Carole Migné

1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes

SCOPE In recent years, there has been a growing body of evidence pointing to an effect of vitamin D on muscle mass and function. Our aim was to investigate the combined effect of 1,25(OH)2-vitamin D3 (1,25(OH)2D3) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube. METHODS AND RESULTS After differentiation, myotubes were cultured in 1,25(OH)2D3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by L-[1-(13) C]valine and puromycin in presence or not of leucine and insulin, and protein FSR was determined by measuring tracer enrichments and puromycin incorporation in proteins, respectively. Protein expression and phosphorylation state of insulin receptor (IR), Akt, GSK3, mTOR, p70 S6 kinase, rpS6, and 4EBP1 were measured by Western blot. Transcript levels of IR and 1,25(OH)2D3 receptor (VDR) were determined by qPCR. 1,25(OH)2D3 (10 nM) with leucine and insulin increased protein FSR in C2C12 myotubes (14-16%). IR and VDR mRNA expression was increased with 1,25(OH)2D3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)2D3. CONCLUSION 1,25(OH)2D3 sensitizes the Akt/mTOR-dependant pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in murine C2C12 skeletal myotubes.

Effect of modification of the O-methyltransferase activity on cell wall composition, ultrastructure and degradability of transgenic tobacco

The effect of O-methyltransferase (OMT) cDNA modulation on cell wall composition, ultrastructure and rumen degradability was measured on transgenic tobacco (Nicotiana tabacum). The expression of OMT cDNA in antisense orientation (AS) inhibited OMT activity by 92% whereas expression of sense constructs led to plants either co-suppressed (CS, 98% inhibition) or overexpressing OMT activity. The cell wall residues of stems were analysed for lignin content, products of nitrobenzene oxidation (NBO) and polysaccharide content. Degradability was determined by a cellulase method. Sections of stem were stained by acid phloroglucinol and Maule reactant. Stem samples were incubated in the rumen for 8, 24 and 48 h and observed by scanning electron microscopy (SEM). Compared to controls, OMT-depleted stems showed decreased hemicellulose content but unchanged lignin content. In contrast, syringyl units decreased by 40 and 90% in AS and CS samples respectively and NBO content followed a similar trend. Dry matter cellulase degradability was significantly improved by 3.5 and 5.6 percentage units in AS and CS samples respectively. SEM showed a greater bacterial colonisation in these samples and indicated a higher rate of rumen degradability in CS tissues than in controls. Overexpressing plants had a composition and a degradability similar to that of controls. For all the plants studied, the improvements in dry matter degradability were closely linked to the syringyl to guaiacyl ratio or to the NBO content. The modifications observed in down-regulated tobacco were similar to those produced by bm3 maize mutation, but without lignin decrease. Genetic modifications should therefore be considered for improving forage digestibility.

Muscle ectopic fat deposition contributes to anabolic resistance in obese sarcopenic old rats through eIF2α activation.

Obesity and aging are characterized by decreased insulin sensitivity (IS) and muscle protein synthesis. Intramuscular ceramide accumulation has been implicated in insulin resistance during obesity. We aimed to measure IS, muscle ceramide level, protein synthesis, and activation of intracellular signaling pathways involved in translation initiation in male Wistar young (YR, 6‐month) and old (OR, 25‐month) rats receiving a low‐ (LFD) or a high‐fat diet (HFD) for 10 weeks. A corresponding cellular approach using C2C12 myotubes treated with palmitate to induce intracellular ceramide deposition was taken. A decreased ability of adipose tissue to store lipids together with a reduced adipocyte diameter and a development of fibrosis were observed in OR after the HFD. Consequently, OR fed the HFD were insulin resistant, showed a strong increase in intramuscular ceramide level and a decrease in muscle protein synthesis associated with increased eIF2α phosphorylation. The accumulation of intramuscular lipids placed a lipid burden on mitochondria and created a disconnect between metabolic and regulating pathways in skeletal muscles of OR. In C2C12 cells, palmitate‐induced ceramide accumulation was associated with a decreased protein synthesis together with upregulated eIF2α phosphorylation. In conclusion, a reduced ability to expand adipose tissues was found in OR, reflecting a lower lipid buffering capacity. Muscle mitochondrial activity was affected in OR conferring a reduced ability to oxidize fatty acids entering the muscle cell. Hence, OR were more prone to ectopic muscle lipid accumulation than YR, leading to decreased muscle protein anabolism. This metabolic change is a potential therapeutic target to counter sarcopenic obesity.

Contrarily to whey and high protein diets, dietary free leucine supplementation cannot reverse the lack of recovery of muscle mass after prolonged immobilization during ageing

Key points  •  During ageing, there is a lack of recovery of muscle mass following immobilization. •  We showed, in old rats, an ‘anabolic resistance’ of muscle protein synthesis to food intake during immobilization and only a slight increase of protein synthesis during the recovery, which explain a poor muscle nitrogen balance that is insufficient to induce a muscle mass gain. •  A supplementation with free leucine, an essential amino acid known to stimulate muscle protein metabolism, was efficient in inducing a greater anabolism but failed to induce muscle mass recovery. •  This discrepancy was explained by a ‘desynchronization’ between the leucine signal and amino acids coming from dietary protein digestion. •  An induction of a larger increase and a longer availability of amino acids in the postprandial state with rich‐protein leucine (i.e. whey) and high protein diets were efficient in inducing a muscle mass recovery after immobilization.

Preferential transfection of adult mouse neural stem cells and their immediate progeny in vivo with polyethylenimine.

The subventricular zone of the adult mammalian brain harbors the neural stem cell population with potential neural regeneration and repair capacity. We describe a nonviral technique to preferentially transfect in vivo the adult neural stem cell population and its immediate progeny based on intraventricular injection of PEI/DNA complexes. The transfected population was identified by cellular and ultra-structural evidence showing their proliferating status and expression of the specific markers GFAP and nestin. Stable activation of the lacZ reporter by cre-recombinase transfection in R26R mice demonstrated survival and migration of stem cell derivatives three months after injection. Apoptosis is thought to be the most common fate of the stem cell progeny. Overexpression of Bcl-X(L) increased number and survival time of transduced progenitors and decreased the frequency of cells immunopositive for activated Caspase-3. This method thus provides selective targeting of the stem cell population and should allow an in-depth understanding of their biology.

Time-course changes of muscle protein synthesis associated with obesity-induced lipotoxicity

•  Prolonged obesity leads to ectopic lipid accumulation in non‐adipose tissues, particularly in skeletal muscles, inducing metabolic dysfunctions (reduced glucose uptake, mitochondria dysfunction, lipotoxicity). •  Several studies in humans and rodents have shown that obesity induces a short‐term increase in fat‐free mass but a long‐term decrease in skeletal muscle mass. •  We investigated the mechanisms potentially involved in muscle loss by measuring simultaneously protein synthesis and lipid infiltration in different types of skeletal muscles, during the development of obesity. •  Our results show that protein synthesis rate in glycolytic muscles increased together with muscle mass during the early phase of obesity development, whereas it decreased later. Reduced protein synthesis rate was associated with a high lipid accumulation in glycolytic muscles. •  These results suggest that lipid accumulation in muscles during prolonged obesity is deleterious for amino acid incorporation in skeletal muscle proteins, and thus indirectly for muscle mass.

Oleate-enriched diet improves insulin sensitivity and restores muscle protein synthesis in old rats

BACKGROUND & AIMS Age-related inflammation and insulin resistance (IR) have been implicated in the inability of old muscles to properly respond to anabolic stimuli such as amino acids (AA) or insulin. Since fatty acids can modulate inflammation and IR in muscle cells, we investigated the effect of palmitate-enriched diet and oleate-enriched diet on inflammation, IR and muscle protein synthesis (MPS) rate in old rats. METHODS Twenty-four 25-month-old rats were fed either a control diet (OC), an oleate-enriched diet (HFO) or a palmitate-enriched diet (HFP) for 16 weeks. MPS using labeled amino acids and mTOR activation were assessed after AA and insulin anabolic stimulation to mimic postprandial state. RESULTS IR and systemic and adipose tissue inflammation (TNFα and IL1β) were improved in the HFO group. Muscle genes controlling mitochondrial β-oxidation (PPARs, MCAD and CPT-1b) were up-regulated in the HFO group. AA and insulin-stimulated MPS in the HFO group only, and this stimulation was related to activation of the Akt/mTOR pathway. CONCLUSIONS The age-related MPS response to anabolic signals was improved in rats fed an oleate-enriched diet. This effect was related to activation of muscle oxidative pathways, lower IR, and a decrease in inflammation.

Fast proteins with a unique essential amino acid content as an optimal nutrition in the elderly: growing evidence.

BACKGROUND & AIMS Adequate protein intake is crucial to maintain body protein content in elderly subjects, but quality of dietary proteins should be also considered since amino acid composition and rate of protein digestion modulate amino acid availability. This study investigates whether the efficacy of optimal protein intake levels for protein retention in the elderly is influenced by protein quality. METHODS We investigated the effect of a 10-day adequate-protein (AP) or high-protein (HP) diet together with the protein source as caseins (CAS) or soluble milk proteins (PRO) on whole-body (WB) protein synthesis (PS) and protein breakdown (PB) in 4 groups of healthy elderly men (mean ± SEM: 71.8 ± 24.4 yr). The study consisted of two periods of 4 h each: a post-absorptive period and a postprandial period. The fed state was defined by consumption every 20 min and for 4 h, of either 15 g or 30 g of PRO or CAS. Steady-state WB and splanchnic leucine kinetics were measured using a continuous infusion of L-[1-13C]leucine in the postabsorptive state and L-[1-13C]leucine infusion plus oral L-[5,5,5-2H3]leucine in the postprandial state. RESULTS WB PS was stimulated by feeding only with HP diets, whereas WB PB corrected for splanchnic extraction showed a similar pattern of post-feeding decrease in all groups. Consequently, net leucine balance was greater in the postprandial state after HP meals than after AP meals, with PRO meals leading to a better postprandial leucine balance (3.63 ± 0.16 μmol kg FFM(-1) min(-1)vs. 2.77 ± 0.21 μmol kg FFM(-1) min(-1) for PRO HP and CAS HP, respectively; P = 0.005). CONCLUSION Postprandial protein retention was better improved in elderly men by an increase in protein intake when the protein supplementation was provided as fast-digesting proteins that induce high leucine availability.

Immunogold labelling of xylans and arabinoxylans in the plant cell walls of maize stem

Summary— Polyclonal antibodies against 4‐O‐methyl‐glucuronoxylan and α L‐1‐3 arabinofuranosyl poly‐β‐d‐1‐4‐xylopyranosyl were raised from rabbits. An immunocytochemical technique was used to localize xylans and arabinoxylans in the plant cell walls of the apical internode of two maize lines of different digestibility. The sclerenchyma, fibres and xylem (lignified tissues) and the parenchyma (non‐lignified tissue) were studied. The arabinoxylans were more heavily labelled than the xylans in the lignified tissues of the less digestible maize whereas in the more digestible line the labelling of the two polysaccharides was similar. The xylans and arabinoxylans were localized in the secondary cell wall. In both maize lines, labelling increased from the base upwards of the apical internode, reflecting the changes in growth stage.

Immunocytochemical localisation of para-coumaric acid and feruloyl-arabinose in the cell walls of maize stem

Two phenolic compounds, p-coumaric acid and feruloyl-arabinose, were localised by immunocytochemistry in the cell walls of the apical internode of two lines of maize (Co125 and W401) of different digestibility. The compounds were detected at two stages of cell maturity in the lignified tissues (sclerenchyma, fibres and xylem) and in the medullary parenchyma, which, in the samples studied, was not lignified. p-Coumaric acid is a phenolic acid associated with lignins, which confer resistance on plant cell walls to microbial degradation in the rumen. Feruloyl-arabinose is a compound associated with xylans, the principal hemicelluloses in Gramineae, which are potentially degradable. Labelling of p-coumaric acid decreased in both maize lines with cell age and as the cell walls became lignified. The mass of lignin deposited in the cell walls masked p-coumaric acid, thereby making it less accessible to the antibodies. There was an inverse relationship in the labelling of p-coumaric acid and feruloyl-arabinose. Feruloyl-arabinose was more heavily labelled as the plant cell walls matured in all the lignified tissues of both maize lines and in the parenchyma of the less digestible line. All tissues except the parenchyma were more heavily labelled with both sera in Co125, the more digestible line.