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Metabolic Signaling

Metabolic Signaling. Describe models of low-force overuse Identify the main energy-dependent signaling molecules and their mechanisms AMPK PGC-1a GSK Reactive oxygen. Low force overuse. Models Chronic stimulation Endurance training Physiological stresses Electrophysiological

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Metabolic Signaling

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  1. Metabolic Signaling • Describe models of low-force overuse • Identify the main energy-dependent signaling molecules and their mechanisms • AMPK • PGC-1a • GSK • Reactive oxygen

  2. Low force overuse • Models • Chronic stimulation • Endurance training • Physiological stresses • Electrophysiological • Oxygen delivery/handling • ATP metabolism • Adaptation • SR swelling • Mitochondrial hypertrophy • “Slow” phenotype expression • Atrophy

  3. Acute changes during contraction • Phosphate redistribution • pCrATP • ATP2 Pi + AMP • pH decline 2 Hz 10 Hz Time (min) Kushmerick & al., 1985

  4. Changes in blood composition 5 min exercise 10 min recovery • Lactate appears ~3 min • pH falls in parallel • Norepinepherine Gaitanos &al 1993

  5. Mechanical performance changes • P0 declines (atrophy) • Vmax declines (slower) • Endurance increases 2 weeks CLFS Control muscle Jarvis, 1993

  6. Cellular energy sensors • AMP kinase: glucose transport, protein balance • PPAR: mitochondrial hypertrophy • GSK: hormonal/systemic integration • ROS: complicated

  7. Endurance adaptation paradigm • Elevated calcium and AMP activate mitochondrial genes • AMPK, PGC-1, pPAR, MEF2 • Elevated calcium activates muscle genes Baar, 2006

  8. AMPK a2 is more sensitive to AMP • AMP activated protein kinase • Catalytic a subunit • Regulatory b subunit • AMP-binding g subunit • AMPK-kinase • Liver Kinase B1 (LKB1) • STE-related adaptor (STRAD) • MOL25 • CaMKK Incubate with phosphatase Add phosphatase inhibitor a2 is more sensitive to phosphorylation, and has stronger autophos Salt & al., 1998

  9. AMPK-Calcium synergy • CaMKK activates AMPK only in the presence of AMP • AMP protects from phosphatase activity (PP2c) • CAMKK, but not LKB1activated by exercise • Starvation vs activity

  10. AMPK analogs • LKB1-STRAD-MOL25 substrates • Tumor suppressor, esp smooth muscle • HeLa cells are LKB1-/- • SNARK • Required for exercise-stimulated glucose uptake • Blocked in insulin-resistant • MARK1-4 LKB1 ko reduces activation of SNARK by exercise SNARK ko reduces activation of GLUT4 by exercise Koh & al 2012

  11. AMPK alters metabolism and growth • Acetyl-coenzyme A carboxylase (ACC, inhibited) • Ac-CoAmalonyl-CoA • Key enzyme in gluconeogenesis • Malonyl-CoA blocks FA import to mitochondria • PFK3B (activated) • F1-p F1,6-pp • TSC2, raptor (inhibited) • mTORC1 control of protein translation • FOXO3a, AREBP, HNF4a (activated) • MafBx, autophagy genes ie: activation of AMPK dis-inhibits FA oxidation, blocks protein translation and activates protein degradation

  12. AMPK metabolic effects • AICAR treatment • AICARZMP≈AMP • 5 days • Inhibits ACC • Upregulates GLUT4 & HK • LKB1-dependent AMPK activation facilitates glucose uptake, glycolysis, and fatty acid transport. ie: production or replenishment of ATP Holmes & al., 1999

  13. FOXO transcription • Counter-regulation by Akt/AMPK • Autophagy: ATG • Atrophy: MuRF MafBx • Arrest: p21, p27 • Apoptosis: BIM, fas • Angiogenesis • Energy: PGC1a, HK • Insulin/IGFAkt • AMPAMPK Salih & Brunet, 2008

  14. PGC-1a • Peroxisome proliferator activated receptor g cofactor 1a • Broad spectrum coordinator of nuclear and mitochondrial transcription • Antioxidant enzymyes: SOD, catalase, GPx1, UCP • Inflammatory response: TNF-a, IL-6 (down) • Mt biogenesis: Tfam, Cytochrome oxidase • Co-factor • MEF2, NFAT, NRF-1

  15. Fast-muscle specific PGC1 overexpression • PGC1 under MCK promoter • Tg muscles: more mt, COX, myoglobin • Tg more MHC-1, but still 90% MHC-2 Lin & al ., 2002

  16. PGC-1a splice variants • PGC-1a1: mitochondrial biogenesis, oxphos • PGC-1a4: IGF-1, myostatin repression

  17. GSK3 • Glycogen synthase kinase 3 (a,b) • Inhibited by phosphorylation: PKB, p38, RSK • Targets mostly primed substrates • Inhibits glycogen synthase • Cell growth control • C-Myc, Bcl2, MDM2, retinoblastoma (Rb) • Wnt, NFAT, CREB

  18. Reactive oxygen species • Oxygen radical (O2-, H2O2, OH∙) signaling/damage Powers & Jackson 2008

  19. Sources of ROS • Electron transport chain • Electron “leakage” through Complex I,III centers • Cytochrome-C, ubiquinone • Antioxidant expression • NAD(P)H oxidase • SR/T-tubules • NADPH + 2 O2NADP+ + 2 O2- • Cell cycle, fibrosis, inflammation • Xanthine oxidase • Plasma membrane • Xanthine+H2O+ O2 Uric acid + H2O2

  20. Targets of ROS • NF-kB • H2O2--|SHIP-1--|NEMOIKKNFkB • Inflammatory • SOD, BIM, p53, SNARK, NOS, Mt biogenesis • p21Ras • Oxidation of cysteine residues increases GTP exchange • PI-3K, MAPKprotein turnover • Src • Oxidation of C245 and C487 increases kinase • Myoblast proliferation • AKAP121-enhanced Mt ATP synthesis

  21. Contractile activity Ca2+ AMP CHO depletion O2- Cn CaMK PKC AMPK GSK Src IKK Ras NFAT MEF2 CREB PGC-1 FOXO TSC2 NF-kb Rb Contractile proteins Mitochondrial proteins Angiogeneis

  22. Combinatorial control of genes • Multiple elements in promoter-proximal region • Cooperative: multiple elements combine to recruit transcription complex • Competitive: overlapping domains block each other • Nonlinear: transcriptosome • Intron elements

  23. MHC control • NFAT isoforms • Intergenic antisense • Intronic miRNA Promoter construct expression combined with knockdown of various NFATs Calabria & al., 2009

  24. Cancer parallels • Proto oncogenes • LKB1, PGC-1, p53, etc • Negative controllers of growth • Defectsuncontrolled growth • Chemotherapy often targets these pathways • Exaggerated muscle loss • Weakness, fatigue

  25. Summary • Prolonged muscle activity stimulates • Persistently elevated calcium • ATP stress • Reactive oxygen stress • Immediate consequences • Increased Ox-phos, FA, and glucose uptake • Suppressed calcium release • Long-term consequences • Mitochondrial biogenesis • Contractile protein isoform switching

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