TRIENNIAL GROWTH AND DEVELOPMENT SYMPOSIUM: Dedifferentiated fat cells: Potential and perspectives for their use in clinical and animal science purpose.

An increasing body of evidences has demonstrated the ability of the mature adipocyte to dedifferentiate into a population of proliferative-competent cells known as dedifferentiated fat (DFAT) cells. As early as the 1970s, in vitro studies showed that DFAT cells may be obtained by ceiling culture, which takes advantage of the buoyancy property of lipid-filled cells. It was documented that DFAT cells may acquire a phenotype similar to mesenchymal stem cells and yet may differentiate into multiple cell lineages, such as skeletal and smooth muscle cells, cardiomyocytes, osteoblasts, and adipocytes. Additionally, recent studies showed the ability of isolated mature adipocytes to dedifferentiate in vivo and the capacity of the progeny cells to redifferentiate into mature adipocytes, contributing to the increase of body fatness. These findings shed light on the potential for use of DFAT cells, not only for clinical purposes but also within the animal science field, because increasing intramuscular fat without excessive increase in other fat depots is a challenge in livestock production. Knowledge of the mechanisms underlying the dedifferentiation and redifferentiation of DFAT cells will allow the development of strategies for their use for clinical and animal science purposes. In this review, we highlight several aspects of DFAT cells, their potential for clinical purposes, and their contribution to adipose tissue mass in livestock.

Review: Animal model and the current understanding of molecule dynamics of adipogenesis.

Among several potential animal models that can be used for adipogenic studies, Wagyu cattle is the one that presents unique molecular mechanisms underlying the deposit of substantial amounts of intramuscular fat. As such, this review is focused on current knowledge of such mechanisms related to adipose tissue deposition using Wagyu cattle as model. So abundant is the lipid accumulation in the skeletal muscles of these animals that in many cases, the muscle cross-sectional area appears more white (adipose tissue) than red (muscle fibers). This enhanced marbling accumulation is morphologically similar to that seen in numerous skeletal muscle dysfunctions, disease states and myopathies; this might indicate cross-similar mechanisms between such dysfunctions and fat deposition in Wagyu breed. Animal models can be used not only for a better understanding of fat deposition in livestock, but also as models to an increased comprehension on molecular mechanisms behind human conditions. This revision underlies some of the complex molecular processes of fat deposition in animals.

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development.

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) ß signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.

Myostatin inhibits porcine intramuscular preadipocyte differentiation in vitro.

This study assessed the effect of myostatin on adipogenesis by porcine intramuscular preadipocytes. Intramuscular preadipocytes were isolated from the longissimus dorsi muscle of newborn pigs. Myostatin inhibited intramuscular preadipocyte differentiation in a dose-dependent manner. Myostatin treatment during preadipocyte differentiation significantly (P < 0.05) inhibited the expression of the adipogenic marker genes CCAAT/enhancer-binding protein ß, CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, fatty acid-binding protein, and adiponectin. Myostatin also significantly (P < 0.05) reduced the release of glycerol and decreased both adipose triglyceride lipase and hormone-sensitive lipase expression in intramuscular adipocytes. Our study suggests that myostatin acts as an extrinsic regulatory factor in regulating intramuscular adipogenesis.

Enhanced mitogenesis in stromal vascular cells derived from subcutaneous adipose tissue of Wagyu compared with those of Angus cattle.

Japanese Wagyu cattle are well known for their extremely high marbling and lower subcutaneous adipose tissue compared with Angus cattle. However, mechanisms for differences in adipose deposition are unknown. The objective of this paper was to evaluate breed differences in the structure of subcutaneous adipose tissue, adipogenesis, and mitogenesis of stromal vascular (SV) cells between Wagyu and Angus cattle. Subcutaneous biopsy samples were obtained from 5 Wagyu (BW = 302 ± 9 kg) and 5 Angus (BW = 398 ± 12 kg) heifers at 12 mo of age, and samples were divided into 3 pieces for histological examination, biochemical analysis, and harvest of SV cells. Adipogenesis of SV cells was assessed by the expression of adipogenic markers and Oil Red-O staining, while mitogenesis was evaluated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium dromide) test, phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB; AKT). Based on histological analysis, Wagyu had larger adipocytes compared with Angus. At the tissue level, protein expression of peroxisome proliferator-activated receptor γ (PPARG) in Wagyu was much lower compared with that of Angus. Similarly, a lower mRNA expression of PPARG was found in Wagyu SV cells. No significant difference was observed for the zinc finger protein 423 (ZNF423) expression between Wagyu and Angus. As assessed by Oil Red-O staining, Wagyu SV cells possessed a notable trend of lower adipogenic capability. Interestingly, higher mitogenic ability was discovered in Wagyu SV cells, which was associated with an elevated phosphorylation of ERK1/2. There was no difference in AKT phosphorylation of SV cells between Wagyu and Angus. Moreover, exogenous fibroblast growth factor 2 (FGF2) enhanced mitogenesis and ERK1/2 phosphorylation of SV cells to a greater degree in Angus compared with that in Wagyu. Expression of transforming growth factor ß 3 (TGFB3) and bone morphogenetic protein 2 (BMP2) in Wagyu SV cells was lower than that of Angus, providing potential clues for breed differences on proliferation of SV cells in these two cattle breeds. The results of this study suggest that subcutaneous adipose-derived SV cells of Wagyu possess a lower trend of adipogenesis but higher mitogenesis compared with those of Angus.

INVITED REVIEW: Evolution of meat animal growth research during the past 50 years: Adipose and muscle stem cells.

If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose- and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed.

Resveratrol induces brown-like adipocyte formation in white fat through activation of AMP-activated protein kinase (AMPK) α1.

OBJECTIVE: Development of brown-like/beige adipocytes in white adipose tissue (WAT) helps to reduce obesity. Thus we investigated the effects of resveratrol, a dietary polyphenol capable of preventing obesity and related complications in humans and animal models, on brown-like adipocyte formation in inguinal WAT (iWAT). METHODS: CD1 female mice (5-month old) were fed a high-fat diet with/without 0.1% resveratrol. In addition, primary stromal vascular cells separated from iWAT were subjected to resveratrol treatment. Markers of brown-like (beige) adipogenesis were measured and the involvement of AMP-activated protein kinase (AMPK) α1 was assessed using conditional knockout. RESULTS: Resveratrol significantly increased mRNA and/or protein expression of brown adipocyte markers, including uncoupling protein 1 (UCP1), PR domain-containing 16, cell death-inducing DFFA-like effector A, elongation of very long-chain fatty acids protein 3, peroxisome proliferator-activated receptor-γ coactivator 1α, cytochrome c and pyruvate dehydrogenase, in differentiated iWAT stromal vascular cells (SVCs), suggesting that resveratrol induced brown-like adipocyte formation in vitro. Concomitantly, resveratrol markedly enhanced AMPKα1 phosphorylation and differentiated SVC oxygen consumption. Such changes were absent in cells lacking AMPKα1, showing that AMPKα1 is a critical mediator of resveratrol action. Resveratrol also induced beige adipogenesis in vivo along with the appearance of multiocular adipocytes, increased UCP1 expression and enhanced fatty acid oxidation. CONCLUSIONS: Resveratrol induces brown-like adipocyte formation in iWAT via AMPKα1 activation and suggest that its beneficial antiobesity effects may be partly due to the browning of WAT and, as a consequence, increased oxygen consumption.

Preadipocyte and adipose tissue differentiation in meat animals: influence of species and anatomical location.

Early in porcine adipose tissue development, the stromal-vascular (SV) elements control and dictate the extent of adipogenesis in a depot-dependent manner. The vasculature and collagen matrix differentiate before overt adipocyte differentiation. In the fetal pig, subcutaneous (SQ) layer development is predictive of adipocyte development, as the outer, middle, and inner layers of dorsal SQ adipose tissue develop and maintain layered morphology throughout postnatal growth of SQ adipose tissue. Bovine and ovine fetuses contain brown adipose tissue but SQ white adipose tissue is poorly developed structurally. Fetal adipose tissue differentiation is associated with the precocious expression of several genes encoding secreted factors and key transcription factors like peroxisome proliferator activated receptor (PPAR)γ and CCAAT/-enhancer-binding protein. Identification of adipocyte-associated genes differentially expressed by age, depot, and species in vivo and in vitro has been achieved using single-gene analysis, microarrays, suppressive subtraction hybridization, and next-generation sequencing applications. Gene polymorphisms in PPARγ, cathepsins, and uncoupling protein 3 have been associated with back fat accumulation. Genome scans have mapped several quantitative trait loci (QTL) predictive of adipose tissue-deposition phenotypes in cattle and pigs.

Maternal overnutrition enhances mRNA expression of adipogenic markers and collagen deposition in skeletal muscle of beef cattle fetuses.

Twenty-four pregnant Nellore cows were randomly assigned into 2 feeding level groups (control [CTL]; fed 1.0 times the maintenance requirement; n = 12; and overnourished [ON]; fed at 1.5 times the maintenance requirement; n = 12) to evaluate effects of maternal overnutrition on fetal skeletal muscle development. Cows were slaughtered at 135, 190, and 240 d of gestation and samples of fetal LM were collected for analysis of mRNA expression analysis and for histological evaluation of collagen content and number of muscle cells. There was no interaction between gestational period and maternal nutrition for the variables evaluated (P > 0.05). The mRNA expression of Cadherin-associated protein, ß 1 (ß-catenin) tended to be greater in fetuses from ON cows (P = 0.08), while myogenic differentiation 1 (MyoD; P = 0.56), myogenin (MyoG; P = 0.70), and the number of muscle cells (P = 0.90) were not affected by maternal overnutrition. Gestational period did not affect the mRNA expression of ß-catenin (P = 0.60) and MyoG (P = 0.21). The mRNA expression of MyoD tended to increase with days of gestation (P = 0.06). The mRNA expression of zinc finger protein 423 (Zfp423; P < 0.0001), C/EBPα (P = 0.01), and PPARγ (P < 0.0001) were enhanced in ON fetuses. No effects of days of gestation were observed for mRNA expression of Zfp423 (P = 0.75) and C/EBPα (P = 0.48). The mRNA expression of PPARγ in fetuses at 190 d of gestation tended to be greater than those at 135 and 240 d of gestation (P = 0.06). The mRNA expression of transforming growth factor ß (TGF-ß; P < 0.0001), collagen type III, α I (COL3A1; P < 0.0001), and collagen content (P = 0.01) were increased in ON fetuses. Gestational period did not affect the mRNA expression of collagen type I, α I (COL1A1; P = 0.65). The mRNA expression of COL3A1 (P = 0.09) in fetuses at 190 d of gestation tended to be greater than fetuses at 135 and 240 d of gestation. The mRNA expression of TGF-ß in fetuses at 190 d of gestation was greater than in fetuses at 135 d of gestation (P = 0.03), and the values observed in fetuses at 240 d of gestation did not differ from the other gestational time points. The least value of collagen content (P = 0.01) was observed in fetuses at 135 d of gestation, and no differences were observed among the other gestational time points. These data shows that maternal overnutrition enhances fibrogenesis and likely adipogenesis without compromising myogenesis in fetal skeletal muscle of cattle.

Enhancement of adipogenesis and fibrogenesis in skeletal muscle of Wagyu compared with Angus cattle.

Intramuscular fat and collagen content are major factors affecting beef quality, but mechanisms regulating intramuscular adipose and connective tissue deposition are far from clear. Japanese Wagyu cattle are well known for their extremely high marbling. The objective of this study was to evaluate intramuscular fat (IMF) and collagen deposition in the muscle of Wagyu compared with Angus cattle. Animals were managed under the same condition and slaughtered at an averaging 585 ± 12.1 kg of BW. Samples of sternomandibularis muscle were collected from Wagyu (n = 3) and Angus (n = 3) for molecular and histological investigations of adipogenesis and fibrogenesis. With exception of C/EBPß (P = 0.2864), the expression of the adipogenic markers C/EBPα (P = 0.008), PPARγ (P = 0.028), and zip finger protein 423 (Zfp423; P = 0.047) in Wagyu were greater than in Angus muscle, which was consistent with greater IMF deposition in Wagyu (P < 0.05). In addition, more adipocytes and preadipocytes were detected intramuscularly in Wagyu cattle. Similarly, fibrogenesis was also enhanced in Wagyu, with a greater expression of fibroblast growth factor (FGF)-2 (P = 0.028), FGF receptor 1 (P = 0.030), transforming growth factor (TGF)-ß (P = 0.028), collagen I (P = 0.012), and collagen III (P = 0.025). Similarly, Wagyu muscle had greater collagen content (P = 0.002) and decreased collagen solubility (P = 0.005). In addition, muscle fiber diameter was larger (P < 0.0001) in Wagyu than in Angus cattle. These results clearly show that both IMF and collagen contents are enhanced in Wagyu cattle and more adipogenic cells are detected in Wagyu muscle, indicating intramuscular adipogenesis is enhanced in Wagyu compared with Angus muscle.

Effects of pregnancy and feeding level on carcass and meat quality traits of Nellore cows.

Carcass and meat quality traits of 16 pregnant and 5 non-pregnant cows fed at 1.2 times maintenance and 16 pregnant and 6 non-pregnant fed ad libitum were evaluated. Pregnancy did not affect final body weight (FBW; P=0.0923), cold carcass yield (CCY; P=0.0513), longissimus muscle area (LMA; P=0.8260), rib fat thickness (RFT; P=0.1873) and shear force (WBSF; P=0.9707). A lower FBW (P=0.0028), LMA (P=0.0048) and RFT (P=0.0001) were observed in feed restricted cows. However, no differences were found for CCY (P=0.7243) and WBSF (P=0.0759) among feeding level groups. These data suggests that carcass and meat quality traits are not affected by pregnancy status in Nellore cows. Moreover, although cows experiencing feed restriction did have reduced deposition of subcutaneous fat and lean tissue, there were no major impacts on meat quality traits.

Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

Cell supermarket: adipose tissue as a source of stem cells.

Adipose tissue is derived from numerous sources, and in recent years this tissue has been shown to provide numerous cells from what seemingly was a population of homogeneous adipocytes. Considering the types of cells that adipose tissue-derived cells may form, these cells may be useful in a variety of clinical and scientific applications. The focus of this paper is to reflect on this area of research and to provide a list of potential (future) research areas.

Meat Science and Muscle Biology Symposium: manipulating mesenchymal progenitor cell differentiation to optimize performance and carcass value of beef cattle.

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.

Bovine mature adipocytes readily return to a proliferative state.

The dynamics of human and animal adipogenesis has been defined using several traditional cell systems including stromal vascular cells and adipocyte-related cell lines. But a relatively new cell system using progeny cells stemming from the dedifferentiation of purified cultures of mature adipocytes may be used for studying the development and biology of adipocytes. In this research, we show that isolated (and purified) mature adipocytes derived from Wagyu cattle dedifferentiate into progeny cells, and that these spindle-shaped, proliferative-competent daughter cells possess ability to proliferate. We outline the optimum cell culture system and offer precautionary thoughts for effective mature adipocyte culture. Collectively, this represents a novel cell model which may provide new insights into cell development, physiology and use as a model for animal production/composition, tissue engineering and disease treatment.

Fatty Acid Composition of Phospholipids and in the Central and External Positions of Triacylglycerol in Muscle and Subcutaneous Fat of Beef Steers Fed Diets Supplemented with Oil Containing n6 and n3 Fatty Acids While Undergoing One of Three 48 h Feed Withdrawal Treatments.

This study was designed to determine the effects of dietary oil and feed withdrawal treatments on fatty acid composition of phospholipids of triacylglycerol in pars costalis diaphragmatis muscle and subcutaneous fat from the brisket. A 2 × 3 factorial experiment was conducted with crossbred steers with an initial body weight of 280.5 ± 5.8 kg. Steers were fed either a control or an oil containing diet where 5% of the control diet was replaced with an equal mixture sunflower and flax oil while undergoing one of three feed withdrawal treatments: no withdrawal, a single 48 h withdrawal before initiation of fattening at one year of age, or 48 h withdrawal at 8 wk intervals from weaning to initiation of fattening. At time of processing samples of muscle and fat were obtained and analyzed to determine fatty acid composition. Disproportionate distribution of the fatty acids was observed by diet, feed withdrawal regimen and whether the sample was from muscle or fat. Differences are discussed in detail, and our data suggests a special function for the fatty acids that accumulate in specific positions of the triacylglycerol due to treatment.

SP-index: the measure of the scientific production of researchers.

Ability to assess how solidly one is participating in their research arena is a metric that is of interest to many persons in academia. Such assessment is not easily defined, and differences exist in terms of which metric is the most accurate. In reality, no single production metric exists that is easy to determine and acceptable by the entire scientific community. Here we propose the SP-index to quantify the scientific production of researchers, representing the product of the annual citation number by the accumulated impact factors of the journals whereby the papers appeared, divided by the annual number of published papers. This article discusses such a productivity measure and lends support for the development of unified citation metrics for use by all participating in science research or teaching.

Obesity, metabolic syndrome, and adipocytes.

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.

Examination of adipose depot-specific PPAR moieties.

Molecular mechanisms of peroxisome proliferator activated receptors (PPARs) are being defined rapidly, as illustrated by the volume of papers published. Much of the research is directed towards a clinical end-point/application; however, the non-homogeneous nature of adipose depots in laboratory animals is spurring similar research in domestic meat animals (such as beef cattle). Moreover, the size of adipose depots in meat animals remains an attractive feature for using them to obtain cells for PPAR research. Examination of meat-animal depot-specific PPAR moieties may provide novel information about adipocyte regulation that might be extrapolated to all animals.

Perspectives on the formation of an interdisciplinary research team.

As research funding becomes more competitive, it will be imperative for researchers to break the mentality of a single laboratory/single research focus and develop an interdisciplinary research team aimed at addressing real world challenges. Members of this team may be at the same institution, may be found regionally, or may be international. However, all must share the same passion for a topic that is bigger than any individual's research focus. Moreover, special consideration should be given to the professional development issues of junior faculty participating in interdisciplinary research teams. While participation may be "humbling" at times, the sheer volume of research progress that may be achieved through interdisciplinary collaboration, even in light of a short supply of grant dollars, is remarkable.