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.

The families of zinc (SLC30 and SLC39) and copper (SLC31) transporters.

The solute carriers families 30 (SLC30; ZnT), 39 (SLC39; ZIP), and 31 (SLC31; CTR) are involved in the essential maintenance of cellular zinc (Zn²âº) and copper (Cu²âº) homeostasis, respectively. ZnTs mediate Zn²âº extrusion from cells (SLC30A1) or transport Zn²âº into organelles and secretory vesicles/granules (SLC30A2-SLC30A8). SLC39 family members are predominantly localized to the cell membrane where they perform Zn²âº uptake and increase the availability of cytosolic Zn²âº. SLC39A1 is ubiquitously expressed, whereas other ZIP transporters (e.g., SLC39A2 and SLC39A3) show a more tissue-restricted expression consistent with organ-specific functions of these proteins. The members A1 (CTR1) and A2 (CTR2) of the SLC31 family of solute carriers belong to a network of proteins that acts to regulate the intracellular Cu²âº concentration within a certain range. SLC31A1 is predominantly localized to the plasma membrane, whereas SLC31A2 is mainly found in intracellular membranes of the late endosome and lysosome. The specific function of SLC31A2 is not known. SLC31A1 is ubiquitously expressed and has been characterized as a high-affinity importer of reduced copper (Cu⁺). Cu²âº transport function of CTR proteins is associated with oligomerization; SLC31A1 trimerizes and thereby forms a channel-like structure enabling Cu²âº translocation across the cell membrane. The molecular characteristics and structural details (e.g., membrane topology, conserved Zn²âº, and Cu²âº binding sites) and mechanisms of translational and posttranslational regulation of expression and/or activity have been described for SLC30 and SLC39 family members, and for SLC31A1. For SLC31A1, data on tissue-specific functions (e.g., in the intestine, heart, and liver) are also available. A link between SLC31A1, immune function, and disorders such as Alzheimer's disease or cancer makes the protein a candidate therapeutic target. In secretory tissues (e.g., the mammary gland and pancreas), Zn²âº transporters of SLC families 30 and 39 are involved in specific functions such as insulin synthesis and secretion, metallation of digestive proenzymes, and transfer of nutrients into milk. Defective or dysregulated Zn²âº metabolism in these organs is associated with disorders such as diabetes and cancer, and impaired Zn²âº secretion into milk.

Substitution p.A350V in Na⁺/Mg²âº exchanger SLC41A1, potentially associated with Parkinson's disease, is a gain-of-function mutation.

Parkinson's disease (PD) is a complex multifactorial ailment predetermined by the interplay of various environmental and genetic factors. Systemic and intracellular magnesium (Mg) deficiency has long been suspected to contribute to the development and progress of PD and other neurodegenerative diseases. However, the molecular background is unknown. Interestingly, gene SLC41A1 located in the novel PD locus PARK16 has recently been identified as being a Na⁺/Mg²âº exchanger (NME, Mg²âº efflux system), a key component of cellular magnesium homeostasis. Here, we demonstrate that the substitution p.A350V potentially associated with PD is a gain-of-function mutation that enhances a core function of SLC41A1, namely Na⁺-dependent Mg²âº efflux by 69±10% under our experimental conditions (10-minute incubation in high-Na⁺ (145 mM) and completely Mg²âº-free medium). The increased efflux capacity is accompanied by an insensitivity of mutant NME to cAMP stimulation suggesting disturbed hormonal regulation and leads to a reduced proliferation rate in p.A350V compared with wt cells. We hypothesize that enhanced Mg²âº-efflux conducted by SLC41A1 variant p.A350V might result, in the long-term, in chronic intracellular Mg²âº-deficiency, a condition that is found in various brain regions of PD patients and that exacerbates processes triggering neuronal damage.

Reduced AgRP activation in the hypothalamus of cows with high extent of fat mobilization after parturition.

Agouti-related protein (AgRP), produced by neurons located in the arcuate nucleus of the hypothalamus stimulates feed intake. During early lactation dairy cows increase their feed intake and additionally mobilize their fat reserves leading to increased plasma non-esterified fatty acid (NEFA) concentrations. Since cows with a higher extent of fat mobilization exhibit the lower feed intake, it seems that high NEFA concentrations confine hyperphagia. To test the involvement of AgRP neurons, we investigated 18 cows from parturition until day 40 postpartum (pp) and assigned the cows according to their NEFA concentration on day 40pp to either group H (high NEFA) or L (low NEFA). Both groups had comparable feed intake, body weight, milk yield, energy balance, plasma amino acids and leptin concentrations. Studies in respiratory chambers revealed the higher oxygen consumption and the lower respiratory quotient (RQ) in H compared to L cows. mRNA abundance of neuropeptide Y, peroxisome proliferator-activated receptor-gamma, AMP-activated protein kinase, and leptin receptor in the arcuate nucleus were comparable between groups. Immunohistochemical studies revealed the same number of AgRP neurons in H and L cows. AgRP neurons were co-localized with phosphorylated adenosine monophosphate-activated kinase without any differences between groups. The percentage of cFOS-activated AgRP neurons per total AgRP cells was lower in H cows and correlated negatively with oxygen consumption and NEFA, positively with RQ, but not with feed intake. We conclude that AgRP activation plays a pivotal role in the regulation of substrate utilization and metabolic rate in high NEFA dairy cows during early lactation.

Plasma ghrelin is positively associated with body fat, liver fat and milk fat content but not with feed intake of dairy cows after parturition.

Ghrelin is a gastrointestinal peptide hormone that is present in blood mostly in a non-posttranslationally modified form, with a minor proportion acylated at Ser(3). Both ghrelin forms were initially assigned a role in the control of food intake but there is accumulating evidence for their involvement in fat allocation and utilization. We investigated changes in the ghrelin system in dairy cows, exhibiting differences in body fat mobilization and fatty liver, from late pregnancy to early lactation. Sixteen dairy cows underwent liver biopsy and were retrospectively grouped based on high (H) or low (L) liver fat content post-partum. Both groups had a comparable feed intake in week -6 (before parturition) and week 2 (after parturition). Only before parturition was preprandial total ghrelin concentration higher in L than in H cows and only after parturition was the basal plasma concentration of non-esterified fatty acids higher in H than in L cows. Both before and after parturition, H cows had higher preprandial plasma concentrations of acyl ghrelin, a higher acyl:total ghrelin ratio, lower plasma triacylglyceride concentrations and a lower respiratory quotient compared with L cows. These group differences could not be attributed to an allelic variant of the acyl ghrelin receptor. Rather, the ratio of acyl:total ghrelin correlated with several aspects of fat metabolism and with respiratory quotient but not with feed intake. These results show that endogenous ghrelin forms are associated with fat allocation, fatty liver, and utilization of fat during the periparturient period.

Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen epithelium to high-energy diet.

Ruminal vacuolar H(+)-ATPase (vH(+)-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH(+)-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH(+)-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH(+)-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger to intracellular pH (pH(i)) regulation was investigated. REC isolated from h/c-fed sheep keep their pH(i) at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pH(i) by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP](i) were reduced after foliomycin-induced vH(+)-ATPase inhibition. Our results provide the first evidence for a role of vH(+)-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pH(i) changes to signaling pathways regulating this process.

Intracellular magnesium detection by fluorescent indicators.

Magnesium is essential for a wide variety of biochemical reactions and physiological functions, but its regulatory mechanisms (both at the cellular and at the systemic level) are still poorly characterized. Not least among the reasons for this gap are the technical difficulties in sensing minor changes occurring over a high background concentration. Specific fluorescent indicators are highly sensitive tools for dynamic evaluation of intracellular magnesium concentration. We herein discuss the main criteria to consider when choosing a magnesium-specific fluorescent indicator and provide examples among commercial as well as developmental sensors. We focus on spectrofluorimetric approaches to quantify Mg(2+) concentration in cell or mitochondria suspensions, and on imaging techniques to detect intracellular magnesium distribution and fluxes by live microscopy, reporting a detailed description of standard protocols for each method. The general guidelines we provide should be applicable to specific issues by any researcher in the field.