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.

Histaminergic regulation of seasonal metabolic rhythms in Siberian hamsters.

We investigated whether histaminergic tone contributes to the seasonal catabolic state in Siberian hamsters by determining the effect of ablation of histaminergic neurons on food intake, metabolic rate and body weight. A ribosomal toxin (saporin) conjugated to orexin-B was infused into the ventral tuberomammillary region of the hypothalamus, since most histaminergic neurons express orexin receptors. This caused not only 75-80% loss of histaminergic neurons in the posterior hypothalamus, but also some loss of other orexin-receptor expressing cells e.g. MCH neurons. In the long-day anabolic state, lesions produced a transient post-surgical decrease in body weight, but the hamsters recovered and maintained constant body weight, whereas weight gradually increased in sham-lesioned hamsters. VO(2) in the dark phase was significantly higher in the lesioned hamsters compared to shams, and locomotor activity also tended to be higher. In a second study in short days, sham-treated hamsters showed the expected seasonal decrease in body weight, but weight remained constant in the lesioned hamsters, as in the long-day study. Lesioned hamsters consumed more during the early dark phase and less during the light phase due to an increase in the frequency of meals during the dark and decreased meal size during the light, and their cumulative food intake in their home cages was greater than in the control hamsters. In summary, ablation of orexin-responsive cells in the posterior hypothalamus blocks the short-day induced decline in body weight by preventing seasonal hypophagia, evidence consistent with the hypothesis that central histaminergic mechanisms contribute to long-term regulation of body weight.

Prokineticin 2 is a hypothalamic neuropeptide that potently inhibits food intake.

OBJECTIVE: Prokineticin 2 (PK2) is a hypothalamic neuropeptide expressed in central nervous system areas known to be involved in food intake. We therefore hypothesized that PK2 plays a role in energy homeostasis. RESEARCH DESIGN AND METHODS: We investigated the effect of nutritional status on hypothalamic PK2 expression and effects of PK2 on the regulation of food intake by intracerebroventricular (ICV) injection of PK2 and anti-PK2 antibody. Subsequently, we investigated the potential mechanism of action by determining sites of neuronal activation after ICV injection of PK2, the hypothalamic site of action of PK2, and interaction between PK2 and other hypothalamic neuropeptides regulating energy homeostasis. To investigate PK2's potential as a therapeutic target, we investigated the effect of chronic administration in lean and obese mice. RESULTS: Hypothalamic PK2 expression was reduced by fasting. ICV administration of PK2 to rats potently inhibited food intake, whereas anti-PK2 antibody increased food intake, suggesting that PK2 is an anorectic neuropeptide. ICV administration of PK2 increased c-fos expression in proopiomelanocortin neurons of the arcuate nucleus (ARC) of the hypothalamus. In keeping with this, PK2 administration into the ARC reduced food intake and PK2 increased the release of alpha-melanocyte-stimulating hormone (alpha-MSH) from ex vivo hypothalamic explants. In addition, ICV coadministration of the alpha-MSH antagonist agouti-related peptide blocked the anorexigenic effects of PK2. Chronic peripheral administration of PK2 reduced food and body weight in lean and obese mice. CONCLUSIONS: This is the first report showing that PK2 has a role in appetite regulation and its anorectic effect is mediated partly via the melanocortin system.

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.

Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin.

Intracerebroventricular (ICV) administration of Neuromedin U (NMU), a hypothalamic neuropeptide, or leptin, an adipostat hormone released from adipose tissue, reduces food intake and increases energy expenditure. Leptin stimulates the release of NMU in vitro, and NMU expression is reduced in models of low or absent leptin. We investigated the role of NMU in mediating leptin-induced satiety. ICV administration of anti-NMU immunoglobulin G (IgG) (5 nmol) to satiated rats significantly increased food intake 4 h after injection, an effect seen for

Hypothalamic cocaine- and amphetamine-regulated transcript (CART) and agouti-related protein (AgRP) neurons coexpress the NOP1 receptor and nociceptin alters CART and AgRP release.

Nociceptin or orphanin FQ (N/OFQ) and its receptor NOP1 are expressed in hypothalamic nuclei involved in energy homeostasis. N/OFQ administered by intracerebroventricular or arcuate nucleus (ARC) injection increases food intake in satiated rats. The mechanisms by which N/OFQ increases food intake are unknown. We hypothesized that N/OFQ may regulate hypothalamic neurons containing peptides involved in the control of food intake such as cocaine- and amphetamine-regulated transcript (CART), alphaMSH, neuropeptide Y (NPY), and agouti-related protein (AgRP). We investigated the ability of N/OFQ to alter the release of CART, alphaMSH, NPY, and AgRP using ex vivo medial basal hypothalamic explants. Incubation of hypothalamic explants with N/OFQ (1, 10, 100 nM) resulted in significant changes in CART and AgRP release. One hundred nanomoles N/OFQ caused a 33% decrease in release of CART (55-102) immunoreactivity (IR) and increased release of AgRP-IR to 163% but produced no change in either alphaMSH-IR or NPY-IR. Double immunocytochemistry/in situ hybridization demonstrated that CART-IR and NOP1 mRNA are colocalized throughout the hypothalamus, in particular in the paraventricular nucleus, lateral hypothalamus, zona incerta, and ARC, providing an anatomical basis for N/OFQ action on CART release. Dual in situ hybridization demonstrated that AgRP neurons in the ARC also express the NOP1 receptor. Our data suggest that nociceptin via the NOP1 receptor may increase food intake by decreasing the release of the anorectic peptide CART and increasing the release of the orexigenic peptide AgRP.