Intracellular signaling pathways regulating net protein balance following diaphragm muscle denervation

Unilateral denervation (DNV) of rat diaphragm muscle increases protein synthesis at 3 days after DNV (DNV-3D) and degradation at DNV-5D, such that net protein breakdown is evident by DNV-5D. On the basis of existing models of protein balance, we examined DNV-induced changes in Akt, AMP-activated protein kinase (AMPK), and ERK½ activation, which can lead to increased protein synthesis via mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K), glycogen synthase kinase-3β (GSK3β), or eukaryotic initiation factor 4E (eIF4E), and increased protein degradation via forkhead box protein O (FoxO). Protein phosphorylation was measured using Western analyses through DNV-5D. Akt phosphorylation decreased at 1 h and 6 h after DNV compared with sham despite decreased AMPK phosphorylation. Both Akt and AMPK phosphorylation returned to sham levels by DNV-1D. Phosphorylation of their downstream effector mTOR (Ser2481) did not change at any time point after DNV, and phosphorylated p70S6K and eIF4E-binding protein 1 (4EBP1) increased only by DNV-5D. In contrast, ERK½ phosphorylation and its downstream effector eIF4E increased 1.7-fold at DNV-1D and phosphorylated GSK3β increased 1.5-fold at DNV-3D ( P < 0.05 for both comparisons). Thus, following DNV there are differential effects on protein synthetic pathways with preferential activation of GSK3β and eIF4E over p70S6K. FoxO1 nuclear translocation occurred by DNV-1D, consistent with its role in increasing expression of atrogenes necessary for subsequent ubiquitin-proteasome activation evident by DNV-5D. On the basis of our results, increased protein synthesis following DNV is associated with changes in ERK½-dependent pathways, but protein degradation results from downregulation of Akt and nuclear translocation of FoxO1. No single trigger is responsible for protein balance following DNV. Protein balance in skeletal muscle depends on multiple synthetic/degradation pathways that should be studied in concert.

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The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.91247.2008 ◽

2009 ◽

Vol 107 (2) ◽

pp. 438-444 ◽

Cited By ~ 41

Author(s):

Heather M. Argadine ◽

Nathan J. Hellyer ◽

Carlos B. Mantilla ◽

Wen-Zhi Zhan ◽

Gary C. Sieck

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Caspase 3 ◽

Diaphragm Muscle ◽

Rat Diaphragm ◽

Ubiquitin Proteasome Pathway ◽

Protein Ubiquitination ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Net Protein

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.

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Differential protein metabolism and regeneration in hypertrophic diaphragm and atrophic gastrocnemius muscles in hibernating Daurian ground squirrels

10.1101/793752 ◽

2019 ◽

Author(s):

Xia Yan ◽

Xuli Gao ◽

Xin Peng ◽

Jie Zhang ◽

Xiufeng Ma ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Muscle Regeneration ◽

Protein Metabolism ◽

Muscle Hypertrophy ◽

Gastrocnemius Muscle ◽

Fiber Type ◽

Ground Squirrels ◽

Diaphragm Muscle ◽

Gastrocnemius Muscles

AbstractWhether differences in regulation of protein metabolism and regeneration are involved in the different phenotypic adaptation mechanisms of muscle hypertrophy and atrophy in hibernators? Two fast-type muscles (diaphragm and gastrocnemius) in summer active and hibernating Daurian ground squirrels were selected to detect changes in cross-sectional area (CSA), fiber type distribution, and protein expression indicative of protein synthesis metabolism (protein expression of P-Akt, P-mTORC1, P-S6K1, and P-4E-BP1), protein degradation metabolism (MuRF1, atrogin-1, calpain-1, calpain-2, calpastatin, desmin, troponin T, Beclin1, and LC3-II), and muscle regeneration (MyoD, myogenin, and myostatin). Results showed the CSA of the diaphragm muscle increased significantly by 26.1%, whereas the CSA of the gastrocnemius muscle decreased significantly by 20.4% in the hibernation group compared with the summer active group. Both muscles displayed a significant fast-to-slow fiber-type transition in hibernation. Our study further indicated that increased protein synthesis, decreased protein degradation, and increased muscle regeneration potential contributed to diaphragm muscle hypertrophy, whereas decreased protein synthesis, increased protein degradation, and decreased muscle regeneration potential contributed to gastrocnemius muscle atrophy. In conclusion, the differences in muscle regeneration and regulatory pattern of protein metabolism may contribute to the different adaptive changes observed in the diaphragm and gastrocnemius muscles of ground squirrels.

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A Clinically Relevant Decrease in Contractile Force Differentially Regulates Control of Glucocorticoid Receptor Translocation in Mouse Skeletal Muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.01064.2020 ◽

2021 ◽

Author(s):

Kirsten R. Dunlap ◽

Jennifer L. Steiner ◽

Michael L. Rossetti ◽

Scot R. Kimball ◽

Bradley S. Gordon

Keyword(s):

Protein Synthesis ◽

Glucocorticoid Receptor ◽

Resistance Exercise ◽

Muscle Atrophy ◽

Nuclear Translocation ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Contractile Force ◽

Force Generation ◽

Protein Balance

Muscle atrophy decreases physical function and overall health. Increased glucocorticoid production and/or use of prescription glucocorticoids can significantly induce muscle atrophy by activating the glucocorticoid receptor thereby transcribing genes that shift protein balance in favor of net protein degradation. While mechanical overload can blunt glucocorticoid-induced atrophy in young muscle, those affected by glucocorticoids generally have impaired force generation. It is unknown whether contractile force alters the ability of resistance exercise to mitigate glucocorticoid receptor translocation and induce a desirable shift in protein balance when glucocorticoids are elevated. In the present study, mice were subjected to a single bout of unilateral, electrically induced muscle contractions by stimulating the sciatic nerve at 100 Hz or 50 Hz frequencies to elicit high force or moderate force contractions of the tibialis anterior, respectively. Dexamethasone was used to activate the glucocorticoid receptor. Dexamethasone increased glucocorticoid signaling, including nuclear translocation of the receptor, but this was mitigated only by high force contractions. The ability of high force contractions to mitigate glucocorticoid receptor translocation coincided with a contraction-mediated increase in muscle protein synthesis, which did not occur in the dexamethasone treated mice subjected to moderate force contractions. Though moderate force contractions failed to increase protein synthesis following dexamethasone treatment, both high and moderate force contractions blunted the glucocorticoid-mediated increase in LC3 II:I marker of autophagy. Thus, these data show that force generation is important for the ability of resistance exercise to mitigate glucocorticoid receptor translocation and promote a desirable shift in protein balance when glucocorticoids are elevated.

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Regulation of cardiac protein balance by hydrocortisone: interaction with insulin.

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1978.234.3.e306 ◽

1978 ◽

Vol 234 (3) ◽

pp. E306

Author(s):

E E Griffin ◽

K Wildenthal

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Cathepsin D ◽

Specific Activity ◽

Branched Chain Amino Acids ◽

Protein Balance ◽

Net Uptake ◽

Specific Activities ◽

Branched Chain ◽

Net Release

In fetal mouse hearts in organ culture the rate of protein synthesis was substantially reduced and the rate of protein degradation slightly increased by hydrocortisone in the absence of insulin, but in the presence of insulin the steroid caused a small increase in protein synthesis and a significant reduction in protein degradation. Hydrocortisone promoted the net uptake (or reduced the net release) of branched-chain amino acids independent of insulin and independent of simultaneous changes in protein balance. The specific activities of the lysosomal enzymes cathepsin D and glucosaminidase were reduced by hydrocortisone in all media, whereas the specific activity of creatine kinase increased when the medium contained insulin but decreased in the absence of insulin. It is concluded that hydrocortisone regulates cardiac protein balance via alterations both in synthesis and in degradation. Some of the hormone's myocardial effects are influenced by insulin so that hydrocortisone is anabolic in its presence but catabolic in its absence.

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The kinase DYRK phosphorylates protein-synthesis initiation factor eIF2Bɛ at Ser539 and the microtubule-associated protein tau at Thr212: potential role for DYRK as a glycogen synthase kinase 3-priming kinase

Biochemical Journal ◽

10.1042/bj3550609 ◽

2001 ◽

Vol 355 (3) ◽

pp. 609-615 ◽

Cited By ~ 184

Author(s):

Yvonne L. WOODS ◽

Philip COHEN ◽

Walter BECKER ◽

Ross JAKES ◽

Michel GOEDERT ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Kinases ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Initiation Factor ◽

Glycogen Synthase Kinase 3 ◽

General Role ◽

Protein Tau ◽

Microtubule Associated Protein Tau ◽

Protein Synthesis Initiation

The substrate specificity of glycogen synthase kinase 3 (GSK3) is unusual in that efficient phosphorylation only occurs if another phosphoserine or phosphothreonine residue is already present four residues C-terminal to the site of GSK3 phosphorylation. One such substrate is the ε-subunit of rat eukaryotic protein-synthesis initiation factor 2B (eIF2Bε), which is inhibited by the GSK3-catalysed phosphorylation of Ser535. There is evidence that GSK3 is only able to phosphorylate eIF2Bε at Ser535 if Ser539 is already phosphorylated by another protein kinase. However, no protein kinases capable of phosphorylating Ser539 have so far been identified. Here we show that Ser539 of eIF2Bε, which is followed by proline, is phosphorylated specifically by two isoforms of dual-specificity tyrosine phosphorylated and regulated kinase (DYRK2 and DYRK1A), but only weakly or not at all by other ‘proline-directed’ protein kinases tested. We also establish that phosphorylation of Ser539 permits GSK3 to phosphorylate Ser535in vitro and that eIF2Bε is highly phosphorylated at Ser539in vivo. The DYRK isoforms also phosphorylate human microtubule-associated protein tau at Thr212in vitro, a residue that is phosphorylated in foetal tau and hyperphosphorylated in filamentous tau from Alzheimer's-disease brain. Phosphorylation of Thr212 primes tau for phosphorylation by GSK3 at Ser208in vitro, suggesting a more general role for DYRK isoforms in priming phosphorylation of GSK3 substrates.

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Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.4.h1056 ◽

2000 ◽

Vol 278 (4) ◽

pp. H1056-H1068 ◽

Cited By ~ 76

Author(s):

Lijun Wang ◽

Xuemin Wang ◽

Christopher G. Proud

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Signaling Pathways ◽

Glycogen Synthase ◽

Mrna Translation ◽

Mitogen Activated Protein Kinase ◽

Initiation Factor ◽

Translation Factors ◽

P70 S6k ◽

Eef2 Kinase

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order α>β>γ. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.

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Anabolic signaling and protein deposition are enhanced by intermittent compared with continuous feeding in skeletal muscle of neonates

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00516.2011 ◽

2012 ◽

Vol 302 (6) ◽

pp. E674-E686 ◽

Cited By ~ 36

Author(s):

Samer W. El-Kadi ◽

Agus Suryawan ◽

Maria C. Gazzaneo ◽

Neeraj Srivastava ◽

Renán A. Orellana ◽

...

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Muscle Protein ◽

Mrna Translation ◽

Initiation Factor ◽

Whole Body ◽

Signaling Proteins ◽

Protein Deposition ◽

Intermittent Bolus ◽

Continuous Feeding

Orogastric tube feeding is indicated for neonates with impaired ability to ingest and can be administered by intermittent bolus or continuous schedule. Our aim was to determine whether feeding modalities affect muscle protein deposition and to identify mechanisms involved. Neonatal pigs were overnight fasted (FAS) or fed the same amount of food continuously (CON) or intermittently (INT; 7 × 4 h meals) for 29 h. For 8 h, between hours 20 and 28, pigs were infused with [2H5]phenylalanine and [2H2]tyrosine, and amino acid (AA) net balances were measured across the hindquarters. Insulin, branched-chain AA, phenylalanine, and tyrosine arterial concentrations and whole body phenylalanine and tyrosine fluxes were greater for INT after the meal than for CON or FAS. The activation of signaling proteins leading to initiation of mRNA translation, including eukaryotic initiation factor (eIF)4E·eIF4G complex formation in muscle, was enhanced by INT compared with CON feeding or FAS. Signaling proteins of protein degradation were not affected by feeding modalities except for microtubule-associated protein light chain 3-II, which was highest in the FAS. Across the hindquarters, AA net removal increased for INT but not for CON or FAS, with protein deposition greater for INT. This was because protein synthesis increased following feeding for INT but remained unchanged for CON and FAS, whereas there was no change in protein degradation across any dietary treatment. These results suggest that muscle protein accretion in neonates is enhanced with intermittent bolus to a greater extent than continuous feeding, mainly by increased protein synthesis.

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Mechanisms of Cancer Cachexia

Physiological Reviews ◽

10.1152/physrev.00016.2008 ◽

2009 ◽

Vol 89 (2) ◽

pp. 381-410 ◽

Cited By ~ 672

Author(s):

Michael J. Tisdale

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Adipose Tissue ◽

Protein Degradation ◽

Initiation Factor ◽

Tissue Loss ◽

Α Subunit ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the α-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NFκB. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-α, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment.

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Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

Circulation Research ◽

10.1161/res.109.suppl_1.ap180 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Kedryn K Baskin ◽

Rebecca Salazar ◽

Wenhao Chen ◽

Heinrich Taegtmeyer

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Fold Increase ◽

Ubiquitin Proteasome System ◽

Initiation Factor ◽

Skeletal Muscle Atrophy ◽

Heart Weight ◽

Cross Sectional

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

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