Review of two adaptive responses in Neuromechanical systems: force-induced atrophy recovery and endurance exercise.

Transcription

1 Review of two adaptive responses in Neuromechanical systems: force-induced atrophy recovery and endurance exercise. Combined isometric, concentric, and eccentric resistance exercise prevents unloading induced muscle atrophy in rats. Journal of Applied Physiology, 103: (2007) and Effects of Endurance Exercise Training on Insulin Signaling in Human Skeletal Muscle: interactions at the level of Phosphatidylinositol 3-kinase, Akt, and AS160 Diabetes, 56(8), 2093 (2007). Bradly Alicea, GEN 800, Fall 2007

2 Part I: force-induced atrophy recovery (Paper #1)

3 Introduction One way to induce hypertrophy (muscle growth due to functional plasticity) is via resistance exercise: * Augment balance between anabolism and catabolism, induces hypertrophy. * hypertrophy offsets muscle wasting prevalent in disease states. * number of reps, relative degree of force generated during contraction, and number of training sessions spanning the program. * these interact to enhance muscle mass and net protein accumulation. * Akt/p70S6k pathway: induces hypertrophic responses in skeletal and cardiac muscle (augmentation of total volume of mechanical stress over the training period).

4 Introduction (con t) Mouse Akt/p70s6K 1 ATK-mTOR pathway: involved in building muscle tissue, inducing hypertrophy. NOTE: p70 = heat shock protein (stressrelated induction of plasticity; e.g. Hsp90 in Drosophila) 2. Mouse Akt/p70s6K is identical to human pathway. Post-translation: a number of growth factors and other proteins upregulated in muscle tissue as a result of an exercise regime

5 Introduction (con t) Unloading: remove ground reaction, gravitational forces via 0-g, physical suspension (all mechanical stimuli). * muscle groups exhibit rapid atrophy (~5-7 days). * reversed via a pure isometric resistance exercise (RE) paradigm: * inhibition of catabolism (i.e. protein synthesis and structural stability), but also inhibits function of Akt/p70s6K signaling pathway. * gastrocnemius muscle does not maintain myofibril protein balance.

6 Training Protocol Objective: duration of each contraction cycle = 3 seconds (effect = 100% increase in force output during each contraction and increase volume of cumulative force activity across training). * NASA recommendation: integrated contraction regimen with dynamic action modes. * impose a systematic combination of isometric (ISO), concentric (CON), and eccentric (ECC) sequence. * position foot at 44 degree angle relative to tibia, induce contraction (ISO), position foot at 55 degrees relative to tibia, induce contraction (CON), position foot back to 44 degrees, induce contraction (ECC). * induces changes similar to that of motor learning (learning, perturbation, relearning).

7 Training Protocol (con t) Experimental Procedure: * 12 Sprague-Dawley rats limb suspended for 5 days. * prior to tail casting, electrical stimulation single hindleg (resistance training) ; repeated on each day of limb suspension. * tissue collection: 24h after last resistance training bout. * measurements: CONTRA (contralateral muscle, control) vs. TRAIN (suspended training, experimental comparison) * 8 additional rats served as sham control (no suspension, resistance training)

8 Molecular Methods RT-PCR (reverse-transcriptase PCR): * 1µg of total RNA for each muscle sample, 20µg total volume for RT reaction. RT produces a cdna (3-5 ) for each mrna transcript type. * numerous primers (previously published) were used to detect peptides involved in muscle growth (growth factors, musclerelated proteins). * primers designed from exon-intron boundary regions, least similarity to other mrnas from same gene family (specificity checked using BLAST search). * each mrna normalized relative to control. In RT-PCR, 18S endogenous control used, often more abundant than transcript (cdna) of interest. Use 18S competimers to attenuate signal appropriately.

9 Molecular Methods (con t) Western Blot: used to analyze expression and determine phosphorylation states of proteins. * Phosphorylation of S6K1 ~ kinase biological activity in vivo. * Hyperphosphorylation of 4E-BP ~ translational inhibition. Enhanced Chemiluminescence (ECL): similar to immunohistochemistry, but designed to work in conjunction with a Western (same principle as luminol). Detects and labels smaller quantities (femtomolar scale) of PCR product. * detected signal: amount of protein loaded on gel (true from µg). * hyperphosphorylation of 4E-BP1 ~ gel migration pattern (higher molecular weight band:total).

10 Results: body weight, muscle mass, protein content Hind Limb suspension: 7% decrease in body weight (vs. non-suspension). * significantly greater muscle mass in suspended TRAIN vs. suspended CONTRA, mean similar to Sham controls. * myofibrillar protein content in medial gastroc: significantly greater in suspended TRAIN vs. suspended CONTRA and Sham controls. RNA concentration and content: significantly increased in suspended TRAIN vs. suspended CONTRA and Sham controls. IGF-1, MGF, IGFBP-4: suspended TRAIN condition also increased production/accumulation of mrnas in medial gastroc (vs. suspended CONTRA).

11 Body Weight, Muscle Weight, Myofibril Content

12 Results: body weight, muscle mass, protein content (con t) Collagen-3 Alpha-1, Cyclin-D: Col-3 mrna increased 5x in suspended TRAIN condition. Also a significant increase in Cyclin-D1 in suspended TRAIN vs. suspended CONTRA. Myogenin, MyoD, Myoferlin, p21 (cyclin-dependent kinase inhibitor): increase in suspended TRAIN vs. suspended CONTRA (markers of myogenesis). Atrogin: Both unloading (suspended) conditions resulted in increase of mrna expression. Unloading combined with resistance exercise ~ further increase in atrogin mrna.

13 Col-3, Cyclin-D, Atogin, Myogenin, p21, and Myoferlin

14 Results: body weight, muscle mass, protein content (con t) Anabolic Signaling: S6K1 and 4E-BP are downstream effectors of mtor signaling. * hyperphosphorylation of 4E-BP and phosphorylation of S6K1 significantly increased in suspended TRAIN vs. suspended CONTRA. * no significant difference for total amounts of 4E-BP and S6K1 across conditions. Regulatory Principle: total and myofibrillar protein content rescued by training, but total weight is reduced among suspended animals. The regulatory response to reduced activity and mechanical loading are independent.

15 MyoD, IGF-BP4, 4E-BP, and S6K1

16 Results: body weight, muscle mass, protein content (con t) Translational capacity of muscles: RNA machinery is sensitive to unloading. * changes in total RNA between conditions ~ amount of ribosomal RNA. * synthetic machinery of muscle protein production maintained by this paradigm. * IGF-1 system (binding proteins, MGF) induced by increased loading; induction is sufficient but not necessary (even when isometric exercise fails to prevent atrophy, increases in IGF-1 mrna). Other factors at play. DNA content of muscle: suspended CONTRA condition muscle weighed less than suspended TRAIN, suggests a net loss of DNA. * unloaded skeletal muscle loses myonuclei (apoptotic-like process), fewer nuclei per muscle cell (hence less DNA content). * training regimen either 1) prevented nuclear apoptosis or 2) promoted cell proliferation.

17 RNA, DNA content; Myostatin, MGF, and IGF-1

18 Discussion Skeletal muscle and phenotypic adaptation: loading = enlargement of tendon tissue and increase in collagen synthesis. * associated with a rapid, early increase in tendom procollagen III (Col-3) mrna. * Col-3 result (5x increase) = robust stimulus for connective tissues and cytoskeleton. Cyclin: changes in Cyclin D1 = regulation of proliferation, may promote cell growth stimulation and ribosomal RNA expression. MyoD, myogenin: involved with cell differentiation and proliferation (acts on satellite cells). P21, myoferlin: expression levels not correlated with MyoD, p21 ~ loading, myoferlin ~ upregulated to mediate fusion of myblasts with myofibers (adults).

19 Discussion (con t) MRF-associated activity: change in mrna levels for cyclin-dependent kinase, change in mrna levels for myoferlin. Increased muscle loading ~ cell differentiation: increased p21 expression (cells leaving mitotic cycle), increased myogenin (myogenic differentiation). Myostatin and p21 negatively co-regulate differentiation of satellite cells. Myogenic regulatory factors: MyoD and Myogenin partake in activation and maturation of satellite cells. MyoD is upregulated in proliferating cells, loaded adult muscle. Regulation and coordination of cell proliferation: cyclin dependent kinases (cdks) pair with cyclin proteins to regulate this process. Cyclin D mrnas expressed when quiescent cells are stimulated, indicator of proliferative behavior in skeletal muscle.

20 Study Limitations/Conclusions Study limited to whole muscle measurements or protein products. Still do not know which components of muscle are responsible for hypertrophy. * do not know whether changes occur in myofibres or satellite cells. * Regular (strong) exercise paradigm tried initially, did not trigger molecularlevel changes in muscle. Resistance exercise protocol ~ suppression of catabolic activity and a maintainance of anabolic processes that blunt atophy. Separate processes that can work in tandem. * applicable to humans? Pathway highly conserved, so perhaps.

21 Part II: endurance exercise and molecular response

22 Introduction Main Question: how do you improve insulin-stimulated glucose uptake in skeletal muscle (insulin action on glucose homeostasis)? Signaling Network cascade (study focuses on downstream adaptations e.g. AS160): 1) delivery of insulin and glucose to muscle cell (via vasculature) 2) binding of insulin to receptor 3) intracellular signaling: * shift in steady state of GLUT4 proteins (vesicle transport to membrane), * increased transport of glucose, * activation of glycogen synthase.

23 Introduction (con t) Skeletal Muscle Adaptations (due to exercise): Mouse Akt/p70s6K a) improved hemodynamic effects of insulin b) elevated gene expression (genes associated with glucose uptake and metabolism). Short-term cycle exercise training: * increases PI3-kinase activity in young subjects only. * AS160 protein (signaling molecule) targeted by Akt2 (protein kinase) given insulin stimulation. * AS160 promotes translocation of GLUT-4 vesicles. * insulin = stimulates glucose uptake in adipocytes and skeletal muscle (important process).

24 Introduction (con t) Molecular variation also plays role in development of muscle mass (determines howindividuals respond to exercise stimulus). Myostatin (GDF-8): negative regulator of muscle mass. Glycoprotein (113 Aas long, 12kDa). TGF-β growth factor. In Belgian Blue cattle, Myostatin mutant = double muscling (hypertrophy). * in texel sheep, point mutation in 3 UTR makes product microrna target, downreg. See McPherron and Lee, PNAS USA, 94, (1997) See Clop et.al, Nature Genetics, 7, 491 (2006). In humans, anti-myostatin therapy for body builders (Myostatin suppression). * ds/dn ratio (natural selection test): positive selection for two variants in human populations ~ functionally significance. See Saunders et.al, American Journal Human Genetics, 79(6), 1089 (2006).

25 Introduction (con t) When exercise-induced changes affect GLUT4 content; is it coordinated with changes in associated proteins or docking and fusion of GLUT4 vesicles at membrane? Example: SNARE proteins (Syntaxin4, Munc18c, and IRAP) * may play a direct role in muscle insulin action. Training Regimen: * one-legged approach. * probed local contraction-induced adaptations (impact minor). * combined hyperinsulinemic-euglycemic clamp/ femoral arterial and venous catheterization technique = measurements of delivery and clearance of insulin and glucose to skeletal muscle.

26 Methods 8 healthy young men (similar age, height, weight, BMI, VO 2 MAX, and peak work load). * 2 days of controlled diet (50% carbs). Assess baseline condition: * 30 minutes of supine rest, needle biopsies from vastus lateralis in both legs. Needle Biopsies: done before first exercise bout and 15h after last exercise bout. Obtained from vastus lateralis in each thigh 10 and 120 minutes after insulin infusion. * tissue frozen in liquid nitrogen (-80C) * Teflon catheters inserted into one femoral artery and both veins.

27 Methods (con t) During measurements, lower leg was occluded using an inflated cuff (one minute in duration). Subjects underwent 120-minutes of a hyperinsulinemic-euglycemic clamp initiated with an injection of insulin. * hyerinsulinemic-euglycemic clamp: direct measure of glucose necessary to compensate for an increased insulin level without causing hypoglycemia. Glucose and insulin are infused to maintain blood sugar levels.

28 Methods (con t) Blood samples taken at 4, 15, 30, 45, 60, 90, 120 minutes before and during insulin injections (no catheter). One-legged training paradigm (gradual increase in training from 1 to 2 hours per session as time progressed): * four sessions during first week, five sessions during second week, six sessions during third week PWL (peak workload) increases in trained leg, but not in untrained leg. * intensity varied between 70 and 80 percent of PWL. * 5-7 minutes of training at 100 percent PWL to ensure the recruitment of muscle fibers in knee extensor region (to measure endurance capacity).

29 Methods (con t) Insulin measurement: plasma insulin concentration ~ radio immunoassay kit. Glucose concentration measurement: glucose analyzer. Muscle lysate protein harvesting: freeze-dried muscle, total protein content ~ bicinchronic acid method. Muscle glycogen measurement: acid hydrolysis of freeze-dried muscle tissue, glycosyl harvested and content (units) determined. SDS-PAGE: (sodium-dodecyl-sulfate polyacrylamide gel electrophoresis) method: separate proteins according to their electrophoretic mobility. * mobility = length of polypeptide chain, molecular weight, protein folding (secondary/tertiary structure), posttranslational modifications. * protein content = expressed in arbitrary units relative to a skeletal muscle standard.

30 Methods (con t) Sampling of methods used: Western Blot: for protein identification and analysis. PI3-kinase measurement: in vivo phosphorylation in phosphatidylinositol (substrate for a large number of enzymes which are involved in cell signaling because it can be phosphorylated by a variety of kinases). * lipids harvested and applied to a silica gel thin-layer chromotography instrument. Spots that co-migrated with a PI-4 standard were quantified. Akt1 measurement: immunopurification using protein G-speharose beads, suspended in lysate buffer (400µg of muscle lysate protein), kinase activity assay run for 30 minutes at 30ºC.

31 General adaptations to training: * no effect seen on glycogen content. Results Significant increases due to training: * PWL of knee extensors (16% in trained leg), citrate synthase (42%), 3- hydroxyacyl-coa dehydrogenase (38%). Insulin infusion: * arterial concentrations ~ 90 µu/ml (above whole-body level needed to maintain euglycemia). * insulin-stimulated glucose uptake increased to a greater extent in trained leg. Training effect seen at 15, 30, 45, 60 minutes after insulin stimulation.

32 Results (con t)

33 Results (con t)

34 Results Western Blot: * none of the proteins that were measured differed between the trained and nontrained legs before intervention. * Akt 1/2, increased 55%. AS160, increased 25% (at p =.08 level). IR, IRS-1, P85α, Akt2, GSK-3α, and GSK-3β, and apkc showed no significant difference. Membrane Fusion Molecules: * GS, Munc18c, Syntaxin4, SNAP-23 do not change. HK2 increased 197%, GLUT4 increased 52%, IRAP increased 65%; all significant at.001 level.

35 Results (con t)

36 Results (con t)

37 Results (con t) Presence of GLUT4 and IRAP (associated functionally), significantly correlated in trained muscle (r 2 =.47, p =.05). Phosphorylation of AS 160: Akt Thr, Akt Ser, GSK-3α Ser, GSK-3β Ser, and Ser/Thr Akt phospho sites. Increase in both trained and untrained muscle tissue significant at.005 level. Apparent at 10 minutes after insulin stimulation, effects seen up to 120 minutes after stimulation. * training did not influence phosphorylation state Akt Ser, Akt Thr, GSK-3α, GSK-3β, Ser, or GS site 3a+b either in basal state or during insulin administration. * phosphorylation of AS160 higher after training before and during insulin stimulation compared with untreated muscle (at.05 level).

38 Results (con t)

39 Results (con t) Activities of insulin signaling molecules: * Insulin stimulation: an increase in activity of IRS-1 associated PI3-K (.005), Akt (.005), Akt2 (.005), and GS (.005) in trained and untrained muscle. Response to insulin at 10 minutes: * no training: level of GS, increase only seen after 120 minutes. * after training: GS was elevated in both basal state and during insulin stimulation. Activity of IRS-1 associated PI3-kinase was significantly reduced at 10 and 120 minutes in trained but not untrained muscle.

40 Results (con t)

41 Discussion Positive main effect for training observed for Akt1 activity: * in mean values, a higher Akt1 activity in trained muscle was only observed after training and during insulin stimulation. Insulin-stimulated glucose uptake markedly increased in trained compared with untrained muscle. * coincided with increases in protein expression of GLUT4, IRAP, HK2. activity/phosphorylation of Akt and AS160 evident after training. * adaptations likely to improve intracellular conditions for uptake and metabolism of glucose.

42 Discussion (con t) Fick principle: measures total venous blood flow and glucose arteriovenous differences across thigh regions. * total venous blood flow higher in trained thigh (increase in hemodynamic response). Several interactions with insulin signaling cascade observed: * significant reduction in IRS-1 associated PI3-kinase activity in basal and insulin-stimulated muscle. Contradictory to findings in rodent muscle. * PI3-kinase may depend on length of exercise training regimen. IRS-1 adaptations at level of PI3-kinase may depend on length of exercise training regimen,

43 Summary Study tracks alterations in protein expression, kinase activation, phosphorylation level of insulin signaling cascade. Training also improves hemodynamic effects insulin (improved delivery of insulin/glucose). * Interactions between insulin and training were observed. Improved transport leads to training adaptations. Activation of Akt in skeletal muscle, phosphorylation of GSK-3, deactivates enzymes insulin signaling to GLUT4 translocation, involves activation of Akt and apkc * although results are problematic when compared with rodents, increase in response to insulin in human seems possible. * large increase in GLUT4 protein expression was observed. Associated with endurance/strength training, closely associated with improved insulin response.