Differential expression of genes in adipose tissue of first-lactation dairy cattle.

Adipose tissue metabolism is an essential factor in establishment of a successful lactation, and we have a good understanding of changes in metabolic flux in relation to lactation, parity, and diet. However, the mechanisms of control of flux are less well understood. To continue our investigations into the control of adipose tissue metabolism, we conducted a transcriptomic analysis of adipose tissue of dairy cattle in late pregnancy and early lactation. Our objective was to determine the changes in gene expression in adipose tissue between 30 d prepartum and 14 d in milk in first-lactation animals, and to determine if changes in expression were related to practical production variables. Animals were Holstein heifers fed the same diet to National Research Council requirements, and adipose tissue was biopsied at 30 d prepartum and 14 DIM. Total RNA was extracted and used to determine gene expression on a bovine gene array. Genes that code for proteins controlling fatty acid transport were highly expressed including fatty acid binding proteins (FABP4 and FABP5) and lipoprotein lipase. Among those genes increasing in expression were those controlling lipolysis, including ADRB2 (52%) and LIPE (23%). Many genes coding for enzymes controlling lipogenesis decreased, including SREBP (-25%), TSHSP14 (-30.8%), LPL (-48.4%), and ACACA (-63.9%). This gene expression array analysis in adipose tissue of lactating dairy cattle identifies several key genes that are components of the adaptation to lactation that can be incorporated into models of nutritional efficiency and may be amenable to genetic or dietary manipulation.

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.

Unidentified cells reside in fish skeletal muscle.

Cell cultures were established from the skeletal muscle tissue of 6-13 months old rainbow trout and 12-14 months old yellow perch. Approximately 27,000 +/- 5,000 cells/g (trout; N = 5) and 5,000 +/- 1,200 cells/g of tissue (perch; N = 4) were obtained. Isolation and propagation were qualitatively greater for both species when the cells (younger fish producer more cells than older fish) were exposed to DMEM + 15% FBS, rather than L-15 + 15% FBS, at 20 degrees C (trout) and at 24 degrees C (yellow perch). Two morphologically distinct cell types were observed in cultures of both species, some of which eventually formed very small myotubes, which displayed immunocytological reactivity for myogenin, myosin heavy chain, and alpha-actinin; the second population of cells remained unstained. Successful cryopreservation was achieved using a 5% DMSO and 95% serum mixture, but post-thawing viabilities were low 5-27% (trout) and 14-30% (perch). Further research is needed in order to determine cell type specificity of isolated cells.

PPARgamma and GLUT-4 expression as developmental regulators/markers for preadipocyte differentiation into an adipocyte.

In this document, we have integrated knowledge about two major cellular markers found in cells of the adipocyte lineage (an adipogenic marker and a metabolic marker). This review provides information as to how differentiation of a cell (such as an adipofibroblast, fibroblast or preadipocyte) to become a viable (and new) adipocyte is under different regulation than that experienced by an immature adipocyte that is just beginning to accumulate lipid. The differentiation, prior to lipid-filling, involves PPARgamma. Subsequently, lipid-filling of the adipocyte relies on a late subset of genes and, depending on depot specificity, involves GLUT-4 or any number of other metabolic markers.

Gaining a solid grip on adipogenesis.

Obesity is presently being combated by fitness regimens, drugs and diet. Increasing our understanding of the physiology of adipocytes, by deducing the regulatory pathways involved in lipid metabolism and all aspects of adipogenesis, will provide alternative strategies to reduce adverse problems of obesity. Research has suggested that mature fat cells may dedifferentiate to form proliferative-competent fat cell precursors. Knowledge of the dedifferentiation process will allow us to gain a solid grip on adipogenesis.

Primary adipocyte culture: adipocyte purification methods may lead to a new understanding of adipose tissue growth and development.

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.

Measuring the effects of phenotype and mechanical restraint on proteolytic degradation and rigor shortening in callipyge lamb longissimus dorsi muscle during extended aging.

The purpose of this study was to determine if tenderness of callipyge (CLPG) longissimus dorsi muscle (LM) could be improved by: (1) extending the aging period to 48 days postmortem or (2) preventing rigor shortening by clamping. In CLPG and normal (NML) chops respectively, initial Warner-Bratzler shear values (WBS) were lower (P<0.05) in clamped (CL) (5.5 and 3.6 kg) compared to unclamped (UCL) (7.4 and 4.9 kg) LM. In CLPG, an acceptable WBS (3.6 kg) was reached at 48 days PM, whereas, NML lambs reached an acceptable level (3.8 kg) by 3 days PM. Sarcomere lengths (SL) of CL (1.68 µm) were longer (P <0.05) than for UCL (1.44 µm) and were negatively correlated with WBS (r=-0.55; P<0.1). The appearance of Troponin-T (TNT) degradation product coincided with tender WBS values; 3 days postmortem in NML UCL and 48 days postmortem in CLPG. In conclusion, clamping reduced WBS possibly by reducing rigor shortening. Extended aging resulted in CLPG LM with acceptable WBS values, concurrent with the appearance of TNT degradation products.

Patterns of expression of muscle-specific markers of differentiation in satellite cell cultures: determination by enzyme-linked immunoculture assay and confocal immunofluorescent assay.

Equine satellite cell clone SE-11 and ovine satellite cell clone I(1)were evaluated for expression of myosin heavy chain, myogenin, desmin, and muscle-specific actin over a 240 h period in culture. An enzyme-linked immunoculture assay (ELICA) was capable of detecting these proteins at all time points evaluated. A linear relationship was demonstrated between the natural logarithm of the absorbance values (corrected for cell number) from the ELICA and percent fusion in both SE-11 and I(1)cultures. The r(2)values for SE-11 cultures were: desmin 0.82, muscle actin 0.81, myogenin 0.78, and myosin 0.70. The r(2)values for I(1)cultures were: desmin 0.77, muscle actin 0.72, myogenin 0.70, and myosin 0.61. Our confocal results support the idea that differences exist between species in the differentiation dynamics of satellite cells. Further, these data suggest that the ELICA may be applied to previously conducted experiments, enabling additional data to be obtained with relation to muscle protein expression.

In vitro model of equine muscle regeneration.

Equine satellite cells are responsible for muscle healing and regeneration in the mature horse. We describe the in vitro cell culture conditions required for clonal populations of equine satellite cells to undergo both proliferation and differentiation. Our hypothesis is that these in vitro conditions model regeneration of muscle and can be used to evaluate potential therapeutics. In this study, 2 areas of satellite cell response were tested: proliferation of clones induced by growth factors, and fusion induced by culture conditions. Equine satellite cell clones showed differences in their response to growth factors as well as accumulation of cellular protein concentrations. Equine satellite cells proliferate in response to both human and bovine FGF. IGF-1, a powerful mitogen of other satellite cell culture systems, was not as effective for inducing equine satellite cell proliferation. Protein concentrations were also measured in satellite cell cultures. Clones differed in cellular protein produced depending on growth conditions. Conditions inducing differentiation into myotubes was also determined for a 96 well assay and can be used to study the final stage of functioning muscle production. This in vitro model is the first step in identifying potential therapeutics to speed wound healing and promote muscle regeneration in horses.

Satellite cell regulation following myotrauma caused by resistance exercise.

It is generally accepted that the primary mechanisms governing skeletal muscle hypertrophy are satellite cell activation, proliferation, and differentiation. Specific growth factors and hormones modulate satellite cell activity during normal muscle growth, but as a consequence of resistance exercise additional regulators may stimulate satellite cells to contribute to gains in myofiber size and number. Present knowledge of the regulation of the cellular, biochemical and molecular events accompanying skeletal muscle hypertrophy after resistance exercise is incomplete. We propose that resistance exercise may induce satellite cells to become responsive to cytokines from the immune system and to circulating hormones and growth factors. The purpose of this paper is to review the role of satellite cells and growth factors in skeletal muscle hypertrophy that follows resistance exercise.

Ten commandments for preventing contamination of primary cell cultures.

Procedures for preventing contamination in primary cell cultures must be carefully defined and strictly followed in order to obtain healthy cells. Protocols have been developed and refined in our laboratory for establishing primary cultures of muscle and fat stem cells without contamination from a variety of animals. Contamination of cell cultures is not only frustrating, but is also very expensive both in time and loss of materials. Through the consistent use of proper aseptic techniques, most instances of contamination may be avoided. We suggest that the basic principles detailed here will find wide applicability in the culturing of primary cells without contamination from many different types of animals and tissues.

Formulation of a defined medium to maintain cell health and viability in vitro.

The first step in formulating a defined medium is to conduct a thorough search of the scientific literature. If a defined medium formulation is located that might be compatible with the intended cell system, a pilot study should be carried out to evaluate the general performance of the medium. Depending on the initial data obtained from this study, individual components of the medium and their concentrations may need to be manipulated (added/subtracted, increased/decreased) to obtain the desired results. Also, sometimes the basal medium or proportions of basal media must be changed. Because the formulation of a defined medium is a circular process, alteration of the basal medium type or ratio of basal media will necessitate redoing all of the previous addition/subtraction and optimization steps. Revalidation must also be done if vendors of components are changed or whenever different cells or cells of other ages are used in the system. This paper presents a brief procedure for formulating a defined media and an overview of the application of two defined media in muscle cell culture.

Methods for animal satellite cell culture under a variety of conditions.

Primary and clonal culture systems have been devised and refined for animal-derived satellite cells. Satellite cell (SC) culture development includes efficient cell isolation techniques, establishment of effective plating and growth conditions, formulation of media requirements and thorough evaluation of experimental limitations. As the field of muscle cell culture has expanded, the number of animal species from which satellite cells have been isolated has increased. The focus of this paper is to compare and contrast SC culture conditions presently used by a variety of researchers and to introduce a new source of SC from wapiti (elk).

Traditional and emerging methods for analyzing cell activity in cell culture.

The selection of appropriate techniques to assay for markers of cell activity is important for obtaining optimal results in cell culture-based research. This paper is intended as a guide to many of the assays currently available and new techniques that have been recently introduced in the literature. This paper addresses both manual assay techniques, including the use of hemocytometers, phase contrast microscopy, cell staining, and the immunofluorescent antibody assay (IFA), and automated assays for cell activity, including stained optical density, proliferating cell nuclear antigen, creatine kinase assay, DNA quantification, electronic cell counting, flow cytometry, magnetic cell sorting, image analysis, chemiluminescence, radioisotope labeling, precursor incorporation, in-situ hybridization/ligand binding, and enzyme-linked immuno-culture assay (ELICA). Advantages/disadvantages and applicability of these assays to different areas of cell culture research are discussed, and guidelines for selecting an appropriate assay are suggested.

Interpretation of cell culture phenomena.

This paper discusses the dilemma of interpreting unusual or abnormal phenomena seen in cell cultures and is not intended to address the statistical design of experiments. Problems that can be encountered when growing cells in experimental situations include low or decreasing cell numbers, abnormal cell morphology, microbial contamination, and detachment of the cell monolayer. If any of these situations occur, it is not realistic to proceed with data analysis until the problem is corrected. The best policy is to attempt to standardize all types of cultures used for analysis and to avoid using any cultures that display atypical characteristics.

Evaluating dot and Western blots using image analysis and pixel quantification of electronic images.

Inexpensive computer imaging technology was used to assess levels of insulin-like growth factor-I (IGF-I) on dot blots (DB) and alpha-Actinin on Western blots (WB). In the first procedure, known IGF-I samples were dotted on nitrocellulose membranes using a vacuum manifold. After the DB were developed and dried, the images were digitized using an HP Deskscan II flat bed scanner, exported into Image-Pro Plus and analyzed by taking the combined mean of 45 degrees and 135 degrees sample lines drawn through each dot. Dot blots corresponding to a linear concentration range from 10 to 300 ng IGF-I were assessed by this method. In the second procedure, WB were scanned with a ScanJet 3c flat bed scanner and their backgrounds were clarified using Image-Pro Plus. A second image analysis program, Alpha Imager 2000, was then used to define the boundaries of protein bands, assess pixel number and density, and to obtain final numerical data for quantifying alpha-Actinin on the WB. Collectively, the results of these two studies suggest that specific proteins may be evaluated by using relatively inexpensive image analysis software systems via pixel quantification of electronic images.

The development and utility of a defined muscle and fat co-culture system.

The utility of co-culture systems in defining the interactions between two different cell types has been well documented in the literature but is a relatively new tool for use in the study of cells derived from normal muscle tissue from meat animals. The majority of myonuclei in postnatal skeletal muscle cells (myofibers) result from satellite cell proliferation, differentiation and fusion with existing myofibers. As such, satellite cell culture systems that mimic postnatal myofibers have great potential for delineating the process of growth and repair of muscle mass. Other ways to simulate the environment of postnatal myofibers might include the development of co-culture systems using satellite cells, or satellite cell-derived myotubes, and other mesodermal cells commonly found associated with muscle tissue in vivo. This brief review describes our initial efforts to develop a defined satellite cell and preadipocyte co-culture system. We provide useful information about defined media requirements and requirements for proper cell orientation and growth on two different growth planes. We present summary data to suggest that differences were found between members of the insulin-like growth factor (IGF) and IGF-binding protein (IGFBP) families of polypeptides, when conditioned media samples were analyzed from co-cultures composed of 3T3-L1 preadipocytes and satellite cells (with different propensities to undergo morphological fusion to form multinucleated myotubes). We also provide information about potential problems to avoid when initiating and conducting co-culture experiments. Such a co-culture system has application in the study of human obesity and also in the regulation of fat deposition in meat animals.

Insulin-like growth factor (IGF)-I and -II and IGFBP secretion by ovine satellite cell strains grown alone or in coculture with 3T3-L1 preadipocytes.

The current study was designed to examine the effects of muscle and fat stem cell coculture on the secretion of insulinlike growth factor (IGF)-I and -II and IGF binding proteins (IGFBP) by these cells. Two sheep satellite cell strains with negligible or high potential for differentiation (10A and 0(1), respectively) were placed in coculture with 3T3-L1 preadipocytes using a filter support to separate the two cell types. Media conditioned by the cells grown alone or in coculture were analyzed for IGFs by RIA or IGFBPs by ligand blotting. The numbers of satellite cells and preadipocytes declined throughout the 5-d culture period, although coculture slowed the 3T3-L1 decline but hastened the satellite cell decline. The satellite cell strains and 3T3-L1 cells secreted small amounts of IGF-I (< or = 2 ng/ml) and IGF-II (< 10 ng/ml) over the 5-d culture period. Coculture did not increase the amount of IGF-I and -II in conditioned media. The lowly differentiating 10A cells secreted barely detectable amounts of the low molecular weight IGFBP-3 subunit (34 kDa), IGFBP-2 (28 kDa), and IGFBP-4 (18 kDa). Coculture of 10A and 3T3-L1 cells potentiated secretion of IGFBP-2 and -3. Strain 0(1), which readily differentiates, secreted high levels of both IGFBP-3 subunits (34 and 39 kDa) and IGFBP-2 (28 kDa), as well as significant amounts of the 18 kDa IGFBP-4. Coculture did not alter IGFBP secretion of 0(1) cells. This study showed that while IGF-I and -II levels in media conditioned by sheep satellite cell strains are low and relatively invariant, the intensity and complexity of IGFBP patterns increases with time in culture and with the potential for differentiation of the satellite cell strains. Coculture with preadipocytes appeared to potentiate IGFBP secretion while reducing satellite cell viability.

Proliferation and differentiation of progeny of ovine unilocular fat cells (adipofibroblasts).

The responsiveness of progency of sheep-derived unilocular fat cells (adipofibroblasts) to dexamethasone, insulin, insulinlike growth factor I (IGF-I), growth hormone (GH), and basic fibroblst growth factor (FGF) was determined in a clonal culture system. Primary cultures of mature adipocytes were obtained from intermuscular adipose tissue (semimembranosus/semitendinosus seam depot) of sheep by ceiling culture techniques. Following degeneration of unilocular fat droplets and re-establishment of fibroblasticlike adipofibroblasts, all adipofibroblasts adhering to upper flask surfaces were collected and isolated away from fibroblasts (which had no multilocular vesicles) by Percoll gradient centrifugation. Progeny derived from a single adipofibroblast were isolated and tested for the ability to proliferate, differentiate, and accumulate lipids. Stock cultures of adipofibroblasts reached confluence in 5 d and were induced to differentiate from 7 to 9 d with dexamethasone-methyl isobutylxanthine-insulin (DMI). Incubation with insulin, IGF-I, GH, or FGF prior to confluence followed by induction with DMI produced no direct (priming) effect on subsequent differentiation. When substituted individually in place of DMI during the 2 d differentiation/induction period, all factors induced differentiation of cultured adipofibroblasts as determined by lipogenesis (P < .05) and lipoprotein lipase activity (P < .05). Thus, isolated adipofibroblasts from sheep muscle may be induced by hormones and growth factors to display mature adipocyte morphology in cell culture. Further definition of the adipofibroblast culture system may aid in the identification of mechanisms regulating adipocyte development in sheep skeletal muscle, as well as in the study of intercommunication between fat and muscle cells.