S. Russ Price

Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression

Skeletal muscle atrophy is a debilitating response to starvation and many systemic diseases including diabetes, cancer, and renal failure. We had proposed that a common set of transcriptional adaptations underlie the loss of muscle mass in these different states. To test this hypothesis, we used cDNA microarrays to compare the changes in content of specific mRNAs in muscles atrophying from different causes. We compared muscles from fasted mice, from rats with cancer cachexia, streptozotocin‐induced diabetes mellitus, uremia induced by subtotal nephrectomy, and from pair‐fed control rats. Although the content of >90% of mRNAs did not change, including those for the myofibrillar apparatus, we found a common set of genes (termed atrogins) that were induced or suppressed in muscles in these four catabolic states. Among the strongly induced genes were many involved in protein degradation, including polyubiquitins, Ub fusion proteins, the Ub ligases atrogin‐1/MAFbx and MuRF‐1, multiple but not all subunits of the 20S proteasome and its 19S regulator, and cathepsin L. Many genes required for ATP production and late steps in glycolysis were down‐regulated, as were many transcripts for extracellular matrix proteins. Some genes not previously implicated in muscle atrophy were dramatically up‐regulated (lipin, metallothionein, AMP deaminase, RNA helicase‐related protein, TG interacting factor) and several growth‐related mRNAs were down‐regulated (P311, JUN, IGF‐1‐BP5). Thus, different types of muscle atrophy share a common transcriptional program that is activated in many systemic diseases.—Lecker, S. H., Jagoe, R. T., Gilbert, A., Gomes, M., Baracos, V., Bailey, J., Price, S. R., Mitch, W. E., Goldberg, A. L. Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression.—Stewart H. Lecker, R. Thomas Jagoe, Alexander Gilbert, Marcelo Gomes, Vickie Baracos, James Bailey, S. Russ Price, William E. Mitch, Alfred L. Goldberg Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J. 18, 39–51 (2004)

Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions

With trauma, sepsis, cancer, or uremia, animals or patients experience accelerated degradation of muscle protein in the ATP-ubiquitin-proteasome (Ub-Psome) system. The initial step in myofibrillar proteolysis is unknown because this proteolytic system does not break down actomyosin complexes or myofibrils, even though it degrades monomeric actin or myosin. Since cytokines or insulin resistance are common in catabolic states and will activate caspases, we examined whether caspase-3 would break down actomyosin. We found that recombinant caspase-3 cleaves actomyosin, producing a characteristic, approximately 14-kDa actin fragment and other proteins that are degraded by the Ub-Psome. In fact, limited actomyosin cleavage by caspase-3 yields a 125% increase in protein degradation by the Ub-Psome system. Serum deprivation of L6 muscle cells stimulates actin cleavage and proteolysis; insulin blocks these responses by a mechanism requiring PI3K. Cleaved actin fragments are present in muscles of rats with muscle atrophy from diabetes or chronic uremia. Accumulation of actin fragments and the rate of proteolysis in muscle stimulated by diabetes are suppressed by a caspase-3 inhibitor. Thus, in catabolic conditions, an initial step resulting in loss of muscle protein is activation of caspase-3, yielding proteins that are degraded by the Ub-Psome system. Therapeutic strategies could be designed to prevent these events.

Etiology of the Protein-Energy Wasting Syndrome in Chronic Kidney Disease: A Consensus Statement From the International Society of Renal Nutrition and Metabolism (ISRNM)

Protein-energy wasting (PEW), a term proposed by the International Society of Renal Nutrition and Metabolism (ISRNM), refers to the multiple nutritional and catabolic alterations that occur in chronic kidney disease (CKD) and associate with morbidity and mortality. To increase awareness, identify research needs, and provide the basis for future work to understand therapies and consequences of PEW, ISRNM provides this consensus statement of current knowledge on the etiology of PEW syndrome in CKD. Although insufficient food intake (true undernutrition) due to poor appetite and dietary restrictions contribute, other highly prevalent factors are required for the full syndrome to develop. These include uremia-induced alterations such as increased energy expenditure, persistent inflammation, acidosis, and multiple endocrine disorders that render a state of hypermetabolism leading to excess catabolism of muscle and fat. In addition, comorbid conditions associated with CKD, poor physical activity, frailty, and the dialysis procedure per se further contribute to PEW.

Evaluation of signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting.

The ubiquitin-proteasome proteolytic system is stimulated in conditions causing muscle atrophy. Signals initiating this response in these conditions are unknown, although glucocorticoids are required but insufficient to stimulate muscle proteolysis in starvation, acidosis, and sepsis. To identify signals that activate this system, we studied acutely diabetic rats that had metabolic acidosis and increased corticosterone production. Protein degradation was increased 52% ( P < 0.05), and mRNA levels encoding ubiquitin-proteasome system components, including the ubiquitin-conjugating enzyme E214k, were higher (transcription of the ubiquitin and proteasome subunit C3 genes in muscle was increased by nuclear run-off assay). In diabetic rats, prevention of acidemia by oral NaHCO3 did not eliminate muscle proteolysis. Adrenalectomy blocked accelerated proteolysis and the rise in pathway mRNAs; both responses were restored by administration of a physiological dose of glucocorticoids to adrenalectomized, diabetic rats. Finally, treating diabetic rats with insulin for ≥24 h reversed muscle proteolysis and returned pathway mRNAs to control levels. Thus acidification is not necessary for these responses, but glucocorticoids and a low insulin level in tandem activate the ubiquitin-proteasome proteolytic system.The ubiquitin-proteasome proteolytic system is stimulated in conditions causing muscle atrophy. Signals initiating this response in these conditions are unknown, although glucocorticoids are required but insufficient to stimulate muscle proteolysis in starvation, acidosis, and sepsis. To identify signals that activate this system, we studied acutely diabetic rats that had metabolic acidosis and increased corticosterone production. Protein degradation was increased 52% (P < 0.05), and mRNA levels encoding ubiquitin-proteasome system components, including the ubiquitin-conjugating enzyme E214k, were higher (transcription of the ubiquitin and proteasome subunit C3 genes in muscle was increased by nuclear run-off assay). In diabetic rats, prevention of acidemia by oral NaHCO3 did not eliminate muscle proteolysis. Adrenalectomy blocked accelerated proteolysis and the rise in pathway mRNAs; both responses were restored by administration of a physiological dose of glucocorticoids to adrenalectomized, diabetic rats. Finally, treating diabetic rats with insulin for >/=24 h reversed muscle proteolysis and returned pathway mRNAs to control levels. Thus acidification is not necessary for these responses, but glucocorticoids and a low insulin level in tandem activate the ubiquitin-proteasome proteolytic system.

Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

Chronic Kidney Disease Causes Defects in Signaling through the Insulin Receptor Substrate/Phosphatidylinositol 3-Kinase/Akt Pathway: Implications for Muscle Atrophy

Complications of chronic kidney disease (CKD) include depressed responses to insulin/IGF-1 and accelerated muscle proteolysis as a result of activation of caspase-3 and the ubiquitin-proteasome system. Experimentally, proteolysis in muscle cells occurs when there is suppression of phosphatidylinositol 3-kinase (PI3-K) activity. Postreceptor signaling through the insulin receptor substrate (IRS)/PI3-K/Akt pathway was evaluated in muscles of acidotic, CKD and pair-fed control rats under physiologic conditions and in response to a dose of insulin that quickly stimulated the pathway. Basal IRS-1-associated PI3-K activity was suppressed by CKD; IRS-2-associated PI3-K activity was increased. The basal level of activated Akt in CKD muscles also was low, indicating that the higher IRS-2-associated PI3-K activity did not compensate for the reduced IRS-1-associated PI3-K activity. Insulin treatment overcame this abnormality. The low IRS-1-associated PI3-K activity in muscle was not due to a decrease in IRS-1 protein, but there was a higher amount of the PI3-K p85 subunit protein without a concomitant increase in the p110 catalytic subunit, offering a potential explanation for the lower IRS-1-associated PI3-K activity. Eliminating the acidosis of CKD partially corrected the decrease in basal IRS-1-associated PI3-K activity and protein degradation in muscle. It is concluded that in CKD, acidosis and an increase in the PI3-K p85 subunit are mechanisms that contribute to suppression of PI3-K activity in muscle, and this leads to accelerated muscle proteolysis.

FOXO3a mediates signaling crosstalk that coordinates ubiquitin and atrogin-1/MAFbx expression during glucocorticoid-induced skeletal muscle atrophy

Muscle atrophy is a consequence of chronic diseases (e.g., diabetes) and glucocorticoid‐induced insulin resistance that results from enhanced activity of the ubiquitin‐proteasome pathway. The PI3K/Akt pathway inhibits the FOXO‐mediated transcription of the muscle‐specific E3 ligase atrogin‐1/MAFbx (AT‐1), whereas the MEK/ERK pathway increases Sp1 activity and ubiquitin (UbC) expression. The observations raise a question about how the transcription of these atrogenes is synchronized in atrophic muscle. We tested a signaling model in which FOXO3a mediates crosstalk between the PI3K/Akt and MEK/ERK pathways to coordinate AT‐1 and UbC expression. In rat L6 myotubes, dexamethasone (≥24 h) reduced insulin receptor substrate (IRS)‐1 protein and PI3K/Akt signaling and increased AT‐1 mRNA. IRS‐2 protein, MEK/ERK signaling, Sp1 phosphorylation, and UbC transcription were simultaneously increased. Knockdown of IRS‐1 using small interfering RNA or adenovirus‐mediated expression of constitutively activated FOXO3a increased IRS‐2 protein, MEK/ERK signaling, and UbC expression. Changes in PI3K/Akt and MEK/ERK signaling were recapitulated in rat muscles undergoing atrophy due to streptozotocin‐induced insulin deficiency and concurrently elevated glucocorticoid production. IRS‐1 and Akt phosphorylation were decreased, whereas MEK/ERK signaling and expression of IRS‐2, UbC and AT‐1 were increased. We conclude that FOXO3a mediates a reciprocal communication between the IRS‐1/PI3K/Akt and IRS‐2/MEK/ERK pathways that coordinates AT‐1 and ubiquitin expression during muscle atrophy.—Zheng, B., Ohkawa, S., Li, H., Roberts‐Wilson, T.‐K., Price, S. R. FOXO3a mediates signaling crosstalk that coordinates ubiquitin and atrogin‐1/MAFbx expression during glucocorticoid‐induced skeletal muscle atrophy. FASEB J. 24, 2660–2669 (2010). www.fasebj.org

Ubiquitin (UbC) Expression in Muscle Cells Is Increased by Glucocorticoids through a Mechanism Involving Sp1 and MEK1

The muscle protein catabolism present in rats with insulin-dependent diabetes and other catabolic conditions is generally associated with increased glucocorticoid production and mRNAs encoding components of the ubiquitin-proteasome system. The mechanisms that increase ubiquitin (UbC) expression have not been identified. We studied the regulation of UbC expression in L6 muscle cells because dexamethasone stimulates the transcription of this gene and others encoding components of the ubiquitin-proteasome pathway. Results ofin vivo genomic DNA footprinting experiments indicate that a protein(s) binds to Sp1 sites ∼50 bp upstream from theUbC transcription start site; dexamethasone changes the methylation pattern at these sites. Sp1 binds to DNA probes corresponding to the rat or human UbC promoter, and treating cells with dexamethasone increases this binding. Deletion and mutation analyses of the rat and human UbC promoters are consistent with an important role of Sp1 in UbC induction by glucocorticoids. Dexamethasone-induced ubiquitin expression is blocked by mithramycin, an inhibitor of Sp1 binding. UO126, a pharmacologic inhibitor of MEK1, also blocks UbCtranscriptional activation by dexamethasone; L6 cells transfected to express constitutively active MEK1 exhibit increased UbCpromoter activity. Thus, glucocorticoids increase UbCexpression in muscle cells by a novel transcriptional mechanism involving Sp1 and MEK1.

Molecular signaling pathways regulating muscle proteolysis during atrophy.

Purpose of reviewAlthough a variety of diverse stimuli induce muscle atrophy, there is a surprising number of similarities in the intracellular responses. One prominent response is an increase in muscle proteolysis resulting from stimulation of the ubiquitin–proteasome pathway. Understanding the intracellular signaling pathways that regulate muscle mass should offer insights into the coordination of cellular responses. This review will discuss recent findings on the molecular signaling pathways regulating proteolysis during muscle atrophy. Recent findingsThe expression of several muscle-specific E3 ubiquitin ligases is consistently increased in conditions causing muscle atrophy. Insulin and insulin-like growth factor-1 act through the phosphoinositide 3-kinase/AKT pathway to suppress the expression of two of these enzymes, MuRF1 and MAFbx/atrogin-1. Efforts to identify targets of the muscle-specific E3 ligases are yielding interesting information. Insulin and insulin-like growth factor-1 also attenuate wasting by inhibiting caspase-3, which cleaves actin to facilitate its destruction by the ubiqutin–proteasome system. Other signaling systems involved in the regulation of muscle mass include the nuclear factor kappa B pathway. SummaryThe maintenance of muscle mass requires a delicate balance between catabolic factors and anabolic factors. These signals inversely modulate the activity of several key regulatory pathways including the phosphoinositide-3 kinase/AKT and nuclear factor kappa B systems, which control the transcription of components of the ubiquitin–proteasome proteolytic pathway activity, the activity of caspase-3, and perhaps other proteolytic functions. When levels of insulin or insulin-like growth factor-1 are insufficient or inflammatory cytokine production is increased, muscle atrophy ensues.

Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation.