Jane E. Pesall

Proliferation of the turkey myogenic satellite cell in a serum-free medium

Abstract 1. In order to determine factors involved in avian skeletal muscle development, a serum-free medium (TSFM) which supports clonal growth of the turkey myogenic satellite cell has been developed. 2. The formulation consists of McCoys 5A medium with added insulin, fibroblast growth factor, Deutsch fetuin, bovine serum albumin, dexamethasone, supplemental minerals and additional organic nutrients. 3. The development of TSFM was made possible by the use of clonal-derived turkey satellite cells. These cells allowed direct assessment of proliferation responses without the confounding effects of nonmyogenic cells in culture.

Variation in fibroblast growth factor response and heparan sulfate proteoglycan production in satellite cell populations.

Variation in the responsiveness of myogenic satellite cell subpopulations to mitogenic stimuli was examined in cells isolated from the turkey pectoralis major muscle. Faster growing clonal cell populations were more responsive to fibroblast growth factor (FGF-2) and expressed greater levels of FGF-2 and FGF receptor-1 at the onset of proliferation than did slower growing cells. Faster growing clones also expressed higher levels of heparan sulfate proteoglycans (HSPG), especially during differentiation, than did slower growing clones. HSPG, which is important in FGF receptor signaling, increased during proliferation of all clones tested and decreased in all but one of the clones during differentiation. Slower growing clones increased their expression of FGF receptor-1 through proliferation and differentiation. However, expression of the receptor in faster growing clones decreased during differentiation. The FGF receptors-2 and -3 were not detected on turkey satellite cells or myotubes by reverse transcriptase-polymerase chain reaction methodology. These results demonstrate that there is heterogeneity in the properties and responsiveness of satellite cell populations derived from single muscles. Satellite cells that differ in proliferation rates differ in responsiveness to FGF-2, and in the expression of FGF-2, FGF receptor-1, and HSPG.

Comparison of the proliferation and differentiation of myogenic satellite cells and embryonic myoblasts derived from the Turkey

Abstract 1. 1. Embryonic and posthatch turkey skeletal muscle development was compared in in vitro studies using clonal-derived embryonic myoblasts and satellite cells. 2. 2. Although population doubling times were similar between the two lines (25.4 hr for satellite cells and 26.4 hr for embryonic myoblasts), embryonic myoblasts consistently began log phase growth 24 hr earlier than satellite cells. 3. 3. Differentiation (fusion) of embryonic myoblasts was maximized by 36 hr in Dulbeccos Modified Eagles Medium containing 1% horse serum compared with 72 hr for satellite cells. 4. 4. When administered a serum-free medium which supports proliferation of turkey satellite cells, embryonic myoblasts differentiated to form myotubes.

Tissue distribution of insulin-like growth factor receptors in the turkey

1. The distribution and relative insulin-like growth factor (IGF) binding capacities of membranes derived from 14 tissues of the turkey were examined. 2. Affinity cross-linking analyses using [125I]IGF-I and [125I]IGF-II with membranes derived from the liver, pectoralis major muscle, gizzard, heart and brain indicated that both IGFs interact with only type-I IGF receptors on these tissues. 3. There was no evidence for the existence of a type-II IGF receptor in any tissue. 4. Although considerable variation was detected in the molecular weights of the IGF receptor alpha subunits between tissues (112.2-132.9 kDa), these differences did not appear to influence receptor-ligand affinities.

Phospho-MAPK as a marker of myogenic satellite cell responsiveness to growth factors.

To determine if differential response to growth factor stimuli between subpopulations of satellite cells was due to variation in the levels of activated intracellular signaling proteins, the levels of phospho-MAPK (phospho-ERK 1/2) were determined in clonal populations of turkey (Meleagris gallopavo) satellite cells. Relative levels of phospho-ERK 1/2 between clones were determined by Western blotting of extracts from satellite cells exposed to growth factor stimuli. Initial measurements using serum mitogenic stimuli showed differences in phospho-MAPK levels between the clonal subpopulations, but the responses did not correlate with proliferation rates of the individual clones (P>0.05). IGF-I alone did not increase phospho-MAPK levels compared to unstimulated controls (P>0.05), whereas FGF-2 did increase levels (P<0.05). A synergistic response was seen in satellite cells as well as embryonic myoblasts administered both IGF-I and FGF-2. When administered FGF-2 and IGF-I, 2 of the slow growing satellite cell clones exhibited lowest levels of phospho-MAPK (P<0.05). One of the slow growing clones had levels of phospho-MAPK similar to the three fast growing clones (P>0.05). The results suggest that variation in responsiveness to growth factor stimuli among satellite cell populations within muscles may be due to several different reasons. Some differences in cell responsiveness appear to be due to variation in phospho-MAPK generation.

Comparison of protein metabolism and glucose uptake in turkey (Meleagris gallopavo) satellite cells and embryonic myoblasts in vitro

Protein synthesis, protein degradation, and glucose uptake were compared in clonal-derived turkey myogenic satellite cells (clone D5-SC) and embryonic myoblasts (EM) and between satellite cell cultures from Nicholas (DN) and Merriams (WM) turkeys. Protein synthesis rates were higher and degradation rates were lower in myotube cultures of D5-SC compared with EM (P < or = 0.05). Protein synthesis and degradation rates did not differ between cultures of DN and WM (P > or = 0.05). Glucose transport rates were significantly higher in EM than D5-SC cultures and did not differ between DN and WM cultures. Insulin-like growth factors and insulin stimulated protein synthesis, decreased protein degradation, and increased glucose uptake in all cell lines.

Comparison of the proliferation and differentiation of myogenic satellite cells derived from Merriam's and commercial varieties of turkeys

1. Myogenic satellite cells were isolated and cloned from the Pectoralis major muscles of 6-week-old Nicholas and Merriams tom turkeys to compare in vitro properties of muscle development between turkeys with markedly different growth rates. 2. Although only small differences (P < or = 0.05) were noted between proliferation rates of the two cell populations in McCoys 5A medium-15% chicken serum, satellite cells derived from the Nicholas variety were more responsive (P < or = 0.05) to mitogenic stimuli from serum at all levels tested. 3. When satellite cells were stimulated by low serum levels to differentiate into multinucleated myotubes, cells from the Merriams turkey fused more rapidly (P < or = 0.05).

Effects of 17β-estradiol on turkey myogenic satellite cell proliferation, differentiation, and expression of glypican-1, MyoD and myogenin.

The hypothesis of this study was that 17β-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10(-9)M following 4days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10(-12)M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10(-9)M and 10(-5)M estradiol following 3days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.

Nampt/visfatin/PBEF affects expression of myogenic regulatory factors and is regulated by interleukin-6 in chicken skeletal muscle cells.

Nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF) has been identified as a rate-limiting NAD(+) biosynthetic enzyme and an adipokine found in the circulation. Human and chicken skeletal muscles are reported to have the highest level of Nampt expression among various tissues whose functional significance remains undetermined. Expression of Nampt is regulated by interleukin-6 (IL-6), an essential cytokine for postnatal muscle growth in mammals. The objective of the current study was to characterize expression of Nampt in chicken (Gallus gallus) myogenic cells and to determine the effect of Nampt on expression of IL-6, myogenic transcription factors, and glucose uptake. We also sought to determine the effect of IL-6 on Nampt expression in chicken myogenic cells. Nampt mRNA and protein were identified in both myoblasts and myocytes, although expression did not differ between the two cell types. Treatment with recombinant human Nampt was found to decrease myoD and mrf4 expression but to increase myf5 expression in myocytes, while glucose uptake was unaffected. In response to treatment with recombinant Nampt, IL-6 expression in myocytes was increased at 24h but decreased when treated for 48 or 72 h. Forced over-expression of chicken Nampt cDNA significantly decreased myf5 expression in myoblasts. Treatment of myogenic cells with lower levels (1 ng.mL(-1)) of recombinant IL-6 increased Nampt expression, whereas a higher IL-6 concentration (100 ng.mL(-1)) decreased Nampt mRNA abundance. Collectively, these results demonstrate that Nampt, regulated in part by IL-6, alters the expression of key myogenic transcription factors and thereby may influence postnatal myogenesis.

Effect of lipids on avian satellite cell proliferation, differentiation and heparan sulfate proteoglycan expression.

The objective of this study was to determine the effects of fatty acids on the proliferation, differentiation, and expression of syndecan-4 and glypican-1 in avian myogenic satellite cells (SC). SC derived from the pectoralis major (PM) and biceps femoris (BF) muscles of the turkey and chicken were individually administered 8 different fatty acids in defined medium during proliferation. A parallel set of turkey SC was induced to differentiate. Highest levels of proliferation of turkey PM and BF SC occurred in cultures containing oleate. Linoleate and oleate were equipotent in supporting proliferation of chicken SC. Microscopic examination revealed that inclusion of docosahexaenoate or eicosapentaenoate was toxic towards both PM and BF SC from both species. Linolenate and arachidonate diminished levels of differentiation. Expression of glypican-1 varied between treatments to a greater extent with turkey BF than with PM SC. Expression in chicken PM and BF SC demonstrated a similar pattern in response to treatments. Turkey PM syndecan-4 expression varied between treatments, whereas expression in turkey BF SC was similar between treatments. Expression in chicken SC varied little between treatments. The results demonstrate species and muscle-specific differences in the parameters examined. It is proposed that changes in lipid raft receptor interactions may contribute to these observed differences.