The intestine is a central regulator of metabolic homeostasis. Dietary inputs are absorbed through the gut, which senses their nutritional value and relays hormonal information to other organs to coordinate systemic energy balance. However, the specific gut hormones that communicate energy availability to target organs to induce appropriate metabolic and behavioral responses are poorly defined. Here we show that the enteroendocrine cells (EECs) of the Drosophila gut sense nutrient stress via the intracellular TOR pathway, and in response secrete the peptide hormone allatostatin C (AstC). Gut-derived AstC induces secretion of glucagon-like adipokinetic hormone (AKH) via its receptor AstC-R2, a homolog of mammalian somatostatin receptors, to coordinate food intake and energy mobilization. Loss of gut AstC or its receptor in the AKH-producing cells impairs lipid and sugar mobilization during fasting, leading to hypoglycemia. Our findings illustrate a nutrient-responsive endocrine mechanism that maintains energy homeostasis under nutrient-stress conditions, a function that is essential to health and whose failure can lead to metabolic disorders.

Objective: Duchenne muscular dystrophy (DMD) patients are often treated with glucocorticoids; yet their precise molecular action remains unknown. Methods: We investigated muscle biopsies from nine boys with DMD (aged: 7,6 +/- 2,8 yrs.) collected before and after three months of deflazacort treatment and compared them to eight healthy boys (aged: 5,3 +/- 2,4 yrs.). mRNA transcripts involved in activation of satellite cells, myogenesis, regeneration, adipogenesis, muscle growth and tissue inflammation were assessed. Serum creatine kinase (CK) levels and muscle protein expression by immunohistochemistry of selected targets were also analysed. Results: Transcript levels for ADIPOQ, CD68, CDH15, FGF2, IGF1R, MYF5, MYF6, MYH8, MYOD, PAX7, and TNF alpha were significantly different in untreated patients vs. normal muscle (p<0.05). Linear tests for trend indicated that the expression levels of treated patients were approaching normal values (p<0.05) following treatment (towards an increase; CDH15, C-MET, DLK1, FGF2, IGF1R, MYF5, MYF6, MYOD, PAX7; towards a decrease: CD68, MYH8, TNFa). Treatment reduced CK levels (p<0.05), but we observed no effect on muscle protein expression. Conclusions: This study provides insight into the molecular actions of glucocorticoids in DMD at the mRNA level, and we show that multiple regulatory pathways are influenced. This information can be important in the development of new treatments.

Objectives. The present study aimed to explore the effect of resistance training in patients with amyotrophic lateral sclerosis (ALS), a disease characterized by progressive motor neuron loss and muscle weakness. Materials and Methods. Following a 12-week “lead-in” control period, a population of ALS patients from Funen, Denmark, completed a 12-week resistance training program consisting of 2-3 sessions/week. Neuromuscular function (strength and power) and voluntary muscle activation (superimposed twitch technique) were evaluated before and after both control and training periods. Physical capacity tests (chair rise and timed up and go), the revised ALS functional rating scale (ALSFRS-R) scores, and muscle cross sectional area (histology) were also assessed. Results. Of twelve ALS patients assessed for eligibility, six were included and five completed the study. Training did not significantly affect the ALSFRS-R score, and loss of neuromuscular function (strength and power) increased following the training period. However, an improved functionality (chair rise) and an increase in greatly hypertrophied type II fibres combined with an increase in atrophied fibres following the training period compared to the control period were observed. Conclusion. In this small study, the present form of resistance training was unable to attenuate progressive loss of neuromuscular function in ALS, despite some changes in physical capacity and morphology.

The phytohormone ethylene plays a central role in development and senescence of climacteric flowers. In ornamental plant production, ethylene sensitive plants are usually protected against negative effects of ethylene by application of chemical inhibitors. In Campanula, flowers are sensitive to even minute concentrations of ethylene.

Depression is a highly heterogeneous disorder presumably caused by a combination of several factors ultimately causing the pathological condition. The genetic liability model of depression is likely to be of polygenic heterogeneity. miRNAs can regulate multiple genes simultaneously and therefore are candidates that align with this model. The habenula has been linked to depression in both clinical and animal studies, shifting interest towards this region as a neural substrate in depression. The goal of the present study was to search for alterations in miRNA expression levels in the medial and lateral habenula of rats exposed to the learned helplessness (LH) rat model of depression. Ten miRNAs showed significant alterations associating with their response to the LH paradigm. Of these, six and four miRNAs were significantly regulated in the MHb and LHb, respectively. In the MHb we identified miR-490, miR-291a-3p, MiR-467a, miR-216a, miR-18b, and miR-302a. In the LHb miR-543, miR-367, miR-467c, and miR-760-5p were significantly regulated. A target gene analysis showed that several of the target genes are involved in MAPK signaling, neutrophin signaling, and ErbB signaling, indicating that neurotransmission is affected in the habenula as a consequence of exposure to the LH paradigm.

Muscle weakness is considered the pivotal sign of amyotrophic lateral sclerosis (ALS). Knowledge about the skeletal muscle degeneration/regeneration process and the myogenic potential is limited in ALS patients. Therefore, we investigate these processes in a time course perspective by analysing skeletal muscle biopsies from ALS patients collected before and after a 12-week period of normal daily activities and compare these with healthy age-matched control tissue. We do this by evaluating mRNA and protein (immunohistochemical) markers of regeneration, neurodegeneration, myogenesis, cell cycle regulation, and inflammation. Our results show morphological changes indicative of active denervation and reinnervation and an increase in small atrophic fibres. We demonstrate differences between ALS and controls in pathways controlling skeletal muscle homeostasis, cytoskeletal and regenerative markers, neurodegenerative factors, myogenic factors, cell cycle determinants, and inflammatory markers. Our results on Pax7 and MyoD protein expression suggest that proliferation and differentiation of skeletal muscle stem cells are affected in ALS patients, and the myogenic processes cannot overcome the denervation-induced wasting.

Objectives: The iPlex (R) ADME PGx Pro Panel was developed to investigate 191 polymorphisms including single nucleotide polymorphisms (SNPs), insertion-deletions (INDELS), and copy number variations (CNV) relevant for absorption, distribution, metabolism, and excretion (ADME) of drugs. The purpose of this study was to perform a technical evaluation of the iPlex (R) ADME PGx Pro Panel by genotyping 50 unrelated Danes and estimate preliminary genotype frequencies among Danes.Design and methods: The investigations were performed by the use of PCR, single base extension (SBE) and Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS).Results: The typing quality of 161 SNP assays was categorized as well performing or acceptable, whereas 22 SNP assays were categorized as either questionable or unacceptable. The frequencies of the genotypes observed in the Danish population were compared to those of the European reference population from the 1000 Genome Project. Three SNPs (rs737865, rs35167514, and rs34305973) showed statistically significantly differences between the frequencies of the 1000 Genomes Europeans and the Danes. The CNV assays could only be used as a guideline.Conclusion: In conclusion, the iPlex (R) ADME PGx Pro Panel is a cost-effective way of genotyping genes relevant for ADME. (C) 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Trapezius myalgia is the most common type of chronic neck pain. While physical exercise reduces pain and improves muscle function, the underlying mechanisms remain unclear. Nitric oxide (NO) signaling is important in modulating cellular function, and a dysfunctional neuronal NO synthase (nNOS) may contribute to an ineffective muscle function. This study investigated nNOS expression and localization in chronically painful muscle. Forty-one women clinically diagnosed with trapezius myalgia (MYA) and 18 healthy controls (CON) were included in the case-control study. Subsequently, MYA were randomly assigned to either 10 weeks of specific strength training (SST, n=18), general fitness training (GFT, n=15), or health information (REF, n=8). Distribution of fiber type, cross-sectional area, and sarcolemmal nNOS expression did not differ between MYA and CON. However, MYA showed increased sarcoplasmic nNOS localization (18.8 ± 12 versus 12.8 ± 8%, P=0.049) compared with CON. SST resulted in a decrease of sarcoplasm-localized nNOS following training (before 18.1 ± 12 versus after 12.0 ± 12%; P=0,027). We demonstrate that myalgic muscle displays altered nNOS localization and that 10 weeks of strength training normalize these disruptions, which supports previous findings of impaired muscle oxygenation during work tasks and reduced pain following exercise.

Skeletal musle disuse has been shown to induce muscle atrophy rapidly in both young and old human individuals (1) as well as extracellular matrix (ECM) remodeling in young individuals (2). However, little is known about the effect of immobilization on ECM remodeling in older individuals. PURPOSE: To study the effect of aging on transcriptional regulatory signaling pathways involved in ECM remodeling with short-term immobilization. METHODS: Myofiber atrophy was induced by application of a knee-brace for a total period of 4 days in young (Y∼20 yrs, n=11) and aged (O∼70 yrs, n=11) individuals. Muscle biopsies of the vastus lateralis (VL) muscle were collected 1 week prior to immobilization and after 24h, 48h and 96h of immobilization. Expression levels of Col1A1, Col3A1, MMP2 and MMP9 mRNAs were determined using real-time RT-PCR and normalized to the Ribosomal Protein Large P0 (RPLP0) mRNA. RESULTS: In both age groups an increase in expression levels of Col1A1, Col3A1, MMP2 and MMP9 mRNA was observed after 24h. In contrast, expression levels of Col1A1 and Col3A1 mRNA were up regulated at 48h and 96h primarily in old individuals and an overall effect of age was observed. The expression level of MMP2 mRNA was not changed from pre at 48h and 96h. The expression level of MMP9 mRNA was up regulated in both age groups at all three time points. CONCLUSIONS: The present data demonstrates early (post 24h) signs of ECM remodeling in both young and old individuals. Moreover, the results point toward an age specific regulation of ECM in response to muscle disuse, with an up regulation of collagen I and III in old only after 48h and 96h of immobilisation. REFERENCES 1) Suetta C et al. Aging affects the transcriptional regulation of human skeletal muscle disuse atrophy. PLoS One. 2012;7(12) 2) Reich KA et al. Forty-eight hours of unloading and 24 h of reloading lead to changes in global gene expression patterns related to ubiquination and oxidative stress in humans. J Appl Physiol 109: 1404-1415, 2010.

Single Muscle Fiber Characteristics of The Oldest-Old Gregory Grosicki, Robert Standley, Kevin Murach, Ulrika Raue, Kiril Minchev, Paul Coen, Anne Newman, Steven Cummings, Tamara Harris, Stephen Kritchevsky, Bret Goodpaster, Scott Trappe, FACSM. Ball State University, Muncie, IN. Translational Research Institute for Metabolism and Diabetes, Orlando, FL. University of Pittsburgh, Pittsburgh, PA. California Pacific Medical Center Research Institute, San Francisco, CA. National Institute on Aging, Bethesda, MD. Wake Forest School of Medicine, Winston-Salem, NC. (Sponsor: Scott Trappe, FACSM) Email: gjgrosicki@gmail.com

The aim was to determine if the metabolic adaptations, particularly PGC‐1α and downstream metabolic genes were affected by restricting CHO following an endurance exercise bout in trained endurance athletes. A second aim was to compare baseline expression level of these genes to untrained. Elite endurance athletes (VO2max 66 ± 2 mL·kg−1·min−1, n = 15) completed 4 h cycling at ~56% VO2max. During the first 4 h recovery subjects were provided with either CHO or only H2O and thereafter both groups received CHO. Muscle biopsies were collected before, after, and 4 and 24 h after exercise. Also, resting biopsies were collected from untrained subjects (n = 8). Exercise decreased glycogen by 67.7 ± 4.0% (from 699 ± 26.1 to 239 ± 29.5 mmol·kg−1·dw−1) with no difference between groups. Whereas 4 h of recovery with CHO partly replenished glycogen, the H2O group remained at post exercise level; nevertheless, the gene expression was not different between groups. Glycogen and most gene expression levels returned to baseline by 24 h in both CHO and H2O. Baseline mRNA expression of NRF‐1, COX‐IV, GLUT4 and PPAR‐α gene targets were higher in trained compared to untrained. Additionally, the proportion of type I muscle fibers positively correlated with baseline mRNA for PGC‐1α, TFAM, NRF‐1, COX‐IV, PPAR‐α, and GLUT4 for both trained and untrained. CHO restriction during recovery from glycogen depleting exercise does not improve the mRNA response of markers of mitochondrial biogenesis. Further, baseline gene expression of key metabolic pathways is higher in trained than untrained.

Cellular senescence is an irreversible arrest of cell division, which could influence the regenerative potential of skeletal muscle stem cells (satellite cells) during aging. The molecular mechanism of senescence is complex and involves epigenetic control of the Polycomb repressive complexes, as well as CDNK2A (p16) and TP53 tumor mediated repression of cyclin dependent kinases and G1 cell cycle arrest. PURPOSE: To investigate the effect of ageing on satellite cell cycle regulation in human skeletal muscle undergoing atrophy and regrowth induced by short-term immobility and subsequent reloading. METHODS: Myofiber atrophy was induced by application of a knee-brace for a period of 4 days in young (Y, ~20 yrs, n=9) and aged (O, ~70 yrs, n=9) individuals. Muscle regrowth after atrophy was induced by 6 days of re-ambulation supplemented by one session of supervised unilateral resistance training for the disused leg 3 days after brace removal. Muscle biopsies (VL) were collected pre and at 1d, 2d and 4d of immobility and after additional 6 days of re-mobilization (10d). Protein and mRNA expression levels of CDNK2A (p16), CDKN1A (p21), CDKN1B (p27), TP53 and PCNA were determined using real-time RT-PCR and Western blotting, respectively. Satellite cell content was determined by immunohistochemical expression of Pax7. RESULTS: Satellite cell content increased in Y at 4d and 10d with no changes in O (p<0.05). p16 mRNA was upregulated at 2d and 4d in O compared to Y and at 10d in Y and O compared to pre (p<0.05). TP53 mRNA was upregulated at 2d in O and at 4d in Y and O compared to pre, while downregulated at 10d in Y and O compared to 4d (p<0.05). p27 mRNA was downregulated in Y and O at 4d and 10d compared to pre (p<0.05). p16 protein increased in O at 1d (7.2-fold) and 2d (3.9-fold) compared to Y and decreased to pre levels in Y and O at 10d (p<0.05). PCNA protein was upregulated in Y (5.5-fold) but blunted in O (1.6-fold) at 10d compared to pre (p<0.05). CONCLUSION: p16 and TP53 early (2-4 days) were selectively upregulated during immobility in O compared with Y subjects, suggesting that cellular senescense and SC cycle arrest could be implicated in the defective regenerative response in O compared to Y. Further analysis of epigenetic modifications may provide further explanation for the present findings.

Vascular endothelial growth factor (VEGF) is traditionally considered important for skeletal muscle angiogenesis. VEGF is released from vascular endothelium as well as the muscle cells in response to exercise. The mechanism and the physiological role of VEGF secreted from the muscle cells remain unclear. However, as VEGF is also considered very important for the regulation of vascular permeability, it is possible that metabolic stress may trigger muscle VEGF release. PURPOSE: To study the role of metabolic stress induced by glycogen-depleting exercise on skeletal muscle VEGF expression. METHODS: Fifteen males (age 27.0±0.8; VO2max 66.0±1.2 ml•kg-1•min-1) carried out 4h of cycling exercise supplied with H2O only followed by 4h of recovery with either carbohydrate (CHO) (n=8) or H2O (n=7) supplementation. Hereafter both groups received CHO. Muscle biopsies were collected pre and post as well as 4 and 24 h following exercise. Muscle glycogen was investigated along with expression levels of mRNA (real time RT-PCR) and protein (immunohistochemistry) of VEGF, VEGF receptor 2 (VEGFR-2) and the molecular chaperones heat shock proteins 27 and 70 (HSP27, HSP70). RESULTS: Double labelling of PAS and myosin heavy chain II (MHC II) or HSP70 in muscle sections demonstrated a pronounced depletion of glycogen in type I fibers and concurrent increase of HSP70 also in type I fibers post and 4h after exercise. Baseline mRNA levels of VEGF, VEGFR-2 and HSP70 correlated positively with percentage of type I fibers (p<0.05). Further, significant increases in mRNA were detected in both CHO and H2O for VEGF, VEGFR-2, and HSP70 (CHO only) post (2.2-3.7 fold, p<0.05) and after 4h of recovery (1.5-2.5 fold, p<0.05). VEGF and HSP70 protein expression increased post (∼35; ∼200 %, respectively, p< 0.05) and after 4h of recovery (∼60; ∼150 %, respectively, p< 0.05) primarily in type I fibers and around nuclei, while VEGFR-2 increased after 4h of recovery (40 %, p<0.05). Immunohistochemistry and electron microscopy revealed co-localization of VEGF and HSP70 after exercise. Generally, all data returned to pre-exercise levels by 24h irrespective of treatment. CONCLUSIONS: Muscle glycogen depletion induced by prolonged exercise leads to up-regulation as well as co-localization of HSP70 and VEGF primarily in type I fibers, thus suggesting that VEGF released from muscle is involved in the maintenance of muscle metabolic homeostasis in response to metabolic stress.

Recovery of skeletal muscle mass from immobilisation-induced atrophy is faster in young than older individuals, yet the cellular mechanisms remain unknown. We examined the cellular and molecular regulation of muscle recovery in young and older human subjects subsequent to 2 weeks of immobility-induced muscle atrophy. Retraining consisted of 4 weeks of supervised resistive exercise in 9 older (OM: mean age) 67.3, range 61-74 yrs) and 11 young (YM: mean age 24.4, range 21-30 yrs) males. Measures of myofibre area (MFA), Pax7-positive satellite cells (SCs) associated with type I and type II muscle fibres, as well as gene expression analysis of key growth and transcription factors associated with local skeletal muscle milieu, were performed after 2 weeks immobility (Imm) and following 3 days (+3d) and 4 weeks (+4wks) of retraining. OM demonstrated no detectable gains in MFA (vastus lateralis muscle) and no increases in number of Pax7-positive SCs following 4wks retraining, whereas YM increased their MFA (P < 0.05), number of Pax7-positive cells, and had more Pax7-positive cells per type II fibre than OM at +3d and +4wks (P < 0.05). No age-related differences were observed in mRNA expression of IGF-1Ea, MGF, MyoD1 and HGF with retraining, whereas myostatin expression levels were more down-regulated in YM compared to OM at +3d (P < 0.05). In conclusion, the diminished muscle re-growth after immobilisation in elderly humans was associated with a lesser response in satellite cell proliferation in combination with an age-specific regulation of myostatin. In contrast, expression of local growth factors did not seem to explain the age-related difference in muscle mass recovery.

OBJECTIVE:This study systematically investigated the effect of chronic mild stress and response to antidepressant treatment in the lateral habenula at the whole genome level.METHODS:Rat whole genome expression chips (Affymetrix) were used to detect gene expression regulations in the lateral habenula of rats subjected to chronic mild stress (mild stressors exchanged twice a day for 8 weeks). Some rats received antidepressant treatment during fifth to eights week of CMS. The lateral habenula gene expression profile was studied through the gene ontology and signal pathway analyses using bioinformatics. Real-time quantitative polymerase chain reaction (RT-PCR) was used to verify the microarray results and determine the expression of the Fcrla, Eif3k, Sec3l1, Ubr5, Abca8a, Ankrd49, Cyp2j10, Frs3, Syn2, and Znf503 genes in the lateral habenula tissue.RESULTS:In particular we found that stress and antidepressant treatment affected intracellular cascades like growth factor receptor signaling, G-protein-coupled receptor signaling, and Wnt signaling - processes involved in the neuroplastic changes observed during the progression of depression and antidepressant treatment.CONCLUSION:The present study suggests an important role of the lateral habenula in the development of depression-like conditions and correlates to previous studies demonstrating a significant role of the lateral habenula in depressive-like conditions and antidepressant treatment.