Beef cattle are raised for their lean tissue, and excessive fat accumulation accounts for large amounts of waste. On the other hand, intramuscular fat or marbling is essential for the palatability of beef. In addition, tender beef is demanded by consumers, and connective tissue contributes to the background toughness of beef. Recent studies show that myocytes, adipocytes, and fibroblasts are all derived from a common pool of progenitor cells during embryonic development. It appears that during early embryogenesis, multipotent mesenchymal stem cells first diverge into either myogenic or adipogenic-fibrogenic lineages; myogenic progenitor cells further develop into muscle fibers and satellite cells whereas adipogenic-fibrogenic lineage cells develop into the stromal-vascular fraction of skeletal muscle where reside adipocytes, fibroblasts, and resident fibro-adipogenic progenitor cells (the counterpart of satellite cells). Strengthening myogenesis (i.e., formation of muscle cells) enhances lean growth, promoting intramuscular adipogenesis (i.e., formation of fat cells) increases marbling, and reducing intramuscular fibrogenesis (i.e., formation of fibroblasts and synthesis of connective tissue) improves overall tenderness of beef. Because the abundance of progenitor cells declines as animals age, it is more effective to manipulate progenitor cell differentiation at an early developmental stage. Nutritional, environmental, and genetic factors shape progenitor cell differentiation; however, up to now, our knowledge regarding mechanisms governing progenitor cell differentiation remains rudimentary. In summary, altering mesenchymal progenitor cell differentiation through nutritional management of cows, or fetal programming, is a promising method to improve cattle performance and carcass value.

In the present manuscript, the methods required to generate purified cultures of mature adipocytes, as well as stromal vascular cells, from the same isolation are detailed. Also, we describe the in vitro conditions for the dedifferentiation of the isolated mature adipocytes. These two types of cells may be used to reevaluate differences between presently available cellular models for lipogenesis/lipolysis and might provide a new cellular physiological system for studies utilizing the proliferative progeny from mature adipocyte dedifferentiation. Alternative possibilities to the dedifferentiation phenomenon are proposed, as this new area of research is novel.Abbreviations: DMEM -Dulbecco's modified Eagle medium; DMEM/F12 -1:1 ratio; Dulbecco's modified Eagle medium + Ham's F12; FBS -fetal bovine serum; HBSS -Hank's balanced salt solution; HS -horse serum; PBS -phosphate buffered saline, pH 7.08; PSG -pigskin gelatin; SC -satellite cell

Thirteen reference genes were investigated to determine their stability to be used as a housekeeping in gene expression studies in skeletal muscle of chickens. Five different algorithms were used for ranking of reference genes and results suggested that individual rankings of the genes differed among them. The stability of the expression of reference genes were validated using samples obtained from the Pectoralis major muscle in chicken. Samples were obtained from chickens in different development periods post hatch and under different nutritional diets. For gene expression calculation the ΔΔCt approach was applied to compare relative expression of pairs of genes within each of 52 samples when normalized to mitochondrially encoded cytochrome c oxidase II (MT-CO2) target gene. Our findings showed that hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are the most stable reference genes while transferrin receptor (TFRC) and beta-2-microglobulin (B2M) ranked as the least stable genes in the Pectoralis major muscle of chickens. Moreover, our results revealed that HMBS and HPRT1 gene expression did not change due to dietary variations and thus it is recommended for accurate normalization of RT-qPCR data in chicken Pectoralis major muscle.

The effect of breed and diet on insulin response to glucose challenge and its relation to intramuscular fat deposition was determined in 36 steers with 12 each of greater than 87% Wagyu (referred to as Wagyu), Wagyu x Limousin, and Limousin breeds. Weaned steers were blocked by weight into heavy, medium, and light calves and placed in six pens with two pens per weight type and with two steers of each breed per pen. Three pens with steers from each weightclass were fed backgrounding and finishing diets for 259 d, while the other three pens were fed the same diets where 6% of the barley grain was replaced with sunflower oil. Prior to initiation of the finishing phase of the study the intravenous glucose tolerance test (VGTIT) was conducted in all steers. Once steers were judged as carrying adequate 12th-rib fat, based on weight and days on feed, they were harvested and graded and samples of the longissimus muscle were procured for determination of fat content and fatty acid composition. Dietary oil improved (P = 0.011; 0.06) ADG and feed conversion efficiency of steers during the latter part of backgrounding and only ADG during early part ofthe finishing period. Generally percent kidney, pelvic, and heart fat was the only adiposity assessment increased (P = 0.003) by dietary oil. The IVGTT results indicated that insulin response to intravenous glucose was lower in Limousin steers than in Wagyu steers. Dietary oil decreased (P = 0.052) fasting plasma insulin concentration in Wagyu steers compared with Limousin steers. The correlation coefficients among the IVGTT measures and intramuscular fat content or marbling score were less than 0.4, and only a negative trend existed between fasting insulin and USDA marbling scores. However, the carcasses of the Wagyu steers graded US Choice, and 66% of the Wagyu carcasses graded US Prime, which were substantially better than the quality grades obtained for the carcasses from the other breed types. Dietary oil did not affect muscle fat content but increased (P = 0.01) conjugated linoleic acid (CLA) concentrations by 339%. Results indicated that IVGTT measures were not appropriate indices of marbling potential in cattle and that dietary oil can enhance CLA content of beef.

Background:The mechanisms eliciting metabolic activation in satellite cells are unclear. Results: Noncanonical Sonic Hedgehog is activated following muscle injury, which activates AMPK␣1 to induce Warburg-like glycolysis and promote satellite cell activation and proliferation. Conclusion: AMPK␣1 is required for Warburg-like glycolysis in satellite cells, which promotes satellite cell activation and muscle regeneration. Significance: AMPK promotes satellite cell activation during muscle regeneration.

fThe link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic ␣ subunits, ␣1 and ␣2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the ␣1 but not the ␣2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPK␣1 and AMPK␣2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPK␣1-specific effect on myogenesis was likely due to the dominant expression of ␣1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPK␣1 subunit for myogenesis in vivo, we prepared both DsRed AMPK␣1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wildtype myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPK␣1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPK␣1. Skeletal muscle, which comprises about 40% of the body mass of adults, is the main peripheral tissue responsive to insulinstimulated uptake of glucose (1) and is critical in the development of type 2 diabetes (2). Proper myogenesis is critical for fetal muscle development (3-5) and postnatal muscle growth and regeneration, which relies heavily on the myogenic differentiation of satellite cells (6-8). In improper myogenesis and muscle regeneration, damaged muscle fibers are replaced with fibric tissue, leading to muscle atrophy and aging (9-11).AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme, composed of ␣, ␤, and ␥ subunits, which plays an important role in energy metabolism (12)(13)(14). In addition to its capacity to acutely regulate the activity of metabolic enzymes through phosphorylation, AMPK is increasingly recognized for its regulatory role in gene expression, cell differentiation, and tissue development (15, 16). The role of AMPK in muscle fiber atrophy has been well defined; a number of studies demonstrated that AMPK promotes muscle protein degradation and autophagy (17) and inhibits protein synthesis (18). To date, however, the role of AMPK in myogenesis (formation of muscle fibers) has been sparsely studied. Our previous studies showed that low AMPK activity due to obesity is correlated with attenuated myogenic differentiation during fetal muscle development (19-21). We further observed that AMPK promotes myogenin expression and myogenesis through phosphorylation of histone deacetylase 5 (HDAC5) (22), suggesting that AMPK has a critical role in myogenesis.The catalytic ␣ subunit of AMPK has two isoforms, ␣1 and ␣2, which display differential...

Obesity and metabolic syndromes are examples whereby excess energy consumption and energy flux disruptions are causative agents of increased fatness. Because other, as yet elucidated, cellular factors may be involved and because potential treatments of these metabolic problems involve systemic agents that are not adipose depot-specific in their actions, should we be thinking of adipose depot-specific (cellular) treatments for these problems? For sure, whether treating obesity or metabolic syndrome, the characteristics of all adipose depot-specific adipocytes and stromal vascular cells should be considered. The focus of this paper is to begin to align metabolic dysfunctions with specific characteristics of adipocytes.

The insulin-independent and combined effects of fatty acids (FA; linoleic and oleic acids) and insulin in modulating lipid accumulation and adipogenesis in 3T3-L1 cells was investigated using a novel protocol avoiding the effects of a complex hormone 'induction' mixture. 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus serum (control) or in DMEM plus either 0.3 mmol/l linoleic or oleic acids with 0.3 mmol/l FA-free bovine serum albumin in the presence or absence of insulin. Cells were cultured for 4 to 8 days and cell number, lipid accumulation, peroxisome proliferator-activated receptor-gamma (PPAR-g) and glucose transporter 4 (GLUT-4) protein expression were determined. Cell number appeared to be decreased in comparison with control cultures. In both oleic acid and linoleic acid-treated cells, notably in the absence (and presence) of insulin, oil-red O stain-positive cells showed abundant lipid. The percentage of cells showing lipid accumulation was greater in FA-treated cultures compared with control cells grown in DMEM plus serum (P , 0.001). Treatment with both linoleic and oleic acid-containing media evoked higher levels of PPAR-g than observed in control cultures (P , 0.05). GLUT-4 protein also increased in response to treatment with both linoleic and oleic acid-containing media (P , 0.001). Lipid accumulation in 3T3-L1 cells occurs in response to either oleic or linoleic acids independently of the presence of insulin. Both PPAR-g and GLUT-4 protein expression were stimulated. Both proteins are considered markers of adipogenesis, and these observations suggest that these cells had entered the physiological state broadly accepted as differentiated. Furthermore, 3T3-L1 cells can be induced to accumulate lipid in a serum-free medium supplemented with FA, without the use of induction protocols using complex hormone mixtures. We have demonstrated a novel model for the study of lipid accumulation that will improve the understanding of adipogenesis in adipocyte lineage cells.Keywords: adipogenesis, fatty acids, GLUT-4, insulin, PPAR-g IntroductionProgression to lipid storage in adipocytes is characterised by two distinct phases. In the first step of hyperplastic expansion, progenitor cells are thought to become committed to the adipocyte lineage, after which they cannot revert back to a less differentiated 'stem-like' cell (Thompson et al., 1998;Boone et al., 2000). Once committed, adipoblasts undergo an exponential replication phase that terminates and the cell cycle arrests at gap 1 (G1). Early markers of differentiation, such as lipoprotein lipase (LPL) are then expressed, and these cells, known as preadipocytes, may then undergo proliferation (Boone et al., 2000). After preadipocytes stop proliferating, late markers of differentiation, such as glycerol-3-phosphate dehydrogenase (GPDH) and fatty acid synthetase (FAS) are detected. Cells then begin lipid accumulation in the cytosol at which time cells are termed adipocytes (Boone et al., 2000).Much of our understanding of these proc...