The effect of estrogen on Akt signalling and protein synthesis in C2C12 mouse skeletal myotubes

The maintenance of skeletal muscle mass is a critical component of health in both chronic wasting diseases and aging. A considerable amount of progress has been made in the understanding of the signalling pathways that mediate skeletal muscle hypertrophy and atrophy. Akt is seen as a key molecular protein involved in the maintenance of skeletal muscle mass as it has the dual ability to positively influence protein syntheses and negatively regulate protein degradation in its active state (Glass, 2003). Potential mechanisms which may assist with maintaining skeletal muscle mass are the estrogen hormones. Estrogens increase the proliferation of mouse and rat myoblasts and can also attenuate immobilization-induced skeletal muscle atrophy in rats in vivo (Kahlert et al., 1997). No studies have investigated the effect of estrogens on the activation of skeletal muscle hypertrophy and atrophy signalling pathways. Estrogens may contribute to maintaining skeletal muscle mass via their activation of the Akt signalling pathways. Therefore, the aims of the present study were to determine if treatment of C2C12 myotubes with either 17β-estrodiol or estrone increases the activity of Akt and its downstream anabolic signalling proteins, GSK, p70s6k and 4E-BP1 and decreases its catabolic stimulating targets, FOXO, atrogin-1 and MuRF-1. A secondary aim was to determine if this was associated with an increased rate of protein synthesis.

C2C12 myotubes were incubated at 37°C in serum free DMEM without phenol red containing 10 000 units/ml penicillin, 10 000 μg/ml streptomycin, and 250μg/ml amphotericin B for 24h. Myotubes were then stimulated with 17-β estradiol (10nM) for 24h. Phosphorylated and total proteins for Akt, p70S6k, GSK3β, 4E-BP1, FOXO and atrogin-1 were measured using western blotting techniques. Atrogin-1 and MuRF1 mRNA levels were measured using real time-PCR. Protein synthesis rates were measured by incorporation of [3H]-tyrosine into the myotubes during the last hour of treatment.

Compared to control myotubes, treatment with 17β-estradiol increased the ratio of phosphorylated to total protein contents for Akt, GSK-3β and P70s6k by, 1.62, 1.53 and 2.2 fold, respectively (n=6 per group; p < 0.05). There was, however, no difference in the ratios of phosphorylated to total 4E-BP1 or Foxo3a or Atrogin-1 and MuRF1 mRNA. Protein synthesis rates remained unchanged.

This study demonstrates that in C2C12 mouse myotubes, 17β-estradiol treatment increases the phosphorylation of the hypertrophy signalling protein, Akt, and its downstream hypertrophy signalling targets, GSK-3β and P70s6k; no associated changes in protein synthesis were observed. Future studies should investigate the ability of 17β-estradiol to activate these proteins in a model of myotube catabolism and to determine if protein degradation is attenuated.

NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle

Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS-/-) or neuronal NOS (nNOS-/-) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS-/-, nNOS-/- and C57Bl6 (CON) mice (16.3 ± 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min-1, 5 grade) for 60 min (n = 8 per group) and were killed 6 h after exercise. Other eNOS-/-, nNOS-/- and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS-/- mice had significantly higher nNOS protein and nNOS-/- mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2α and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS-/- mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1α mRNA higher in the EDL. Also, eNOS-/- mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC-1α mRNA in soleus and EDL and NRF2α mRNA in the EDL to a similar extent in all genotypes. In addition, short-term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.

Developmental changes in the expression of creatine synthesizing enzymes and creatine transporter in a precocial rodent, the spiny mouse

Background : Creatine synthesis takes place predominately in the kidney and liver via a two-step process involving AGAT (L-arginine:glycine amidinotransferase) and GAMT (guanidinoacetate methyltransferase). Creatine is taken into cells via the creatine transporter (CrT), where it plays an essential role in energy homeostasis, particularly for tissues with high and fluctuating energy demands. Very little is known of the fetal requirement for creatine and how this may change with advancing pregnancy and into the early neonatal period. Using the spiny mouse as a model of human perinatal development, the purpose of the present study was to comprehensively examine the development of the creatine synthesis and transport systems.

Results : The estimated amount of total creatine in the placenta and brain significantly increased in the second half of pregnancy, coinciding with a significant increase in expression of CrT mRNA. In the fetal brain, mRNA expression of AGAT increased steadily across the second half of pregnancy, although GAMT mRNA expression was relatively low until 34 days gestation (term is 38–39 days). In the fetal kidney and liver, AGAT and GAMT mRNA and protein expression were also relatively low until 34–37 days gestation. Between mid-gestation and term, neither AGAT or GAMT mRNA or protein could be detected in the placenta.

Conclusion : Our results suggest that in the spiny mouse, a species where, like the human, considerable organogenesis occurs before birth, there appears to be a limited capacity for endogenous creatine synthesis until approximately 0.9 of pregnancy. This implies that a maternal source of creatine, transferred across the placenta, may be essential until the creatine synthesis and transport system matures in preparation for birth. If these results also apply to the human, premature birth may increase the risk of creatine deficiency.

A cetylcholinesterase is increased in mouse neuronal and astrocyte cultures after treatment with b-amyloid peptides

The cellular origin of the acetylcholinesterase (AChE) associated with amyloid plaques in the Alzheimer’s disease (AD) brain is unknown. In this study we report that amyloid β-peptides (Aβ) increased AChE levels in both neuronal and astrocytic primary cultures, supporting the possibility that both neurons and glia may make a direct contribution to the pool of AChE seen around amyloid deposits in the AD brain.

Mouse B7-H3 induces antitumor immunity

Members of the B7 family costimulate the proliferation of lymphocytes during the initiation and maintenance of antigen-specific humoral and cell-mediated immune responses. While B7-1 and -2 are restricted to lymphoid tissues, and activate naïve T cells, recently identified members including B7-H2 and -H3 are widely expressed on nonlymphoid tissues, and regulate effector lymphocytes in the periphery. B7-H3 has properties that suggested it may display antitumor activity, including the ability to stimulate Th1 and cytotoxic T-cell responses. Here, we test this notion by determining whether intratumoral injection of an expression plasmid encoding a newly described mouse homologue of B7-H3 is able to eradicate EL-4 lymphomas. Intratumoral injection of a mouse B7-H3 pcDNA3 expression plasmid led to complete regression of 50% tumors, or otherwise significantly slowed tumor growth. Mice whose tumors completely regressed resisted a challenge with parental tumor cells, indicating systemic immunity had been generated. B7-H3-mediated antitumor immunity was mediated by CD8(+) T and NK cells, with no apparent contribution from CD4(+) T cells. In summary, the results indicate that B7-H3 interactions may play a role in regulating cell-mediated immune responses against cancer, and that B7-H3 is a potential therapeutic tool.

Molecular physiology of water balance in the Spinifex hopping mouse

Many animals live in the deserts of Australia and survive without drinking water. This research examined desert adaptations in the Spinifex hopping mouse (Notomys alexis), and is the first study to demonstrate how many genes and exzymes in the kidney are controlled to maintain water balance in the absence of drinking water.

Conservation genetics of the New Holland mouse Pseudomys novaehollandiae, Waterhouse, 1843 (Rodentia: Muridae) : an analysis of mitochondrial DNA control region variation

An investigation of the genetic diversity of New Holland mouse populations using DNA. Ten distinct restriction enzyme fragment patterns or haplotypes were detected. From the fragment patterns, estimates of genetic divergence between the haplotypes revealed a degree of genetic structuring within New Holland mouse with four population assemblages apparent.

Research on human embryos and cloning : difficulties of legislating in a changing environment and model approaches to regulation

It is difficult to regulate rapidly changing fields of science. New technologies are not anticipated and legislation becomes inadequate. Legislative definitions are also problematic. This article begins with consideration of such difficulties in the context of research on human embryos and cloning. It considers problems with past legislative definitions in Australia, the new regulatory regime, and whether that regime now sets clear boundaries. It is found that problems still exist – some terms are not adequately defined and boundaries for research prove unclear. Three regulatory approaches are therefore discussed. Legislation based on strict definitions is compared to a legislative model that leaves terms undefined. The third model – which combines framework legislation with the oversight of a regulatory authority – is seen as most suitable. However, problems with this model are recognised and suggestions made regarding how to ensure the “framework” remains workable and effective.

Regulatory design strategies and enforcement approaches for research involving human embryos and cloning in Australia and the United Kingdom – time for a change.

This paper examines regulatory design strategies and enforcement approaches in the context of the UK and Australia’s regulation of research involving human embryos and cloning. The aim is to discuss current regulation in view of the impending review of the Research Involving Human Embryos Act 2002 (Cth) and the Prohibition of Human Reproductive Cloning Act 2002 (Cth). It is argued that the type of regulation used in relation to those who are licensed to research in Australia is unsuitable due to an over-emphasis on deterrence and the authoritarian approach taken by regulatory bureaucracies. The cost and efficiency of the current system is also questioned. The central thesis is that a co-regulatory system that combines the existing framework legislation with self-regulation should be adopted for licence holders. Such regulation of licence holders should include responsive regulatory strategies. ‘Command and control’ design strategies and deterrence approaches present in the current regulatory systems for breaches of legislation by non-licence holders and serious breaches by licence holders should be maintained.