Janet L. Vierck

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.

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.

The effects of ergogenic compounds on myogenic satellite cells.

PURPOSE A series of studies were conducted in which compounds commonly shown to be ergogenic aids for strength athletes if taken orally were evaluated for their ability to directly induce postnatal muscle stem cell proliferation or differentiation/fusion in vitro. METHODS Compounds tested were creatine monohydrate, creatine pyruvate, L-glutamine, dehydroepiandrosterone (DHEA), androstenedione, Ma Huang (Ephedra sinensis) extract, and Zhi Shi (Citrus aurantium) extract. Dulbeccos modified eagle medium, supplemented with minimal levels of serum and antibiotics, was used as the initial vehicle for the test compounds. Subsequently, a defined treatment medium termed ITTC was used. Satellite cells were exposed to the test compounds for the indicated times and then evaluated by counting mononucleated and multinucleated (fused) cells. RESULTS In serum-containing media, none of the treatment groups displayed increased proliferation over that of the control. However, in the differentiation cultures, 0.10% creatine monohydrate increased differentiation over that of the control cultures. When 0.10% creatine monohydrate was added to defined media formulations, all treatments but one demonstrated increased differentiation over the 0.5% serum control. Time course experiments, which followed the effect of 0.10% creatine monohydrate contained in ITTC defined media over 120 h, suggested that cells exposed to this treatment differentiated earlier and to a greater level than cells exposed to ITTC alone. CONCLUSIONS Creatine in the monohydrate form induced differentiation of myogenic satellite cells. Other agents examined did not increase satellite cell proliferation or differentiation. These results provide initial evidence for a mechanistic understanding of observed effects in vivo of increased muscular size and strength from creatine supplementation.

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

SummaryThe responsiveness of progeny of sheep-derived unilocular fat cells (adipofibroblasts) to dexamethasone, insulin, insulinlike growth factor I (IGF-I), growth hormone (GH), and basic fibroblast 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.

Adipocytes may not be a terminally differentiated cell type: implications for animal production

Grazing animals cause major alterations to vegetation structure and botanical composition through their selective grazing, trampling and excretal deposition (Hester et al. , 2005). Through these effects they modify habitats and thus the populations of invertebrates and other organisms at higher trophic levels. Herbivores are thus key drivers of ecosystem function and nutrient dynamics within grazed plant communities. Changes in grazing intensity and the species mix of grazing livestock can therefore have important implications for resulting biodiversity. Ongoing reform of the European Union Common Agricultural Policy (CAP) will lead to a shift in the way financial support for livestock is distributed and hence to changes in grazing management practices. Farmers will increasingly receive financial support subject to cross-compliance with various environmental conditions and for delivery of specific environmental and social objectives. In some areas, livestock are likely to be increasingly viewed as tools for habitat management rather than solely as producers of food and other commodities. Against this backdrop of policy change the British Society of Animal Science and the British Ecological Society organized a joint symposium at the BSAS Annual Meeting in April 2005 on the links between farm animals and biodiversity. Four papers from this symposium are presented as mini-reviews in the current issue of Animal Science. David Oglethorpe set the policy context in his review of the environmental implications of CAP reform (Oglethorpe, 2005). His paper highlighted the likely changes in the livestock sector that will ensue including a polarization of agriculture into intensive producers versus environmental managers, increasing extensification in the uplands and some substitution of beef with sheep. Jerry Tallowin then presented a review of the impact of grazing management on grassland biodiversity (Tallowin et al. , 2005). His paper showed that lenient grazing pressure by cattle in species-rich grassland was sufficient to maintain botanical diversity but did not enhance it over a 5-year period. For species-poor grassland, grazing management could alter sward structure but, in the absence of seed sources, botanical diversity was resistant to change. There is obviously much research still be done in this area to support the development of suitable agri-environment measures under Pillar 2 support mechanisms. David Buckingham went on to consider the extent to which grassland management might influence habitat quality for farmland birds (Buckingham and Peach, 2005). His paper showed that the exacting requirements of declining granivorous birds pose the greatest challenges while the needs of soil invertebrate feeding species are more easily met under agri-environmental schemes. In the final paper by Bruno Martin, the influence of pasture diversity on cheese quality was the theme (Martin et al. , 2005). This paper reviewed recent work, primarily from France, which has examined the links between the diet of grazing animals and the sensory characteristics of various Protected Designation of Origin cheeses. The review highlighted the sometimes subtle, but none the less important influence of the grazing environment on food quality. The purpose of the symposium was to draw together animal scientists, conservation biologists, ecologists and socio-economists to consider the changing role of farm livestock within the new ‘decoupled’ economic environment. Judging by the popularity of the symposium and the vibrant nature of the discussion that followed each paper, there are plenty of issues still to consider and the hope is that some of the contacts made at the meeting will yield fruitful collaborations in the future.

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.

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.

Assessing a Non-Traditional View of Adipogenesis: Adipocyte Dedifferentiation – Mountains or Molehills?

Based on our studies we propose the following hypothesis: mature, lipid-containing adipocytes possess the ability to undergo symmetrical or asymmetrical cell division, without losing lipid. While our research to discern the mechanism(s) involved in what we have termed ‘dedifferentiation’ of adipocytes is ongoing, we have identified several roadblocks to our work in this area. However, due to the newness of this research, we believe that none of these problems discounts the potential importance of our initial observations, or the excitement of contributing knowledge in the area. In this manuscript we address some of these problems and suggest possible solutions in an attempt to make ‘molehills’ out of ‘mountains.’

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.

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.