Bovine Intramuscular Adipocytes Research Articles

SERPINE1AS2 regulates intramuscular adipogenesis by inhibiting PAI1 protein expression

Antisense long non-coding RNAs (lncRNAs) played a crucial role in the precise regulation of essential biological processes and were abundantly present in animals. Many of these antisense lncRNAs have been identified as key roles in adipose tissue accumulation in livestock, underscoring their vital role in the regulation of animal physiology. Nonetheless, the functional roles of these antisense lncRNAs in regulating adipogenesis and the specific molecular mechanisms these processes were still unclear, which was a significant gap in current scientific research.In this study, we identified and characterized SERPINE1AS2, a novel natural antisense lncRNA, was highly expressed in the fat tissues of adult cattle and calves. Its expression gradually increased during the differentiation of intramuscular adipocytes. Through functional studies, we observed that knockdown of SERPINE1AS2 inhibited the proliferation and adipogenesis of intramuscular adipocytes, while overexpression of SERPINE1AS2 produced the opposite effect. RNA sequencing (RNA-seq) analysis following SERPINE1AS2 knockdown revealed that differential expression genes (DEGs) were significantly enriched in key signaling pathways, notably the MAPK, Wnt, and mTOR signaling pathways. Furthermore, SERPINE1AS2 interacted with Plasminogen Activator Inhibitor-1 (PAI1), forming RNA dimers through complementary base pairing and consequently influencing PAI1 expression. Interestingly, studies on PAI1 suggested that reduced expression facilitated adipogenesis and the downregulation of PAI1 alleviated the inhibitory effect of reduced SERPINE1AS2 on adipogenesis. In summary, this study suggested that SERPINE1AS2 played a crucial role in the adipogenesis of bovine intramuscular adipocytes by modulating the expression of PAI1. SERPINE1AS2 also regulated adipogenesis by engaging in the MAPK, Wnt, and mTOR signaling pathways. Our results suggested that SERPINE1AS2 had a complex regulatory mechanism on adipogenesis in intramuscular adipocytes.

RNA-seq analysis reveals the critical role of the novel lncRNA BIANCR in intramuscular adipogenesis through the ERK1/2 signaling pathway

BackgroundLong non-coding RNAs (lncRNAs) regulate numerous biological processes, including adipogenesis. Research on adipogenesis will assist in the treatment of human metabolic diseases and improve meat quality in livestock, such as the content of intramuscular fat (IMF). However, the significance of lncRNAs in intramuscular adipogenesis remains unclear. This research aimed to reveal the lncRNAs transcriptomic profiles in the process of bovine intramuscular adipogenesis and to identify the lncRNAs involved in the adipogenesis of bovine intramuscular adipocytes.ResultsIn this research, a landscape of lncRNAs was identified with RNA-seq in bovine intramuscular adipocytes at four adipogenesis stages (0 d, 3 d, 6 d, and 9 d after differentiation). A total of 7035 lncRNAs were detected, including 3396 novel lncRNAs. Based on the results of differential analysis, co-expression analysis, and functional prediction, we focused on the bovine intramuscular adipogenesis-associated long non-coding RNA (BIANCR), a novel lncRNA that may have an important regulatory function. The knockdown of BIANCR inhibited proliferation and promoted apoptosis of intramuscular preadipocytes. Moreover, BIANCR knockdown inhibited intramuscular adipogenesis by regulating the ERK1/2 signaling pathway.ConclusionThis study obtained the landscape of lncRNAs during adipogenesis in bovine intramuscular adipocytes. BIANCR plays a crucial role in adipogenesis through the ERK1/2 signaling pathway. The results are noteworthy for improving beef meat quality, molecular breeding, and metabolic disease research.Graphical

Lubabegron fumarate acts as a β-adrenergic receptor antagonist in cultured bovine intramuscular and subcutaneous adipocytes.

We hypothesized that lubabegron fumarate (LUB) (Experior, Elanco Animal Health, Greenfield, IN) would act as an antagonist to β-adrenergic receptor (β-AR) subtypes in primary bovine subcutaneous (s.c.) and intramuscular (i.m.) adipocytes differentiated in culture. This study employed LUB, dobutamine (DOB, a selective β1-agonist), salbutamol (SAL, a selective β2-agonist), and propranolol (PRO, a non-selective β-AR antagonist). Preadipocytes were isolated by standard techniques from bovine longissimus muscle and overlying s.c. adipose tissue and differentiated to adipocytes for 14 d. The adipocyte source x stage of differentiation interaction was significant for β-adrenergic receptors-1 (ADRB1) (P = 0.001) and ADRB2 (P = 0.01) in that expression of ADRB1 and ADRB2 was greater in s.c. adipocytes than in s.c. preadipocytes; expression of the ADRB1-3 did not change after differentiation of i.m. adipocytes. CCATT/enhancer-binding protein alpha (CEBPA) expression increased upon differentiation in both s.c. and i.m. adipocytes (P = 0.006). The source x stage of differentiation interaction was significant for peroxisome proliferator-activated receptor gamma (PPARG) (P ≤ 0.001) and fatty acid binding protein-4 (FABP4) (P = 0.004). Expression of PPARG increased after differentiation of s.c. preadipocytes to adipocytes, but PPARG expression did not change with differentiation of i.m. preadipocytes to adipocytes. FABP4 expression increased after differentiation of both s.c. and i.m. adipocytes, but FABP4 expression increased to a greater extent in s.c. adipocytes. In s.c. adipocytes, DOB elevated cAMP and glycerol production and protein kinase A (PKA) activity, and SAL increased PKA activity; these effects were abolished by LUB and PRO (P < 0.001). Incubation of i.m. adipocytes with SAL increased cAMP production and PKA activity, which was attenuated by LUB and PRO (P ≤ 0.006). In s.c. adipocytes, SAL, LUB + SAL, and LUB + DOB upregulated hormone sensitive lipase (HSL) (P < 0.001) and perilipin (P = 0.002) gene expression. In i.m. adipocytes, DOB and LUB + DOB increased HSL gene expression (P = 0.001) and LUB + SAL depressed adipose triglyceride lipase expression below control levels (P = 0.001). These results demonstrate that LUB is a β-AR antagonist at the β1-AR and β2-AR subtypes in s.c. adipocytes, and that s.c. and i.m. exhibit different responses to β-AA and LUB.

HMGR overexpression and interference affect the expression of steroidogenic genes and cholesterol content in bovine intramuscular adipocytes

Previously, we found that mevalonic acid stimulates 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) expression in bovine intramuscular adipocytes to influence adipocyte differentiation. However, any direct links among HMGR, steroidogenic genes, and cholesterol content remain unclear. RNA-Seq was conducted to determine the differences between the gene expression profiles of bovine adipocytes containing different HMGR expression constructs. In total, 10,234 differentially expressed genes (DEGs) were found. Of these, 35 and 6 DEGs between the control and the overexpression groups were functionally related to lipid and energy metabolism, respectively. In addition, 43 and 8 DEGs between the control and the HMGR inhibition groups were related to lipid and energy metabolism, respectively. Several DEGs related to lipid and energy metabolism were also identified between the HMGR overexpression group and the HMGR interference group, and many DEGs were correlated positively or negatively with the overexpression or inhibition of HMGR. We also found that, following the activation or inhibition of the HMGR gene, AMP-activated protein kinase (AMPK) and sirtuin type 1 (SIRT1) had opposite expression patterns in bovine intramuscular adipocytes. Interestingly, the HMGR gene was downregulated when HMGR was overexpressed, and upregulated when HMGR was inhibited. Our findings establish a theoretical understanding of signaling pathways involved in cholesterol synthesis by elucidating the relationships between key genes.

Chromium acetate stimulates adipogenesis through regulation of gene expression and phosphorylation of adenosine monophosphate-activated protein kinase in bovine intramuscular or subcutaneous adipocytes

ObjectiveWe hypothesized that Cr source can alter adipogenic-related transcriptional regulations and cell signaling. Therefore, the objective of the study was to evaluate the biological effects of chromium acetate (CrAc) on bovine intramuscular (IM) and subcutaneous (SC) adipose cells.MethodsBovine preadipocytes isolated from two different adipose tissue depots; IM and SC were used to evaluate the effect of CrAc treatment during differentiation on adipogenic gene expression. Adipocytes were incubated with various doses of CrAc: 0 (differentiation media only, control), 0.1, 1, and 10 μM. Cells were harvested and then analyzed by real-time quantitative polymerase chain reaction in order to measure the quantity of adenosine monophosphate-activated protein kinase-α (AMPK-α), CCAAT enhancer binding protein-β (C/EBPβ), G protein-coupled receptor 41 (GPR41), GPR43, peroxisome proliferator-activated receptor-γ (PPARγ), and stearoyl CoA desaturase (SCD) mRNA relative to ribosomal protein subunit 9 (RPS9). The ratio of phosphorylated-AMPK (pAMPK) to AMPK was determined using a western blot technique in order to determine changing concentration.ResultsThe high dose (10 μM) of CrAc increased C/EBPβ, in both IM (p = 0.02) and SC (p = 0.02). Expression of PPARγ was upregulated by 10 μM of CrAc in IM but not in SC. Expression of SCD was also increased in both IM and SC with 10 μM of CrAc treatment. Addition of CrAc did not alter gene expression of glucose transporter 4, GPR41, or GPR43 in both IM and SC adipocytes. Addition of CrAc, resulted in a decreased pAMPKα to AMPKα ration (p<0.01) in IM.ConclusionThese data may indicate that Cr source may influence lipid filling in IM adipocytes via inhibitory action of AMPK phosphorylation and upregulating expression of adipogenic genes.

Comparison of apoptosis between bovine subcutaneous and intramuscular adipocytes by resveratrol via SIRT1

A better understanding of the differential mechanisms regulating the deposition and release of fat between intramuscular and external adipose tissues is very important to the quality of beef. Resveratrol is a natural activator of sirtuin type 1 (SIRT1), a NAD-dependent deacetylase involved in regulating the cell cycle, energy homeostasis and apoptosis in adipose tissue. To compare the molecular mechanisms underlying differential apoptosis in bovine intramuscular and subcutaneous adipocytes, we evaluated the effect of resveratrol on differentiated adipocytes. We found that resveratrol-induced apoptosis in bovine adipocytes by regulating SIRT1 activity. In addition, we report that bovine intramuscular and subcutaneous adipocytes exhibited differential responses to resveratrol. In particular, gene and protein expression of Bcl-2 was higher, whereas that of SIRT1, AMPKα, FOXO1, Bax and caspase-3 were lower in bovine subcutaneous adipocytes than in intramuscular adipocytes. After resveratrol-treatment, the extent of up- or down-regulation was higher in subcutaneous adipocytes than in intramuscular adipocytes. These data indicate that bovine subcutaneous adipocytes are more sensitive to apoptosis than intramuscular adipocytes following treatment with resveratrol by regulating SIRT1 activity.

Effect of simvastatin on bovine intramuscular and subcutaneous adipocytes proliferation and gene expression in vitro

Simvastatin (SIM) is a widely used anticholesterolemic drug that blocks the biosynthesis of cholesterol. However, SIM also has pleiotropic effects on 3-hydroxy-3-methyglutary-CoA reductase (HMGR), cholesteryl ester transfer protein (CETP), and lipoprotein lipase (LPL), which are important genes in the cholesterol biosynthesis and transport processes. We investigated the effects of different concentrations of SIM on the mRNA expression of these genes in bovine intramuscular and subcutaneous adipocytes from the longissimus dorsi muscle and subcutaneous fat tissues of Luxi Yellow cattle. The results showed that SIM treatment showed dose-dependent toxicity on normal adipose cells, but no effect on cell proliferation. SIM decreased HMGR expression in a dose-dependent manner but showed no significant effect on CETP and LPL expression. Thus, SIM may lower the cholesterol content by decreasing the HMGR expression level, but CETP and LPL may be regulated through other mechanisms, which require further investigation.

Molecular characterization of ABHD5 gene promoter in intramuscular preadipocytes of Qinchuan cattle: Roles of Evi1 and C/EBPα

The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/β hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of –109/+307 was identified as proximal minimum core promoter region of ABHD5 in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.

Resveratrol induces apoptosis and inhibits adipogenesis by stimulating the SIRT1-AMPKα-FOXO1 signalling pathway in bovine intramuscular adipocytes.

Sirtuin type 1 (SIRTl) and AMP-activated protein kinase (AMPK) play important roles in regulating energy metabolism, cell proliferation and differentiation, ageing, apoptosis, and metabolism. The effect of 100, 200, and 400μm Resveratrol (RES), an activator of SIRT1, on apoptosis of bovine intramuscular adipocytes was investigated by nuclear staining, flow cytometry, quantitative real-time polymerase chain reaction, and western blotting. Results show that RES inhibited adipogenesis, decreased cell viability, and increased apoptotic rates in a dose-dependent way. RES up-regulated SIRT1, AMPKα, forkhead box O1 (FOXO1), hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), caspase-3, and Bax; and down-regulated peroxisome proliferator-activated receptor-gamma (PPARγ), fatty acid synthase (FAS), and Bcl-2, at both mRNA and protein level. The effect of RES was abolished by addition of sirtinol (an inhibitor of SIRT1). This is the first study demonstrating a role for AMPK-SIRT1-FOXO1 signalling pathway in regulating apoptosis in bovine intramuscular adipocytes. Our findings provide important information on the mechanism by which RES controls deposition of cattle intramuscular fat via adipocyte apoptosis.

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes.

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells' lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

Oleic acid enhances G protein coupled receptor 43 expression in bovine intramuscular adipocytes but not in subcutaneous adipocytes1

We hypothesized that fatty acids would differentially affect G protein coupled receptor (GPR) 43 mRNA expression and GPR43 protein concentrations in bovine intramuscular (IM) and subcutaneous (SC) adipocytes. The GPR43 protein was detected in bovine liver, pancreas, and semimembranosus (MUS) muscle in samples taken at slaughter. Similarly, GPR43 protein levels were similar in IM adipose tissue and SM muscle but was barely detectable in SC adipose tissue. Primary cultures of IM and SC stromal vascular cells were isolated from bovine adipose tissues. Oleic acid (100 μ) stimulated PPARγ gene expression and decreased stearoyl-CoA desaturase (SCD) gene expression but had no effect on GPR43 gene expression, which was readily detectable in both IM and SC adipocytes. Differentiation cocktail (Diff; 10 μ insulin, 4 μ dexamethasone, and 10 μ ciglitizone) stimulated CCAAT/enhancer-binding protein β (C/EBPβ) and PPARγ gene expression in SC but not IM adipocytes, but Diff increased SCD gene expression in both cell types. Linoleic acid (10 µ) increased PPARγ gene expression relative to Diff cocktail in SC adipocytes, whereas linoleic acid and α-linolenic decreased SCD gene expression relative to control adipocytes and adipocytes incubated with Diff ( < 0.05). Increasing concentrations of oleic acid (1, 10, 100, and 500 μM) increased GPR43 protein and mRNA expression in IM but not SC adipocytes. These data indicated that oleic acid alters mRNA and protein concentrations of GPR43 in bovine IM adipocytes.

Subcellular Localization and Polymorphism of Bovine FABP4 in Bovine Intramuscular Adipocytes

ABSTRACTFatty acid binding protein 4 (FABP4) I74 V, a gene polymorphism associated with unsaturated fatty acid contents, was discovered in Japanese Black cattle. Individuals with FABP4 I/I genotype contain a significantly high level of palmitoleic acid compared to those with FABP4 V/V genotype. It remains unknown how the FABP4 polymorphism leads to different palmitoleic acid contents.We overexpressed FABP4 of different genotypes in bovine intramuscular preadipocytes and examined whether the intracellular localization of FABP4 and the expression levels of lipid metabolism-related genes were different among cells expressing different genotypes. Nuclear localization was observed for the FABP4 V/V, while the FABP4 I/I almost did not. The cells expressing FABP4 of different genotypes were comparable in terms of the expression levels of genes involved in lipid metabolism. FABP4 I/I was localized in most of the lipid droplets 4 days after differentiation induction, whereas approximately 25% lipid droplet co-localized with FABP4 in cells expressing FABP4 V/V. The lipid droplet size increased when palmitoleic acid was added compared to the size observed when palmitic acid was added. These results suggest that lipid droplet enlargement caused by palmitoleic acid and genotype-dependent differences in the fatty acid transporting capacity underlie variations in palmitoleic acid content among FABP4 polymorphisms.

Effect of mevalonic acid on cholesterol synthesis in bovine intramuscular and subcutaneous adipocytes.

Mevalonic acid (MVA) is a key material in the synthesis of cholesterol; indeed, intracellular cholesterol synthesis is also called the mevalonic acid pathway. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is an essential enzyme in cholesterol biosynthesis. This study suggests that MVA may play an important role in the differentiation of bovine adipose tissue in vivo. We investigated differential mRNA expression in bovine intramuscular preadipocytes (BIPs) and bovine subcutaneous preadipocytes (BSPs) by culturing cells from the longissimus dorsi muscle and subcutaneous fat tissues of Luxi yellow cattle. The morphology of lipid accumulation of bovine preadipocytes was detected by Oil Red O staining, and total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and high-density lipoprotein cholesterol (HDLC) levels were measured. Temporospatial expression of HMGR was investigated by real-time quantitative polymerase chain reaction (PCR). The TC, LDLC, and HDLC content did not significantly differ over time but increased slowly with increasing MVA concentration. HMGR expression increased over time and with increasing concentrations of MVA. MVA increased adipose cell proliferation in a dose-dependent and time-dependent manner. MVA stimulated HMGR expression in two cell types and its influence on adipocyte differentiation.

REGULATION OF RETINOL BINDING PROTEIN 4 EXPRESSION AND ITS RELATION TO ADIPOGENESIS IN BOVINE ADIPOCYTES

Adipogenesis is of great importance in beef cattle. Recent findings indicate that glucose, a substrate for fatty acid biosynthesis and retinoic acid enhance a dipogenesis in bovine intramuscular adipocytes. However, other recent findings indicate that Retino l-Binding Protein 4 (RBP4) interferes with glucose uptake and utilization by rodents’ adipocytes. In t his study we examined the regulation of RBP4 expression and its relation to adipogenesis in bovi ne adipocytes. Stromal vascular cells were prepared by collagenase digestion from subcutaneous and intr amuscular adipose tissues of Japanese black steers. RT-PCR revealed that RBP4 mRNA was expressed in bovine adipose tissue. Northern and Western Blot analysis showed that RBP4 was highly expressed and secreted from bovine preadipocytes. However, RBP4 expression and secretion were significantly reduced by induction of the adipogenic differentiation of preadipocytes into ma ture adipocytes. Glucose and retinoic acid have a suppressive effect on RBP4 expression and secretion from intramuscular adipocytes. Retinoic acid significantly decreased RBP4 expression in Japanese black steer subcutaneous adipocytes. Retinoic acid itself had no effect on lipid accumulation in subcu taneous adipocytes however, retinoic acid enhanced lipid accumulation in these adipocytes after additi on of acetate, a substrate for fatty acid biosynthe sis in subcutaneous adipocytes. This study indicated a neg ative correlation between adipogenesis and RBP4 expression in bovine adipocytes and suggests possib le inhibitory effect of RBP4 on adipogenesis.

Effects of Testosterone, 17β-estradiol, and Progesterone on the Differentiation of Bovine Intramuscular Adipocytes

The aim of this study was to investigate the effects of testosterone, 17β-estradiol, and progesterone on the differentiation of bovine intramuscular adipocytes (BIA). Stromal-vascular (SV) cells were obtained from M. longissimus dorsi of 20 months old Korean (Hanwoo) steers, and were cultured in DMEM containing 5% FBS. The proliferated BIA were induced to differentiate with 0.25 µM dexamethasone, 0.5 mM 1-methyl-3-isobutyl-xanthine and 10 µg/ml insulin. During differentiation, the cells were treated with testosterone, 17β-estradiol, and progesterone at concentrations of 10 -10 , 10 -9 , and 10 -8 M, respectively, for 12 days. Regardless of its concentration, testosterone remarkably reduced lipid droplets in the cytosol of BIA. On the other hand, 17β-estradiol and progesterone increased the accumulation of lipid droplets in BIA. Testosterone significantly (p<0.05) decreased GPDH activities with a dose-dependent pattern. 17β-Estradiol treatment onto BIA during differentiation, however, increased GPDH activity showing the highest activity (11.3 nmol/mg protein/min) at 10 -10 M. Treatment of BIA with progesterone also increased (p<0.05) GPDH activity with the highest activity (13.8 nmol/mg protein/min) at 10 -9 M. In conclusion, the results in the current study suggest that testosterone inhibits differentiation of BIA by suppressing GPDH activity while 17β-estradiol and progesterone have adverse effects. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 11 : 1589-1593)

Effects of Zinc on Lipogenesis of Bovine Intramuscular Adipocytes

Zinc (Zn) is a micromineral and functions as a cofactor of many enzymes and its deficiency induces retardation of growth and dysfunction of the immune system in animals. This study was conducted to determine lipogenic activity of Zn in bovine intramuscular adipocytes. Preadipocytes were isolated from intramuscular fat depots of 26 month old Korean (Hanwoo) steers and cultured in media containing Zn. At confluence, the cells were treated with insulin, dexamethasone, and 1-methyl-3-isobutyl-xanthine to induce differentiation (accumulation of lipid droplets in cells). The sources of Zn were zinc chloride (ZnCl2) and zinc sulfate (ZnSO4), and the final concentrations of both Zn sources were 0, 5, 25, 50 and 100 µM. Glycerol-3-phosphate dehydrogenase (GPDH) activity, an index of adipocyte differentiation, was increased as the concentration of Zn in media increased showing the highest activity (25.74 ng/min/mg protein) at 25 µM of ZnSO4. Supplementation of Zn during differentiation of bovine intramuscular adipocytes tended to decrease the production of nitric oxide (NO). Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) gene expression was increased 10 days after differentiation induction. The current results indicate that Zn has a strong lipogenic activity in cultured bovine intramuscular adipocytes with remarkable suppression of NO production. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 10 : 1378-1382)

Differential expression of NAT1 translational repressor during development of bovine intramuscular adipocytes.

This study was undertaken to test for differential gene expression in intramuscular adipocytes during fat deposition of feedlot steers. Angus x Hereford steers (n = 50) were fed a high-energy concentrate ration ad libitum for 20 (n = 5), 86 (n = 15), 121 (n = 15), and 146 days (n = 15) to obtain various degrees of intramuscular adipocyte development. Carcass traits were significantly different (P < 0.05) between the groups. Intramuscular adipose tissue was excised from the longissimus dorsi and snap frozen in liquid nitrogen. Pooled samples of total RNA representing each group were analyzed by differential-display polymerase chain reaction using 200 primer combinations comprising 20 arbitrary (5') and 10 anchor (3') oligonucleotides. Bands (n = 70) representing putative differences among treatment groups were excised, sequenced, and subjected to BLAST homology search. From these, 40 contained significant homology to known genes. One was of particular interest, the translational repressor NAT1 (novel APOBEC-1 target-1). NAT1 mRNA was quantified in individual animals to confirm differential expression among treatment groups. Results indicate that NAT1 message is more abundant (P < 0.05) in intramuscular adipocytes of younger/leaner animals.

Positive effect of collagen V and VI on triglyceride accumulation during differentiation in cultures of bovine intramuscular adipocytes

Ethyl-3,4-dihydroxybenzoate (EDHB), a specific inhibitor of collagen synthesis, was used to study the role of collagen in the differentiation of bovine intramuscular preadipocytes (BIP). Triglyceride (TG) accumulation levels of BIP cells were dose-dependently inhibited by EDHB and were reduced to 50 % at a 0.1 mM concentration. EDHB addition prevented the accretion of collagens (types I–VI) on the cell surface, which generally increases during adipose conversion. Western blotting and immunofluorescence studies showed in detail that triple-helical conformation of procollagen molecules was drastically interrupted by EDHB, and as a result, their matrix assembly was not performed in the extracellular space of adipocytes. Particularly, the development of collagen types IV, V and VI during differentiation was severely damaged. When exogenous collagens were supplied to make up for the lack of endogenous products, cultured EDHB-treated cells on type V and VI collagen-coated dishes were the only ones among six collagens to accumulate more TG, although their TG content did not reach that of normal adipocytes. This result implies the importance and the active role of collagens V and VI for adipogenesis. However, these findings also indicate that collagen newly synthesized and organized by the adipocyte itself during differentiation is still necessary for the growth of adipose tissue.