid uptake and adipogenesis. A deficiency in STAT5 signaling under HFD feeding deregulates hepatic and body glucose and lipid metabolism, leading to the development of hepatic steatosis. Our study indicates that low STAT5 signaling, due to low GH secretion, may increase a chance for NAFLD development in elderly people.

Effects of Ambient Temperature on Growth Performance, Blood Metabolites, and Immune Cell Populations in Korean Cattle Steers

Exposure to cold may affect growth performance in accordance with the metabolic and immunological activities of animals. We evaluated whether ambient temperature affects growth performance, blood metabolites, and immune cell populations in Korean cattle. Eighteen Korean cattle steers with a mean age of 10 months and a mean weight of 277 kg were used. All steers were fed a growing stage-concentrate diet at a rate of 1.5% of body weight and Timothy hay ad libitum for 8 weeks. Experimental period 1 (P1) was for four weeks from March 7 to April 3 and period 2 (P2) was four weeks from April 4 to May 1. Mean (8.7°C) and minimum (1.0°C) indoor ambient temperatures during P1 were lower (p<0.001) than those (13.0°C and 6.2°C, respectively) during P2. Daily dry matter feed intake in both the concentrate diet and forage groups was higher (p<0.001) during P2 than P1. Average daily weight gain was higher (p<0.001) during P2 (1.38 kg/d) than P1 (1.13 kg/d). Feed efficiency during P2 was higher (p = 0.015) than P1. Blood was collected three times; on March 7, April 4, and May 2. Nonesterified fatty acids (NEFA) were higher on March 7 than April 4 and May 2. Blood cortisol, glucose, and triglyceride concentrations did not differ among months. Blood CD4+, CD8+, and CD4+CD25+ T cell percentages were higher, while CD8+CD25+ T cell percentage was lower, during the colder month of March than during May, suggesting that ambient temperature affects blood T cell populations. In conclusion, colder ambient temperature decreased growth and feed efficiency in Korean cattle steers. The higher circulating NEFA concentrations observed in March compared to April suggest that lipolysis may occur at colder ambient temperatures to generate heat and maintain body temperature, resulting in lower feed efficiency in March.

Effects of Castration on Expression of Lipid Metabolism Genes in the Liver of Korean Cattle

Castration induces the accumulation of body fat and deposition of intramuscular fat in Korean cattle, resulting in improved beef quality. However, little is known about the metabolic adaptations in the liver following castration. To understand changes in lipid metabolism following castration, hepatic expression levels of lipid metabolism genes were compared between Korean bulls and steers. Steers had higher (p<0.001) hepatic lipids contents and higher (p<0.01) mRNA levels of lipogenic acetyl-CoA carboxylase. This differential gene expression may, in part, contribute to increased hepatic lipid content following the castration of bulls. However, we found no differences in the hepatic expression levels of genes related to triglyceride synthesis (mitochondrial glycerol-3-phosphate acyltransferase, diacylglycerol O-acyltransferase 1 and 2) and fatty acid (FA) oxidation (carnitine palmitoyltransferase 1A, C-4 to C-12 straight chain acyl-CoA dehydrogenase, very long chain acyl-CoA dehydrogenase) between bulls and steers. No differences in gene expression for very-low-density lipoprotein (VLDL) secretion, including apolipoprotein B mRNA and microsomal triglyceride transfer protein (MTTP) protein, were observed in the liver although MTTP mRNA levels were higher in steers compared to bulls. In conclusion, FA synthesis may contribute to increased hepatic lipid deposition in steers following castration. However, hepatic lipid metabolism, including triglyceride synthesis, FA oxidation, and VLDL secretion, was not significantly altered by castration. Our results suggest that hepatic lipid metabolism does not significantly contribute to increased body fat deposition in steers following castration.