Methionine Sources Differently Affect Production of Reactive Oxygen Species, Mitochondrial Bioenergetics, and Growth of Murine and Quail Myoblasts In Vitro.

An optimal supply of L-methionine (L-Met) improves muscle growth, whereas over-supplementation exerts adverse effects. To understand the underlying mechanisms, this study aims at exploring effects on the growth, viability, ROS production, and mitochondrial bioenergetics of C2C12 (mouse) and QM7 (quail) myoblasts additionally supplemented (100 or 1000 µM) with L-Met, DL-methionine (DL-Met), or DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). In both cell lines, all the supplements stimulated cell growth. However, in contrast to DL-Met, 1000 µM of L-Met (C2C12 cells only) or DL-HMTBA started to retard growth. This negative effect was stronger with DL-HMTBA and was accompanied by significantly elevated levels of extracellular H2O2, an indicator for OS, in both cell types. In addition, oversupplementation with DL-HMTBA (1000 µM) induced adaptive responses in mitochondrial bioenergetics, including reductions in basal (C2C12 and QM7) and ATP-synthase-linked (C2C12) oxygen consumption, maximal respiration rate, and reserve capacity (QM7). Only QM7 cells switched to nonmitochondrial aerobic glycolysis to reduce ROS production. In conclusion, we found a general negative effect of methionine oversupplementation on cell proliferation. However, only DL-HMTBA-induced growth retardation was associated with OS and adaptive, species-specific alterations in mitochondrial functionality. OS could be better compensated by quail cells, highlighting the role of species differences in the ability to cope with methionine oversupplementation.

Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles.

Muscle stem cells, termed satellite cells (SC), and SC-derived myogenic progenitor cells (MPC) are involved in postnatal muscle growth, regeneration, and muscle adaptability. They can be released from their natural environment by mechanical disruption and tissue digestion. The literature contains several isolation protocols for porcine SC/MPC including various digestion procedures, but comparative studies are missing. In this report, classic trypsinization and a more complex trypsin, collagenase, and DNase (TCD) digestion were performed with skeletal muscle tissue from 4- to 5-d-old piglets. The two digestion procedures were compared regarding cell yield, viability, myogenic purity, and in vitro cell function. The TCD digestion tended to result in higher cell yields than digestion with solely trypsin (statistical trend p = 0.096), whereas cell size and viability did not differ. Isolated myogenic cells from both digestion procedures showed comparable proliferation rates, expressed the myogenic marker Desmin, and initiated myogenic differentiation in vitro at similar levels. Thus, TCD digestion tended to liberate slightly more cells without changes in the tested in vitro properties of the isolated cells. Both procedures are adequate for the isolation of SC/MPC from juvenile porcine muscles but the developmental state of the animal should always be considered.

Separation of functionally divergent muscle precursor cell populations from porcine juvenile muscles by discontinuous Percoll density gradient centrifugation.

BACKGROUND: Satellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity. Several studies have provided evidence that SC and MPC represent a heterogeneous population differing in their biochemical and functional properties. The identification and characterization of functionally divergent SC subpopulations should help to reveal the precise involvement of SC/MPC in these myogenic processes. The aim of the present work was therefore to separate SC subpopulations by using Percoll gradients and to characterize their myogenic marker profiles and their functional properties (adhesion, proliferation, and differentiation). RESULTS: SC/MPC from muscles of 4-day-old piglets were isolated by trypsin digestion and enriched by Percoll density gradient centrifugation. A mixed myogenic cell population was obtained from the 40/70% interface (termed: mixed P40/70) of a 25/40/70% Percoll gradient. Thereafter, by using a more stepped 25/40/50/70% Percoll gradient, mixed P40/70 was divided into subpopulation 40/50 (SP40/50) collected from the 40/50% interface and subpopulation 50/70 (SP50/70) collected from the 50/70% interface. All three isolated populations proliferated and showed a myogenic phenotype characterized by the ability to express myogenic markers (Pax7, MyoD1, Desmin, and MyoG) and to differentiate into myotubes. However, compared with mixed P40/70, SP40/50 and SP50/70 exhibited distinct functional behavior. Growth kinetic curves over 90 h obtained by the xCELLigence system and proliferation assays revealed that SP40/50 and mixed P40/70 constituted a fast adhering and fast proliferating phenotype. In contrast, SP50/70 showed considerably slower adhesion and proliferation. The fast-proliferating SP40/50 showed the highest myogenic differentiation potential with higher fusion rates and the formation of more middle-sized and large myotubes. CONCLUSIONS: The described Percoll density gradient centrifugation represents a useful tool for subdividing pig SC/MPC populations with divergent myogenic functions. The physiological role of SC subpopulations during myogenesis and the interaction of these populations can now be analyzed to a greater extent, shedding light on postnatal growth variations in pigs and probably in other animals.

Molecular and functional heterogeneity of early postnatal porcine satellite cell populations is associated with bioenergetic profile.

During postnatal development, hyperplastic and hypertrophic processes of skeletal muscle growth depend on the activation, proliferation, differentiation, and fusion of satellite cells (SC). Therefore, molecular and functional SC heterogeneity is an important component of muscle plasticity and will greatly affect long-term growth performance and muscle health. However, its regulation by cell intrinsic and extrinsic factors is far from clear. In particular, there is only minor information on the early postnatal period which is critical for muscle maturation and the establishment of adult SC pools. Here, we separated two SC subpopulations (P40/50, P50/70) from muscle of 4-day-old piglets. Our results characterize P40/50 as homogeneous population of committed (high expression of Myf5), fast-proliferating muscle progenitors. P50/70 constituted a slow-proliferating phenotype and contains high numbers of differentiated SC progeny. During culture, P50/70 is transformed to a population with lower differentiation potential that contains 40% Pax7-positive cells. A reversible state of low mitochondrial activity that results from active down-regulation of ATP-synthase is associated with the transition of some of the P50/70 cells to this more primitive fate typical for a reserve cell population. We assume that P40/50 and P50/70 subpopulations contribute unequally in the processes of myofiber growth and maintenance of the SC pool.

Sex differences in body composition, fat storage, and gene expression profile in Caenorhabditis elegans in response to dietary restriction.

The metabolic and health-promoting effects of dietary restriction (DR) have been extensively studied in several species. The response to DR with respect to sex is essentially unknown. To address this question, we used the model organism Caenorhabditis elegans to analyze body composition and gene expression in males and hermaphrodites in response to DR. Unexpectedly, DR increased the fat-to-fat-free mass ratio and enlarged lipid droplets in both sexes to a similar extent. These effects were linked to a downregulation of the lipase-like 5 (lipl-5) gene in both sexes at two developmental stages. By contrast, the reductions in body size, protein content, and total RNA content in response to DR were more pronounced in hermaphrodites than in males. Functional enrichment analysis of gene expression data showed a DR-induced downregulation of several embryogenesis-associated genes concomitant with an ongoing expression of sperm-associated genes in hermaphrodites. In conclusion, DR increases fat stores in both sexes of C. elegans in the form of large and possibly lipolysis-resistant lipid droplets and markedly alters the reproductive program in hermaphrodites but not in males.

Sex differences in carbohydrate metabolism are linked to gene expression in Caenorhabditis elegans.

The male and the hermaphrodite forms of the nematode Caenorhabditis elegans (C. elegans) differ markedly in anatomy, nervous system and behavior at adulthood. Using the male mutants fog-2, him-5, and him-8, we compared body proportions and composition, and aspects of carbohydrate metabolism and gene expression between the C. elegans sexes in three adult stages. In all experiments, both sexes were grown on the same plate and separated using flow cytometry. The fat to fat-free mass ratio and the body volume-adjusted fat mass is similar between the sexes, although the body size is more than 50% smaller in adult males than in age-matched hermaphrodites. The volume-adjusted total RNA content is approximately 2-fold lower in males. Biochemical and NMR-based analyses reveal higher trehalose levels and much lower glucose levels in males than in hermaphrodites. The resulting trehalose-to-glucose ratio is 5.4-fold higher in males. These sex differences are reflected in gene expression data because the genes encoding key enzymes of the glycolysis and trehalose synthesis pathways are more highly expressed in males than in hermaphrodites. Notably, expression of the phosphofructokinase gene (C50F4.2) is 29-fold higher in males. Comparative analysis of gene expression data identifies 285 male-specific and 160 hermaphrodite-specific genes. These include transcription factor and C-type lectin-encoding genes. More than 35% of all C-type lectin genes are more highly expressed in males. The expression of many C-type lectin genes differs by a factor of >100 between the sexes. In conclusion, we found sex differences in carbohydrate metabolism that are linked to gene expression and identified certain lectin genes that are differentially expressed by the C. elegans sexes.

Paternal dietary restriction affects progeny fat content in Caenorhabditis elegans.

Epidemiological data from human populations and few reports in rodents suggested that the paternal diet affects offspring adiposity and its related diseases. We tested whether this nongenetic and intergenerational inheritance depends on paternal treatment dose. Using the model organism Caenorhabditis elegans, males undergoing several dietary restriction regimes were crossed with ad libitum fed females. We found an inverted U-shaped relationship between the extent of paternal dietary restriction and the level of fat content of progeny. The relationship was evident in both sexes. Body proportions were not affected in offspring. Overall, our findings extent the concept of developmental and adaptive plasticity to include the extent of paternal food consumption in the origin of phenotypic alterations.