Key Components of Human Myofibre Denervation and Neuromuscular Junction Stability are Modulated by Age and Exercise

The decline in muscle mass and function with age is partly caused by a loss of muscle fibres through denervation. The purpose of this study was to investigate the potential of exercise to influence molecular targets involved in neuromuscular junction (NMJ) stability in healthy elderly individuals. Participants from two studies (one group of 12 young and 12 elderly females and another group of 25 elderly males) performed a unilateral bout of resistance exercise. Muscle biopsies were collected at 4.5 h and up to 7 days post exercise for tissue analysis and cell culture. Molecular targets related to denervation and NMJ stability were analysed by immunohistochemistry and real-time reverse transcription polymerase chain reaction. In addition to a greater presence of denervated fibres, the muscle samples and cultured myotubes from the elderly individuals displayed altered gene expression levels of acetylcholine receptor (AChR) subunits. A single bout of exercise induced general changes in AChR subunit gene expression within the biopsy sampling timeframe, suggesting a sustained plasticity of the NMJ in elderly individuals. These data support the role of exercise in maintaining NMJ stability, even in elderly inactive individuals. Furthermore, the cell culture findings suggest that the transcriptional capacity of satellite cells for AChR subunit genes is negatively affected by ageing

Mutual stimulatory signaling between human myogenic cells and rat cerebellar neurons

Abstract Insight into the bidirectional signaling between primary human myogenic cells and neurons is lacking. For this purpose, human myogenic cells were derived from the semitendinosus and gracilis muscles of five healthy individuals and co‐cultured with cerebellar granule neurons from two litters of 7‐day‐old Wistar rat pups, in muscle medium or neural medium, alongside monocultures of myogenic cells or neurons. RT‐PCR was performed to determine human mRNA levels of GAPDH, Ki67, myogenin, and MUSK, and the acetylcholine receptor subtypes CHRNA1, CHRNB1, CHRNG, CHRND, and CHRNE, and rat mRNA levels of GAPDH, Fth1, Rack1, vimentin, Cdh13, and Ppp1r1a. Immunocytochemistry was used to evaluate neurite outgrowth (GAP43) in the presence and absence of myogenic cells. Co‐culture with primary neurons lead to higher myogenic cell gene expression levels of GAPDH, myogenin, MUSK, CHRNA1, CHRNG, and CHRND, compared to myogenic cells cultured alone. It appeared that neurons preferentially attached to myotubes and that neurite outgrowth was enhanced when neurons were cultured with myogenic cells compared to monoculture. In neural medium, rat mRNA levels of GAPDH, vimentin, Cdh13, and Ppp1r1a were greater in co‐culture, versus monoculture, whereas in muscle medium co‐culture lead to lower levels of Fth1, Rack1, vimentin, and Cdh13 than monoculture. These findings demonstrate mutually beneficial stimulatory signaling between rat cerebellar granule neurons and human myogenic cells, providing support for an active role for both the neuron and the muscle cell in stimulating neurite growth and myogenesis. Bidirectional muscle nerve signaling

mTORC2 and AMPK differentially regulate muscle triglyceride content via Perilipin 3.

OBJECTIVE: We have recently shown that acute inhibition of both mTOR complexes (mTORC1 and mTORC2) increases whole-body lipid utilization, while mTORC1 inhibition had no effect. Therefore, we tested the hypothesis that mTORC2 regulates lipid metabolism in skeletal muscle. METHODS: Body composition, substrate utilization and muscle lipid storage were measured in mice lacking mTORC2 activity in skeletal muscle (specific knockout of RICTOR (Ric mKO)). We further examined the RICTOR/mTORC2-controlled muscle metabolome and proteome; and performed follow-up studies in other genetic mouse models and in cell culture. RESULTS: Ric mKO mice exhibited a greater reliance on fat as an energy substrate, a re-partitioning of lean to fat mass and an increase in intramyocellular triglyceride (IMTG) content, along with increases in several lipid metabolites in muscle. Unbiased proteomics revealed an increase in the expression of the lipid droplet binding protein Perilipin 3 (PLIN3) in muscle from Ric mKO mice. This was associated with increased AMPK activity in Ric mKO muscle. Reducing AMPK kinase activity decreased muscle PLIN3 expression and IMTG content. AMPK agonism, in turn, increased PLIN3 expression in a FoxO1 dependent manner. PLIN3 overexpression was sufficient to increase triglyceride content in muscle cells. CONCLUSIONS: We identified a novel link between mTORC2 and PLIN3, which regulates lipid storage in muscle. While mTORC2 is a negative regulator, we further identified AMPK as a positive regulator of PLIN3, which impacts whole-body substrate utilization and nutrient partitioning

Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger

The expression of inulinolytic genes in Aspergillus niger is co-regulated and induced by inulin and sucrose. We have identified a positive acting transcription factor InuR, which is required for the induced expression of inulinolytic genes. InuR is a member of the fungal specific class of transcription factors of the Zn(II)2Cys6 type. Involvement of InuR in inulin and sucrose metabolism was suspected because of the clustering of inuR gene with sucB, which encodes an intracellular invertase with transfructosylation activity and a putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene resulted in a strain displaying a severe reduction in growth on inulin and sucrose medium. Northern analysis revealed that expression of inulinolytic and sucrolytic genes, e.g., inuE, inuA, sucA, as well as the putative sugar transporter gene (An15g00310) is dependent on InuR. Genome-wide expression analysis revealed, three additional putative sugar transporters encoding genes (An15g04060, An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR dependent way. In silico analysis of the promoter sequences of strongly InuR regulated genes suggests that InuR might bind as dimer to two CGG triplets, which are separated by eight nucleotides

Resistance training and insulin action in humans: effects of de-training

Aerobic endurance training increases insulin action in skeletal muscle, but the effect of resistance training has not been well described. Controversy exists about whether the effect of resistance training is merely due to an increase in muscle mass. We studied the effect of cessation of resistance training in young, healthy subjects by taking muscle biopsies and measuring insulin-mediated whole body and leg glucose uptake rates after 90 days of heavy resistance training (T) and again after 90 days of de-training (dT). Data on leg glucose uptake were expressed relative to accurate measures of leg muscle mass by MRI scanning. Muscle strength (239 ± 43 vs. 208 ± 33 N m), quadriceps area (8463 ± 453 vs. 7763 ± 329 mm2) and glycogen content (458 ± 22 vs. 400 ± 26 mmol (kg dry weight muscle)−1) decreased, while myosin heavy chain isoform IIX increased 4-fold in dT vs. T, respectively (all P < 0.05). GLUT4 mRNA levels and enzyme activities and mRNA levels of glycolytic, lipolytic and glyconeogenic enzymes did not change with de-training. Likewise, capillary density did not change. Whole body glucose uptake decreased 11 % and leg glucose uptake decreased from 75 ± 11 (T) to 50 ± 6 (dT) nmol min−1 (mm muscle)−2 (P < 0.05) at maximal insulin, the latter decrease being due to decreased arterio-femoral venous glucose extraction. The decrease was mainly due to reduced non-oxidative glucose disposal. We have thus shown that 90 days after the termination of heavy resistance training, insulin-mediated glucose uptake rates per unit of skeletal muscle have decreased significantly