In vitro Effects of Biologically Active Vitamin D on Myogenesis: A Systematic Review.

Vitamin D (VD) deficiency is associated with muscle weakness. A reduction in the incidence of falls in the elderly following VD supplementation and identification of the VD receptor within muscle cells suggests a direct effect of VD on muscle, but little is known about the underlying mechanisms. Here we systematically searched the literature to identify effects of active VD [1,25(OH)2D3] on skeletal muscle myogenesis in vitro, with no restriction on year of publication. Eligibility was assessed by strict inclusion/exclusion criteria and agreed by two independent investigators. Twelve relevant pa-pers were identified using four different cell types (C2C12, primary mouse satellite cells, primary chick myoblasts, and primary human myoblasts) and a range of myogenic markers (myoD, myogenin, creatine kinase, myosin heavy chain, and myotube size). A clear inhibitory effect of 1,25(OH)2D3 on proliferation was reported, while the effects on the different stages of differentiation were less consistent probably due to variation in cell type, time points and doses of 1,25(OH)2D3 used. However, myotube size was consistently increased by 1,25(OH)2D3. Overall, the evidence suggests that 1,25(OH)2D3 inhibits proliferation and promotes differentiation of myoblasts, but future studies should use time courses to gain a clearer understanding.

Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain.

VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well as in the adult.

The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo.

INTRODUCTION: The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. NEW METHOD: To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. RESULTS: Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. COMPARISON WITH OLD METHOD: The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. CONCLUSION: The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects.

Effects of manipulating hypothalamic triiodothyronine concentrations on seasonal body weight and torpor cycles in Siberian hamsters.

Siberian hamsters display photoperiodically regulated annual cycles in body weight, appetite, and reproduction. Previous studies have revealed a profound up-regulation of type 3 deiodinase (DIO3) mRNA in the ventral ependyma of the hypothalamus associated with hypophagia and weight loss in short-day photoperiods. DIO3 reduces the local availability of T(3), so the aim of this study was to test the hypothesis that decreased hypothalamic T(3) availability underlies the short-day-induced catabolic state. The experimental approach was to determine whether a local increase in T(3) in the hypothalamus of hamsters exposed to short days could reverse the behavioral and physiological changes induced by this photoperiod. In study 1, microimplants releasing T(3) were placed bilaterally into the hypothalamus. This treatment rapidly induced a long-day phenotype including increased appetite and body weight within 3 wk of treatment and increased fat mass and testis size by the end of the 10-wk study period. In study 2, hypothalamic T(3) implants were placed into hamsters carrying abdominal radiotelemetry implants. Again body weight increased significantly, and the occurrence of winter torpor bouts was dramatically decreased to less than one bout per week, whereas sham-implanted hamsters entered torpor up to six times a week. Our findings demonstrate that increased central T(3) induces a long-day metabolic phenotype, but in neither study was the molt cycle affected, so we infer that we had not disrupted the initial detection of photoperiod. We conclude that hypothalamic thyroid hormone availability plays a key role in seasonal regulation of appetite, body weight, and torpor.

VGF-derived peptide, TLQP-21, regulates food intake and body weight in Siberian hamsters.

The Siberian hamster survives winter by decreasing food intake and catabolizing abdominal fat reserves, resulting in a sustained, profound loss of body weight. VGF gene expression is photoperiodically regulated in the hypothalamus with significantly higher expression in lean Siberian hamsters. The aim of this study was to investigate the role of VGF in regulating these seasonal cycles by determining the effects of a VGF-derived peptide (TLQP-21) on food intake and body weight. Acute intracerebroventricular administration of TLQP-21 decreased food intake, and chronic treatment caused a sustained reduction in food intake and body weight and decreased abdominal fat depots. Behavioral analysis revealed that TLQP-21 reduced meal size but not the frequency of feeding bouts, suggesting a primary action on satiety. Hamsters treated with TLQP-21 lost a similar amount of weight as a pair-fed group in which food intake was matched to that of the TLQP-21-treated group. Central or peripheral treatment with TLQP-21 did not produce a significant effect on resting metabolic rate. We conclude that the primary action of TLQP-21 is to decrease food intake rather than increase energy expenditure. TLQP-21 treatment caused a decrease in UCP-1 mRNA in brown adipose tissue, but hypothalamic expression of orexigenic and anorexigenic neuropeptide genes remained unchanged after TLQP-21 treatment, although compensatory increases in NPY and AgRP mRNA were observed in the pair-fed hamsters. The effects of TLQP-21 administration are similar to those in hamsters in short days, suggesting that increased VGF activity may contribute to the hypophagia that underlies the seasonal catabolic state.

Effects of fatty acids on skeletal muscle cell differentiation in vitro.

Previous studies have shown stimulatory effects of linoleic acid (LA, C18:2) on differentiation of rat muscle cells in culture (Allen et al. 1985), but there appears to be little investigation of the effects of other fatty acids. The present study therefore compared the effects of different fatty acids on muscle cell differentiation in vitro. L6 myoblasts were cultured (Dulbecco's Modified Eagles Medium + 10 % fetal calf serum) in six-well plates until 80 % confluent (day 0). Cells were then either harvested or the medium switched to differentiation medium (Dulbecco's Modified Eagles Medium+2 % horse serum), supplemented with fatty acid or drug treatments. Cells were harvested on days 0-5 and assayed for creatine kinase (CK), protein and DNA contents, to give a measure of differentiation (CK/DNA). Initial studies indicated a stimulatory effect of the cis9,trans11 (c9,t11) isomer of conjugated linoleic acid (CLA) relative to control. By contrast, the trans10,cis12 (t10,c12) isomer of CLA inhibited differentiation. Further experiments indicated that inhibition of differentiation by the t10,c12 CLA isomer was dose-dependent (up to 200 microm) and may be via increased cell proliferation. LA and c9,t11 CLA stimulated differentiation at low concentrations (up to 50 microm), but inhibited differentiation at high concentrations (200 microm). In contrast, oleic acid stimulated differentiation at all concentrations, whereas the saturated fatty acid, palmitic acid, had no effect. The mechanism appeared not to involve either peroxisome proliferator-activated receptors alpha or gamma. The data suggest that only unsaturated fatty acids have an effect and the presence or absence of a cis-9 double bond may be important.