Articles in PresS. Am J Physiol Endocrinol Metab (March 10, 2015). doi:10.1152/ajpendo.00006.2015

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Dysregulation of Skeletal Muscle Protein Metabolism by Alcohol

Jennifer L. Steiner Charles H. Lang

Cellular & Molecular Physiology Pennsylvania State College of Medicine Hershey, PA 17033

Running title: Alcohol and Skeletal Muscle Protein Metabolism

Send correspondence to: Charles H. Lang, PhD Cell & Molec Physiology Penn State College Medicine 500 University Drive Hershey, PA 17033 Phone: 717.531.5538 Fax: 717.531.7667 [email protected] 1

Copyright © 2015 by the American Physiological Society.

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ABSTRACT Alcohol abuse, either by acute intoxication or prolonged excessive consumption, leads to

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pathological changes in many organs and tissues including skeletal muscle. As muscle protein

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serves not only a contractile function, but also as a metabolic reserve for amino acids which are

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used to support the energy needs of other tissues, its content is tightly regulated and dynamic.

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This review focuses on the etiology by which alcohol perturbs skeletal muscle protein balance

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and thereby over time produces muscle wasting and weakness. The preponderance of data

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suggest alcohol primarily impairs global protein synthesis, under basal conditions as well as in

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response to several anabolic stimuli including growth factors, nutrients and muscle contraction.

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This inhibitory effect of alcohol is mediated, at least in part, by a reduction in mTOR kinase

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activity via a mechanism which remains poorly defined but likely involves altered protein-protein

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interactions within mTOR complex 1. Furthermore, alcohol can exacerbate the decrement in

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mTOR and/or muscle protein synthesis present in other catabolic states. In contrast, alcohol-

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induced changes in muscle protein degradation, either global or via specific modulation of the

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ubiquitin-proteasome or autophagy pathways, are relatively inconsistent and appear to be model

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dependent. Herein, changes produced by acute intoxication versus chronic ingestion are

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contrasted in relation to skeletal muscle metabolism, and limitations as well as opportunities for

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future research are discussed. As the proportion of more economically developed countries ages

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and chronic illness becomes more prevalent, a better understanding of the etiology of biomedical

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consequences of alcohol use disorders is warranted.

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Key words: ethanol; alcohol use disorder; skeletal muscle; protein synthesis; proteolysis

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I.

INTRODUCTION

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Estimates from a national health survey show more than half of the adult population in the

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United States consume ethyl alcohol and approximately 5% can be classified as “heavy” drinkers

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(e.g., more than 7 standard drinks per week for women and more than 14 drinks per week for

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men) (19). Moreover, demographic data also suggest an increase in episodic heavy (binge)

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drinking (12) and which is associated with a higher odds ratio of mortality (37). Multi-criteria

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decision analysis of drug use in the United Kingdom reveals that alcohol (aka ethanol) is the drug

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which causes the greatest combined harm to the user and the wider society (101). While light to

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moderate alcohol consumption has a beneficial or protective effect on some organ systems (e.g.,

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cardiovascular), both excessive chronic alcohol consumption and acute intoxication adversely

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affect multiple organ systems and ultimately increases mortality (49). While most studies and

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reviews have focused on the effect of alcohol on brain or liver, the potential toxic effects of

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alcohol on striated muscle are among the earliest recognized derangements, and represent one

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of the most common forms of skeletal muscle myopathy (114).

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This review focuses on the molecular etiology for the derangements in skeletal muscle

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protein metabolism and/or mass under conditions of acute alcohol intoxication and chronic

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alcohol abuse. The acute and chronic nature of the alcohol exposure, where possible, will be

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separated and defined, as the mechanisms for the observed changes often times differ.

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Moreover, although alcoholic cardio-myopathy is an important clinical manifestation of prolonged

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alcohol abuse (110), discussion has been restricted specifically to skeletal muscle to minimize

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any potential confusion related to the tissue-specific effects of alcohol. Further, other conditions

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(e.g., cancer, aging, disuse, sepsis, trauma, diabetes) are also associated with the erosion of lean

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body mass (LBM) and are not reviewed as they may proceed via different mechanisms. The

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reader is referred to recent excellent reviews on these topics (6, 50, 71, 98). The final section

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does, however, provide analysis of pertinent studies where the interaction of alcohol with other

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catabolic states has been examined. Finally, we have highlighted limitations of various 3

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approaches which may complicate data interpretation and provide commentary on future

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research opportunities.

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II.

ALCOHOL DECREASES BASAL MUSCLE PROTEIN SYNTHESIS Following early reports of patients presenting with skeletal muscle symptoms in greater

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frequency and independent of other alcohol-related pathologies (i.e., cirrhosis, cardiomyopathy or

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neuropathy) attention turned to determining how alcohol causes such a substantial loss of muscle

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function and size (15, 17, 20, 33, 90). Early investigations in rats showed chronic alcohol

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consumption reduced muscle protein content and protein synthesis in type II myofibers, and

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paved the way for subsequent studies elucidating how alcohol alters various components of the

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protein synthetic machinery and signal transduction pathways (92, 112). As protein degradation

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appears to be minimally impacted in this disease (see Section III), the regulation of protein

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synthesis has been of primary importance. This first section highlights recent advances as well as

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past work which laid the foundation for these discoveries. Unless specifically noted, results were

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generated from work performed in animal models using either rats or mice, and refer to skeletal

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muscle rich in type II myofibers like the gastrocnemius and/or plantaris.

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Chronic alcoholic consumption One advantage of rodent models is the ability to tightly match nutritional intake so that all

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animals are provided an isocaloric, isonitrogenous diet differing only in the presence of alcohol

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(65, 85). Therefore, metabolic differences between control and alcohol-fed rodents can be solely

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attributed to alcohol intake. Rats ingesting the alcohol-containing diet typically derive up to 36% of

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their total daily caloric intake from ethanol. Extrapolation to humans would necessitate an intake

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of ~700 Kcal ethanol /day or the ingestion of approximately 7 standard drinks. While the total

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caloric intake from alcohol is higher for rats compared to humans, rodents also have a greater

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rate of alcohol metabolism. Hence, the blood alcohol level (BAL) achieved in chronic alcohol-fed

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rodents is quite comparable to that observed in humans (100-200 mg/dL) (18, 51).

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Muscle mass and protein synthesis. The protracted imbalance in protein homeostasis

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resulting from excessive chronic alcohol consumption manifests as a decrease in muscle mass

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and cross-sectional area (CSA) of type II-fiber rich muscle, and the development of progressive

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proximal myopathy (23, 33, 90, 147). The prolonged imbalance between skeletal muscle protein

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accretion and breakdown represents the basic mechanism of alcohol-induced myopathy.

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However, the reduced rate of muscle protein synthesis per se appears to be the primary

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contributor to this catabolic condition. After being first reported in skeletal muscle from alcoholic

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patients (105), several independent investigators have modeled this pathology using a variety of

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alcohol feeding regimes in rats (62, 69, 72, 80, 113).

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The synthesis of new proteins is highly regulated and controlled by multiple signals

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integrated by mTOR (mammalian/mechanistic target of rapamycin) which resides as part of two

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distinct protein complexes – mTORC1 and mTORC2 (83). While a primary role of mTORC1 is

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regulating protein synthesis, this protein complex also mediates autophagy, lysosome biogenesis

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and lipid biosynthesis thereby broadly integrating and regulating cellular energy metabolism

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(Figure 1). The role of mTORC2 is less well understood but includes effects on cytoskeletal

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organization as well as growth, differentiation and survival (31). However, the recent literature

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suggests mTORC2 may have an important role in metabolic reprogramming (45, 94). The

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proteins forming mTORC1 (in addition to mTOR) include DEP domain-containing mTOR-

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interacting protein (Deptor), regulatory-associated protein of mTOR (Raptor), mammalian lethal

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with SEC13 protein 8 (MLST8), and proline-rich Akt substrate of 40 kD (PRAS40). In mTORC2

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the rapamycin-insensitive companion of mTOR (Rictor) replaces Raptor, while PRAS40 is

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replaced by the mammalian stress-activated protein kinase interacting protein 1 (SIN1) and

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Protor. In general, chronic alcohol intake does not alter the total content of mTORC1 proteins in

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skeletal muscle (62). However, alcohol does disrupt protein-protein interactions within mTORC1 5

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which may account for impaired kinase activity. Chronic alcohol consumption decreases the auto-

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phosphorylation of mTOR (Ser2481) (135), an indicator of mTOR catalytic activity, in conjunction

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with enhanced binding of Raptor to mTOR and Deptor (62). This latter association is posited to be

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of particular importance as Deptor is a recognized mTOR inhibitory protein (109) and decreasing

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Deptor content in muscle partially prevents muscle wasting (55).

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Protein synthesis is stimulated by events initiated by mTORC1 including phosphorylation

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of its two primary substrates, ribosomal protein S6 kinase (S6K) 1 and eukaryotic initiation factor

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4E binding protein-1 (4E-BP1). Phosphorylation of S6K1 enhances its kinase activity to

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subsequently phosphorylate multiple downstream proteins capable of stimulating mRNA

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translation (129). As chronic alcohol consumption suppresses ribosomal protein S6 (rpS6)

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phosphorylation in skeletal muscle (62), it is possible that the phosphorylation of other

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downstream targets (e.g., programed cell death protein 4 and eIF4B) is also impaired; however,

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this has not yet been assessed. Chronic alcohol also impairs 4E-BP1-dependent initiation of cap-

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dependent mRNA translation (69, 72, 80). The decreased phosphorylation of 4E-BP1 in response

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to chronic alcohol consumption enhances its association with eIF4E which decreases binding of

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eIF4E with eIF4G and ultimately prevents the formation of the active eIF4F complex necessary

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for mRNA binding to the 43S pre-initiation complex (2, 129). Conversely, these alcohol-induced

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effects on mTOR kinase activity are reversed in rats when alcohol is removed from the diet (155).

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Chronic alcohol intake also impairs translational efficiency (i.e., functioning of existing

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protein synthetic molecules); however, whether this occurs in addition to a reduction in ribosome

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number remains equivocal (80, 115, 120). While it was originally reported that alcohol

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consumption for 6 wks decreased total RNA content (assumed to be an indicator of ribosome

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number as 80-90% of cellular RNA in muscle is ribosomal), subsequent work failed to confirm

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such an effect, even when the duration of alcohol feeding was prolonged for several months (67,

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68, 80, 81, 115, 153). Further support for the impact of impaired translational efficiency on

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alcoholic myopathy is garnered from reports of suppressed eIF2B activity in muscle of chronically 6

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fed rats (69, 80). Binding of met-tRNAiMet to the 40S ribosomal subunit, necessary for 43S pre-

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initiation complex formation, is enhanced by eIF2B as it catalyzes the exchange of eIF2-bound

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GDP for GTP and regenerates the active eIF2•GTP complex to which the met-tRNAiMet binds.

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Peptide chain elongation and termination/release of the new protein complete translation

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and represent potential sites for alcohol action. Chronic alcohol feeding has divergent effects on

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various markers of peptide chain elongation. Alcohol decreases the distribution of 40S and 60S

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subunits in the type II psoas muscle indicating the movement of ribosomes along the mRNA (i.e.,

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elongation) and subsequent release of the mRNA (i.e., termination) is unable to keep pace with

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binding of mRNA to the ribosome (i.e., initiation) (80). Similarly, a decrease in the protein content

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of eukaryotic elongation factor (eEF)-1A occurred in the gastrocnemius of alcohol-fed rats

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implicating a potential decrease in the transfer of aminoacyl-tRNAs to the A-site of the ribosome.

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However, it appears alcohol does not slow the movement of the tRNA to the P-site of the

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ribosome as no change in eEF2 Thr56-phosphorylation was detected (154). The effect of chronic

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alcohol intake on termination and release of the polypeptide chain from the ribosome has not yet

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been assessed.

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Regulation of mTORC1. Progress has been made towards elucidating the mechanism by

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which chronic alcohol intake decreases muscle protein synthesis and how this relates to the

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developing myopathy. Nonetheless, gaps in our understanding remain including exactly how

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alcohol induces the mTOR-associated decrease in translation initiation. The mTORC1 pathway is

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regulated by several upstream inputs including (but not limited to) AMP-activated protein kinase

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(AMPK), regulated in development and DNA response (REDD1) protein, nutrients (e.g., amino

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acids), and growth factors (e.g., insulin). Activation of AMPK and REDD1 content in skeletal

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muscle are unchanged by chronic alcohol intake in adult male rats (62, 70) and therefore appear

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unlikely mediators of the suppression of basal mTORC1 activity. With regard to nutrients, chronic

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alcohol consumption does not alter plasma levels of the branch chain amino acids (BCAAs),

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leucine, isoleucine, and valine, which are the most potent stimulators of protein synthesis (57, 62, 7

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130). Similarly, chronic alcohol does not lower circulating levels of insulin (74). While these

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findings indicate the prevailing concentrations of amino acids and insulin are not causally related

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to the alcohol-induced decrease in protein synthesis, they do not exclude the possibility that

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alcohol antagonizes the anabolic effects of these stimuli (see Section V).

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In contrast, a significant decrease in the anabolic hormone insulin-like growth factor (IGF)-

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I is detected in both plasma and muscle, and correlates with the decrease in muscle protein

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synthesis and development of alcohol myopathy (65, 69). Accordingly, administration of a binary

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complex consisting of IGF-I and IGF binding protein-3 (IGFBP-3) – which delays clearance and

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extends bioactivity – restored muscle protein synthesis to basal control values when given during

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the final week of chronic alcohol feeding (65, 69). However, unexpectedly, 4E-BP1

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phosphorylation remained decreased in alcohol-fed rats treated with the binary complex

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indicating alcohol may have mTORC1-independent effects on protein synthesis or that the IGF-

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induced increase in synthesis was mediated by either mTORC1-induced phosphorylation of S6K1

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or an mTOR-independent mechanism (69).

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While these findings represent the generally accepted mechanism of how chronic alcohol

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consumption impairs muscle protein metabolism, differences in the sex and age of the

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experimental animals may complicate data interpretation. For example, in contrast to human

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studies where women are more susceptible than men to developing alcoholic myopathy (148),

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female rats did not exhibit overt disease after 26 wks of alcohol consumption; no reduction in

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gastrocnemius weight, total protein or rates of protein synthesis were observed (72). Further,

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plasma IGF-I and the phosphorylation and/or binding of eIF4E, eIF4G and 4E-BP1 was not

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altered in alcohol-fed female rats (72). Gender differences may be affected by muscle fiber type

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composition as the RNA-to-protein ratio was decreased more in the plantaris than gastrocnemius

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or soleus of female compared with male rats (47). Strain differences may also influence the

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response of female rats to chronic alcohol as adult Fischer F344 rats (62) show a reduction in

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protein synthesis and mTOR signaling whereas Sprague Dawley (72) and Wistar (47) rats did not 8

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exhibit an overt myopathy. These findings are highlighted to underscore the importance of the

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recent NIH initiative towards the inclusion of both male and female animals in all experimental

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studies. This work emphasizes the need for models specifically mimicking the physiological

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impact of chronic heavy alcohol consumption in females where variations in the hormonal milieu

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and smaller overall muscle mass with potentially different fiber type characteristics may influence

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disease development. Elucidating the cause for this sexual dimorphic response to alcohol could

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also potentially identify novel causative factors.

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The age of the animal when alcohol feeding is initiated may also influence the

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development of myopathy. The use of young or immature rats (i.e., ~6 wks of age; 100-150 g

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BW) is common and allows for a shorter period of alcohol feeding prior to the development of

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myopathy (116). However, whether the myopathy present in young rodents results from a

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blunting of normal muscle growth and protein accretion or authentic muscle atrophy as seen in

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adult human subjects remains ill-defined. Accordingly, the muscle catabolic response to chronic

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alcohol intake was exaggerated in 18-month old compared to 4-month old mature rats, which was

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ascribed to greater inhibition of mTORC1 activity via activation of the stress sensing proteins,

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AMPK and REDD1 (62). Therefore, alcohol may have differential effects at both ends of the age

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continuum and a consensus on the age of initiation and duration of feeding may prove effective

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for producing consistent results across studies utilizing this model.

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Acute alcohol intoxication Acute ingestion of large doses of alcohol (i.e., binge drinking) antagonize muscle protein

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synthesis in a dose- and time-dependent manner. The most commonly employed model of acute

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intoxication uses either intraperitoneal (I.P.) injection or oral gavage to deliver a single dose of

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alcohol in rats or mice. The standard dose administered to rats is 75 mmol/kg (~3.5 g/kg) which

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produces a BAL of ~300 mg/dL (~65 mM) after 2.5 hr (78). However, doses as low as 20 mmol/kg

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(78) and as high as 90 mmol/kg (79) have been reported. In mice, doses typically range from 3 to 9

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5 g/kg body weight and produce a BAL of ~275-350 mg/dL after 1 hr (77, 140); a lower dose of

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1.2 g/kg of alcohol is cleared from circulation within an hour (54). Although oral gavage is more

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physiological, both routes of administration yield comparable BALs (86) and have similar

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inhibitory effects on muscle protein synthesis (78).

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Acute alcohol intoxication (75 mmol/kg) in rats decreases muscle protein synthesis,

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translational efficiency and mTORC1 signaling (66, 72, 78, 79). Phosphorylation of rpS6, 4E-BP1,

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mTOR and eIF4G is decreased while the association of 4E-BP1 with eIF4E and mTOR with

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Raptor is increased by acute intoxication (63, 66, 72, 78, 79). Although some studies have also

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reported an alcohol-induced decrease in S6K1 phosphorylation (62, 78), this reduction is not

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consistently detected (63, 66). However, when observed, the alcohol-induced reduction in S6K1

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phosphorylation was independent of the sex of the animal, route of alcohol administration (e.g.,

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intraperitoneal vs. intragastric) and nutritional status (fed vs. fasted) (78). Lastly, despite

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decreases in translational efficiency, alcohol does not acutely alter the expression or activity of

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eIF2α, which mediates the formation of the 43S pre-initiation complex, or eIF2B which catalyzes

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the exchange of eIF2-bound GDP for GTP to regenerate the active eIF2•GTP complex necessary

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for translation initiation (68).

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Similar changes following acute alcohol intoxication (5 g/kg, 1 hr) are observed in mice as

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the rate of muscle protein synthesis, 4E-BP1 Thr37/46-phosphorylation and the binding of 4E-

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BP1 to Raptor are decreased (77). Further, Raptor Ser792-phosphorylation was increased along

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with binding of mTOR to Raptor likely contributing to the suppression of mTORC1 activity (77).

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Raptor is a scaffolding protein for mTORC1 and is a direct substrate for AMPK implicating it as an

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essential component of AMPK-mediated suppression of mTORC1 during situations of cellular

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stress. ERK1/2 phosphorylation is also suppressed indicating that alcohol antagonizes pathways

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that can alter protein synthesis independent of mTORC1 (141, 157). Finally, the inhibitory effect

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of acute alcohol on muscle protein synthesis is relatively long-lasting (>13 hr) and persists for

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hours even after clearance of alcohol from the circulation and normalization of mTORC1 signaling 10

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(140). Therefore, acute alcohol may damage the synthetic process in muscle to a greater extent

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than predicted by solely monitoring mTORC1 signaling at a single time point.

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Supporting work has also been reported using the C2C12 myoblast model. Culturing

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C2C12 myoblasts with alcohol (80-100 mM) for 24 hrs reduces protein synthesis and mTORC1

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signaling including phosphorylation of its substrates 4E-BP1 and S6K1 among others (41, 44). In

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this system there appears to be a greater role for AMPK-mediated changes of the mTORC1

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pathway or at least a greater ability to detect and/or manipulate activation of this protein. For

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example, alcohol-induced impairment of elongation assessed by eEF2 phosphorylation was

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mediated by AMPK (43). In vitro work has also permitted a greater mechanistic understanding of

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the direct effects of alcohol on muscle synthetic signaling and has revealed a potential role for

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mTORC2 signaling (42). Incubation of C2C12 myoblasts with alcohol (100 mM) increases

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mTORC2 activity assessed by increased phosphorylation of its substrate Akt (Ser473), as well as

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decreases Rictor phosphorylation and its association with Deptor and the 14-3-3 protein (41, 42).

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These changes occur concomitantly with the alcohol-induced decrease in mTORC1 activity and

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protein synthesis indicating a potential interaction between the two mTOR complexes. In this

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regard, Rictor knock-down using shRNA increases S6K1 Thr389-phosphorylation (i.e., mTORC1

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activity) in control cells and prevents the alcohol-induced decrease in protein synthesis (41, 42).

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The importance of these alcohol-induced changes in mTORC2 signaling will need to be verified in

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vivo. For a more thorough description and analysis of the in vitro effects of alcohol on myocytes

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protein synthesis the reader is referred to the review by Hong-Brown et al. (45).

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Limitations and opportunities One limitation to this field of study is the lack of mechanistic human data available from

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individuals with different types of alcohol use disorder (AUD) (34). Therefore, experimental

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evidence garnered from rat and mouse models are assumed to provide an accurate

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representation of the human disease. Moreover, while chronic alcohol related myopathy has been 11

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consistently reported in various strains of rats, there is a paucity of data as to whether mice

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experience similar consequences. To our knowledge only one report exists showing significant

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loss of muscle mass and CSA in female C57BL/6 mice after 6 wk on an alcohol-containing liquid

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diet, but as this work focused on increased autophagy (e.g., protein degradation) it is unknown if

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protein synthesis was reciprocally suppressed (144). Verification that mice develop chronic

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alcoholic myopathy similarly to humans is important to expand mechanistic research as more

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transgenic models are available (compared with rats) and genetic manipulations are easier to

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perform. Similarly, increased availability of muscle-specific and/or inducible knock-out models

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should aid in the future identification or validation of the importance of specific components of the

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mTORC1 (and mTORC2) signaling pathway in alcoholic myopathy.

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Emerging evidence supports the role of another signaling pathway upstream of mTORC1

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in the control of muscle atrophy; the Smad family of proteins are activated in response to

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transforming growth factor (TGF)-β, activin or select growth and differentiation factors like

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myostatin, and are essential mediators of muscle atrophy (30). For instance, Smad signaling is

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required for the myostatin-mediated inhibition of Akt/mTORC1 signaling and myotube atrophy, as

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well as for TGF-β-induced fiber atrophy (30, 128, 146). Nevertheless, mTORC1-independent

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effects of myostatin signaling may also occur (159). Smad signaling in muscle has not been

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assessed following alcohol intoxication; however, both TGF-β and myostatin mRNA are increased

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in response to alcohol feeding (69, 102). Collectively, the increase in myostatin and TGF-β

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suggests the Smad pathway may be activated and contributing to the impairment of mTORC1.

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This increased TGF-β also suggests that alcohol may increase fibrosis and remodeling in the

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skeletal muscle over time which could also potentially impair muscle function (38, 119).

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Appropriate translocation of mTOR to the lysosomal surface is necessary for its activation

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and is a rapidly evolving area in the study. Positioning of mTOR at or near the lysosomal

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membrane is essential to its interaction with and subsequent activation by GTP-loaded Rheb. The

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GTP/GDP loading of Rheb is regulated by the TSC1/TSC2/TBC complex (aka the Rhebulator), 12

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which when phosphorylated by Akt, dissociates from the lysosome and allows Rheb to remain

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GTP-loaded and active (14, 162). Currently, the effects of alcohol on the lysosome in general, or

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translocation of mTOR to the lysosomal membrane specifically, remain unknown. Modulation of

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the Rhebulator complex and Rheb GDP/GTP loading state by alcohol also remain unstudied. As

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the lysosomal positioning of mTOR is at the crux of amino acid-dependent activation of protein

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synthesis (125), the importance of these observations cannot be underscored. Should alcohol

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prevent the appropriate interactions of mTOR at the lysosomal surface it could be inferred that

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alcohol may also prevent the normal stimulation by amino acids. Such a defect may contribute to

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the development of myopathy in chronic alcoholics in the absence of overt malnutrition. As

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discussed in Section III, alcohol may adversely impact other important lysosome functions,

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including autophagy (118).

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Finally, a major shortcoming in the literature associated with the prevalent use of animal

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models to mimic chronic alcohol myopathy is the lack of accompanying functional endpoints.

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Much of the seminal work describing alcoholic myopathy was based upon functional deficits in

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man which included muscle pain and weakness (114); however, muscle function per se is rarely

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measured in animals likely due to methodological difficulties or limitations. Being able to quantify

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the deleterious effects of alcohol on contractile function in the same muscle for which

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corresponding synthetic signaling data are available would be invaluable.

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III.

PATHWAYS FOR MUSCLE PROTEIN DEGRADATION Tissue protein content is also governed by protein degradation which is primarily

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distributed between the ubiquitin-proteasome pathway (UPP) and the autophagic-lysosomal

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system (6, 126). The role of intracellular protein degradation in the development of alcoholic

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myopathy as well as the relative contribution of these two pathways are ill-defined and an issue of

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some debate. The paucity of data in this area relates at least in part to the lack of a convenient

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method for assessing in vivo rates of proteolysis causing data to be limited by their inferential 13

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nature. In this regard, studies have used urinary 3-methylhistidine (3-MH) excretion as a marker

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of whole-body protein breakdown with the understanding that release of this amino acid can

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originate from myofibrillar degradation in both skeletal and smooth (i.e., intestine) muscle (35).

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Although not extensive, there are reports that 3-MH excretion is either decreased (91) or

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unchanged (122) in humans chronically consuming alcohol, and increased in alcohol consuming

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(6 wk) mice (100). Hence, data on 3-MH excretion provide little definitive insight into the effect of

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alcohol on muscle proteolysis, thereby necessitating other approaches.

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Ubiquitin-proteasome pathway (UPP) and proteases In mature rats, acute alcohol intoxication decreased activity of the cytoplasmic proteases

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alanyl aminopeptidase, arginyl amino peptidase and leucyl aminopeptidase at 2.5 hr post-alcohol

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administration (59). Furthermore, while dipeptidyl aminopeptidase II was decreased by acute

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alcohol, there was no alcohol-induced change in dipeptidyl aminopeptidase I (59). As these

373

relatively modest alcohol-induced changes were transient in nature and their activity did not differ

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in muscle from chronic alcohol-fed and pair-fed control rats (59, 121), the contribution of these

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enzymes to the development of alcoholic myopathy appears limited. In addition, the cysteine

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proteases referred to as calpains, which are calcium but not ATP-dependent, could potentially

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contribute to the degradation of cytoplasmic proteins (138). However, the activity of calpain 1 and

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2 (µ- and m-calpains, respectively), and their inhibitor calpastatin, did not differ in alcohol-fed and

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control rats (59). Chronic alcohol consumption also did not alter muscle caspase-3 activity which

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contributes to apoptotic cell death by both the extrinsic (death ligand) and intrinsic (mitochondrial)

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pathway (133). In this regard, a surrogate marker for caspase-3-mediated cleavage of

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actomyosin in skeletal muscle, a 14-KDa degradation product of actin, did not differ between

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control and alcohol-fed rats (152). Likewise, neither acute or chronic alcohol abuse altered other

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endpoints related to apoptosis [i.e., DNA fragmentation or TUNEL (terminal deoxynucleotidyl

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transferase mediated dUTP nick end labelling) assays] in rat skeletal muscle (106). 14

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The role of the UPP as a major regulator of overall muscle proteolysis and the

387

degradation of myofibrillar proteins has been an area of recent focus, and the specifics of this

388

ATP-dependent proteolytic system have been reviewed elsewhere (126). A diverse array of

389

atrophic stimuli increase two muscle-specific ubiquitin E3 ligases – muscle atrophy F-box (MAFbx

390

or atrogin-1) and muscle RING finger-1 (MuRF1) – and the mRNA content of these proteins is

391

routinely used as a biomarker of UPP activity (7, 29). These “atrogenes” have been reported to

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be increased in fast-twitch muscles in chronic alcohol-fed rats (62, 104). Moreover, acute alcohol

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intoxication increased atrogene mRNA expression in a dose- and time-dependent manner in

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skeletal muscle (152). However, independent lines of investigation suggest these changes do not

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increase global proteolysis in the presence of alcohol. First, in vitro-determined proteasome

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activity was either unchanged (62, 152) or decreased (59) in muscle from alcohol-treated

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compared to control rats. Second, alcohol did not alter the rate of protein degradation of in vitro

398

incubated epitrochlearis muscle (143, 152). Third, tyrosine and 3-MH release from the isolated

399

perfused hindlimb was the same in control and alcohol-treated rats (152). Finally, protein

400

breakdown in vitro did not differ between control and alcohol (100 mM) treated C2C12 myotubes

401

(44). Hence, the alcohol-induced increase in atrogene mRNA is not associated with a

402

corresponding increase in muscle proteolysis, a discordant observation which has been reported

403

in other catabolic conditions (3, 82). One caveat to this general conclusion might involve

404

conditions where a secondary stress is combined with alcohol leading enhanced proteasome

405

activity (84).

406 407 408

Autophagy Cellular organelles as well as specific intracellular proteins can undergo autophagy-

409

mediated degradation in the lysosome. Early studies detected no change in the lysosomal

410

cathepsins B, D, H and L in muscle from control and alcohol-fed mature rats or following acute

411

alcohol intoxication (59). Thapaliya et al. (143) have reported increased autophagy in skeletal 15

412

muscle of alcohol-fed mice as indicated by increased LC3B-II, autophagy-related gene (Atg)-7

413

mRNA, and BECN1/Beclin1 protein expression; however, other work shows no change in LC3-II,

414

p62 or ULK-1 phosphorylation between mice fed an alcohol and time-matched control values

415

(142). Increased LC3B lipidation was also detected in muscle from alcoholic patients with

416

cirrhosis in association with a loss of muscle mass. Furthermore, complementary in vitro studies

417

indicated short-term (e.g., 6 h) incubation of C2C12 myotubes with alcohol increased expression

418

of autophagy-related genes, enhanced global proteolysis and decreased myocyte size. These

419

latter two alcohol-induced changes were largely prevented in myocytes by knockdown of Atg7 or

420

by chemical inhibition of autophagy. Such an alcohol-induced increase in autophagy would be

421

consistent with the above mentioned inhibition of mTOR by alcohol as mTOR activation is a

422

potent inhibitor of autophagy (32). While these data demonstrate the ability of alcohol to stimulate

423

autophagy in skeletal muscle, it is difficult to reconcile these data with the numerous in vivo

424

studies where alcohol intoxication or long-term consumption has not been shown to increase

425

overall muscle proteolysis (62, 152). Further, the alcohol-induced activation of the autophagy

426

pathway observed in mice contrasts with the lack of change in autophagic markers in alcohol

427

consuming rhesus macaques (131).

428 429

Limitations and opportunities

430

Many of the data in this area are inferential, with the majority suggesting acute alcohol

431

intoxication and chronic alcohol consumption do not accelerate global protein breakdown. The

432

inability to easily quantitate in vivo muscle proteolysis remains a limiting factor in assessing its

433

contribution to the associated wasting syndrome. Alcohol-induced changes in the mRNA content

434

of atrogenes and other UPP components are often reported; however few studies provide

435

corroborating evidence related to protein levels for these factors. As a discordance between

436

alcohol-induced changes in mRNA and protein might be anticipated under circumstances where

437

mRNA translation is impaired (74), extrapolating mRNA changes in atrogenes or components of 16

438

the UPP to global protein degradation should be avoided. Utilizing mice with a tissue-specific and

439

inducible promoter to knock down individual elements of the proteolytic signaling network (e.g.,

440

MuRF1 or atrogin-1) could provide more definitive mechanistic information regarding the relative

441

importance of proteolytic processes as mediators of alcohol-induced muscle wasting.

442

Cell culture systems are commonly employed to overcome the challenges associated with

443

in vivo research; however, relatively high concentrations (e.g., 100 mM) of ethanol over a long

444

period of time (e.g., 24 hrs) are often required to induce changes in protein balance thereby

445

raising concerns regarding the physiological significance of this model. Moreover, the use of

446

myoblasts versus differentiated myotubes further complicates the clinical relevance of this

447

system. The concept of cross-talk between the various proteolytic pathways (e.g., activation of

448

one pathway leading to a compensatory inhibition of another) has been proposed but largely

449

unexplored in relation to alcohol (107). It is also paramount to determine exactly which cellular

450

proteins undergo altered rates of proteolysis in response to alcohol. Finally, it remains to be

451

assessed whether therapeutic inhibition of autophagy and/or the UPP in an attempt to minimize

452

alcoholic muscle wasting may actually prove problematic as this could lead to the accumulation of

453

damaged proteins and enhanced cellular stress (127), and even greater muscle wasting (93) and

454

increased mortality (73).

455 456 457

IV.

MITOCHONDRIAL ENERGY PRODUCTION AND ROS PRODUCTION Muscle protein balance and function rely heavy on normal mitochondrial function and

458

hence deficits in ATP production may be causally related to the development of alcoholic

459

myopathy (36). Early light and electronic microscopic studies demonstrated diffuse morphological

460

changes in mitochondria in both acute and chronic alcoholic myopathy, though these were not

461

quantified and their functional significance not assessed (58, 149). Other studies reported

462

mitochondrial size (136, 150) and/or number (56) may be decreased in humans chronically

463

abusing alcohol. However, functional studies in both humans and rats detected either few or no 17

464

alcohol-induced change in the oxidation rate of various substrates, activity of individual

465

respiratory chain complexes, or cytochrome content in skeletal muscle (8, 9). These data are

466

consistent with the observation that ATP content and energy charge in the gastrocnemius did not

467

differ between control and alcohol-fed rats (80).

468

In vivo muscle energy metabolism studied using 31P magnetic resonance spectroscopy

469

(MRS) indicated the intracellular muscle pH and phosphocreatine (PCr) content did not differ

470

between controls and humans challenged acutely with alcohol under both resting basal conditions

471

and after exercise (160). Similarly, chronic alcohol consumption without accompanying hepatic

472

disease did not alter basal pH or PCr in muscle. However, alcohol ingestion was associated with

473

a greater reduction in both endpoints during exercise and a slower recovery of pH (but not PCr)

474

post-exercise. These data suggest chronic alcohol abuse may alter muscle blood flow and/or

475

produce a yet to be defined defect in mitochondrial function. While similar MRS results were

476

reported for chronic alcoholic patients with mild cirrhosis (Child-Pugh class A), alcoholics with

477

more extensive liver derangements (class B and C) showed a lower PCr/Pi ratio and decreased

478

maximal rate of mitochondrial ATP synthesis (48). These data imply that cumulative alcohol

479

consumption may have a dose-dependent effect (either direct or indirect) on muscle

480

mitochondrial function. Overall, the lack of an alcohol-induced change in basal ATP content or

481

synthesis in response to chronic intake is consistent with the absence of an increase in the

482

phosphorylation of the muscle energy sensor AMPK (62).

483

The possibility that chronic alcohol consumption induces mitochondrial changes

484

independent of ATP production cannot be excluded. For example, mitochondrial fusion and

485

connectivity is inhibited in muscle from alcohol-fed rats and this may result in part from a

486

reduction in the outer mitochondrial membrane fusion protein Mitofusin-1 (Mfn1) (16). Such

487

changes may be causally related to the observed calcium dysregulation and thereby impair both

488

bioenergetics and excitation-contraction coupling. Mitochondria also play a pivotal role in

18

489

regulating apoptosis, but as noted above, there is little definitive evidence of alcohol-induced

490

apoptosis in skeletal muscle.

491

In addition to their energy producing function, mitochondria are a major source of reactive

492

oxygen species (ROS). While the current review is not intended to provide a comprehensive

493

description of alcohol-induced oxidative damage, there are some studies have examined the

494

potential role of oxidants in the developing myopathy which are pertinent. In general, tissue

495

damage after alcohol could be due to either an increased ROS production and/or decreased

496

antioxidant mechanisms. In this regard, the decrease in CSA in chronic alcohol-fed rats was

497

associated with a decrease in total and free glutathione (GSH) content as well as reduced activity

498

of glutathione reductase and glutathione peroxidase and the mRNA for the mitochondrial form of

499

the superoxide scavenging enzyme, superoxide dismutase (SOD)-2 (Mn-SOD2) (102, 104).

500

Moreover, some studies have reported that alcoholic patients have a reduction in the circulating

501

concentration of antioxidants such as alpha-tocopherol (158). Consistent with these changes,

502

redox damage in muscle from alcohol-fed rats was evidenced by an increase in protein carbonyl

503

(61, 102) and cholesterol hydroperoxide content (1), and malondialdehyde (MDA) which primarily

504

reflecting lipid oxidation (156). In contrast, other studies have reported no alcohol-induced change

505

in MDA in muscle from rats (61), and no change in alpha-tocopherol, ascorbic acid or retinol (22),

506

or muscle antioxidant changes in alcohol consuming patients with myopathy (21). Finally, while

507

exogenous treatment with alpha-tocopherol failed to blunt the decrease in muscle protein

508

synthesis seen with either alcohol or chronic alcohol treatment (60), administration of the anti-

509

oxidant GSH precursor, procysteine, to chronic alcohol-fed rats ameliorated some of the oxidant

510

stress response and prevented at least part of the loss of muscle CSA (103, 104). Hence,

511

evaluation of the current data does not unequivocally support a causative role for oxidative stress

512

in the etiology of alcoholic myopathy. However, the advent of mitochondria-targeted anti-oxidants

513

may yet prove useful in limited alcohol-induced mitochondrial dysfunction, increased production

514

of ROS and the resultant myopathy (123). 19

515

Limitations and opportunities

516

To date the extent and physiological importance of alcohol-induced changes in

517

mitochondrial bioenergetics in muscle remain enigmatic. There is increasing evidence indicating

518

discordant results obtained using isolated (in vitro) mitochondria compared to studies using in

519

situ- or in vivo-based approaches (64). As alterations in mitochondrial structure and function are

520

common during aging, it remains to be determined whether defects in this organelle might be

521

responsible for the increased sensitivity of muscle to the catabolic effects of alcohol in aged rats

522

(62). Systematic studies on mitochondrial fission and the mitochondrial stress response are

523

needed, as the ability of alcohol to increase the latter via inhibition of mTOR signaling might be

524

predicted (111). Finally, the relative importance and the connection between alcohol-induced

525

mitochondrial dysfunction and tissue injury produced by ROS production specifically in skeletal

526

muscle requires clarification as this represents a nexus for potential therapeutic intervention.

527 528 529 530

V.

MODULATION OF ANABOLIC RESPONSIVENESS IN MUSCLE

531

administration of an anabolic agent designed to reverse and/or protect muscle from the catabolic

532

insult. While in theory this approach appears poised for success, independent reports show

533

alcohol antagonizes the desired anabolic response to several exogenously administered stimuli.

534

As presented below, alcohol-induced anabolic resistance would be expected to limit the

535

therapeutic effectiveness of exogenously administered agents as well as the accretion of muscle

536

protein by these same mediators when they are synthesized endogenously.

A strategy to prevent or attenuate the development of muscle atrophy involves

537 538 539

Hormone resistance IGF-I is a well-characterized, growth promoting hormone which activates the canonical

540

AKT/mTORC1 signaling pathway to increase protein synthesis (25). Chronic alcohol consumption

541

has been consistently shown to decrease plasma and muscle IGF-I (65, 75, 99, 137). Short-term 20

542

(4 day) treatment of alcohol-fed rats with an IGF-I/IGFBP3 binary complex reversed the decrease

543

in plasma IGF-I and restored muscle protein synthesis, translational efficiency, eIF4E•eIF4G

544

binding, eIF4G Ser1108-phosphorylation and myostatin back to levels seen in untreated control

545

rats (69). However, chronic alcohol feeding prevented the maximal response IGF-I/IGFBP3

546

stimulation as these endpoints remained below those observed in the control rats treated with the

547

binary complex (69). Furthermore, the binary complex did not reverse the alcohol-induced

548

decrease in 4E-BP1 phosphorylation or the increase in 4E-BP1•eIF4E binding (69).

549

Endogenously produced IGF-I can also been acutely increased with recombinant human growth

550

hormone (GH) in chronic alcohol-fed rats; however, it is unknown whether this GH-induced

551

increase of IGF-I is physiological meaningful as neither protein synthesis nor mTORC1 activity

552

were assessed (75).

553

A more systematic evaluation of IGF-I action in muscle has been pursued using acute

554

alcohol intoxication. While these alcohol models do not acutely lower blood borne IGF-I, alcohol

555

intoxication dose-dependently impairs IGF-I stimulated muscle protein synthesis and mTORC1

556

signaling (63, 78). Resistance to IGF-I, as assessed by phosphorylation of S6K1 and rpS6, was

557

present 2.5 hr after administration of 20, 50 or 75 mmol/kg of alcohol; however, partial recovery

558

was noted at the lowest dose (63, 78). Furthermore, at the highest alcohol dose, partial IGF-I

559

resistance was still detected 8 hr after alcohol administration and the normal anabolic response

560

only fully recovered at 24 hr. This alcohol-induced IGF-I resistance was independent of animal

561

sex, nutritional status (fasted/fed) or the route of alcohol administration (oral gavage/IP) (78).

562

Acute alcohol also blunted the IGF-I stimulated increase in 4E-BP1 phosphorylation in mice (76)

563

but not rats (63), and the reason for this difference is not known. In vitro data do not clarify this

564

issue as neither S6K1 nor 4E-BP1 were measured in a previous report showing that 3 days of

565

alcohol exposure suppressed IGF-I stimulation of protein synthesis in myoblasts (44). Similarly,

566

acute alcohol intoxication also decreased insulin-stimulated phosphorylation of S6K1 and S6 (but

567

again not 4E-BP1), which was independent of a change in insulin receptor phosphorylation (63). 21

568 569

The effect of alcohol, whether acute or chronic, on muscle could be direct or regulated

570

indirectly by secondary mediators released into the systemic circulation. This issue was

571

addressed using an isolated perfused muscle preparation and the data revealed the inhibition

572

was attributable to the local actions of alcohol on muscle rather than by increased concentrations

573

of glucocorticoid or acetaldehyde (63, 78). Moreover, as little alcohol metabolism occurs in this

574

muscle preparation, these data strongly support the concept that alcohol, and not one of its

575

metabolites (e.g., acetaldehyde) is responsible for inhibiting protein synthesis. When assessing

576

the mechanism of alcohol action we emphasize that caution should be exercised when

577

extrapolating data from other tissues. For example, in the liver, alcohol is actively metabolized

578

producing high levels of acetaldehyde, which are only present in low concentrations in skeletal

579

muscle because of the nominal capacity to oxidatively metabolize ethanol. Overall, alcohol

580

produces a growth factor resistance related to protein synthesis in skeletal muscle which is in part

581

mediated by a reduction in mTOR kinase activity and may contribute to development of myopathy

582

either alone or in conjunction with nutrient stimulation (see next section).

583 584 585

Feeding/nutrients/amino acids Feeding or nutrient intake stimulates muscle protein synthesis as mTORC1 is activated

586

both directly by select amino acids and indirectly by the coordinated rise in circulating growth

587

factors. Further, these stimuli are translational in nature as food and alcohol consumption often

588

occur in close temporal proximity. Alcohol blunted the stimulation of muscle protein synthesis and

589

mTORC1 activity when a nutritionally complete supplement was administered intragastrically in

590

24 hr-fasted rats (134). In contrast, alcohol did not antagonize the stimulation of protein synthesis

591

when nutrition was infused intravenously. While these data may suggest that alcohol inhibits

592

nutrient absorption from the gut, subsequent studies showed that alcohol does not slow leucine

593

distribution from the intestine or the secondary increase in plasma insulin (66). 22

594

Stimulation of mTORC1 signaling by feeding is dependent not only on hormonal activation

595

of the canonical AKT pathway (26), but also amino acid-specific stimulation of a separate

596

pathway composed of the Rag GTPases, v-ATPase, Ragulator, GATOR1 and GATOR2 protein

597

complexes (4). It has been shown that amino acids induce GATOR (GAP activity towards Rags)-

598

2-mediated inhibition of GATOR1 while the Ragulator and v-ATPase mediate GTP binding of

599

RagA and GDP binding of RagC (5). As a result, mTORC1 is recruited to the lysosomal surface

600

where it colocalizes with and is activated by Rheb-GTP. While addition of leucine to C2C12

601

myoblasts after a 24-hr exposure to alcohol returned protein synthesis, binding of mTOR to Rheb

602

and the phosphorylation of mTOR and its substrates 4E-BP1, S6K1, rpS6 back to untreated

603

control levels (41), these changes were all attenuated when compared to the response of control

604

(no alcohol) cells challenged with leucine. Acute alcohol intoxication in rats also blunted the

605

leucine-induced increase in muscle protein synthesis and mTORC1 kinase activity (66). Just as

606

exogenous leucine does not overcome the inhibitory actions of alcohol, increasing circulating

607

levels of leucine (and the other BCAAs) via disruption of the gene encoding the mitochondrial

608

branched-chain aminotransferase isozyme (BCATm) also does not prevent the alcohol-induced

609

decrease in protein synthesis and mTORC1 substrate phosphorylation (77). It is also noteworthy

610

that intracerbroventricular injection of alcohol decreased basal muscle protein synthesis and

611

recapitulated the leucine resistant state observed when alcohol was administered systemically,

612

suggesting some of the muscle metabolic effects of alcohol may be mediated via the central

613

nervous system (117).

614

The effect of alcohol on Rag protein-mediated mTOR activation is not well characterized

615

in muscle despite the recognized importance of this pathway. The simultaneous constitutive

616

activation of RagA and RagC increased the phosphorylation of S6K1 and 4E-BP1 back to control

617

levels in myocytes cultured with alcohol (41). However, alcohol did not impair the leucine-induced

618

increase in RagA/C binding to mTOR (41). Supporting data have not been obtained in animals

619

following alcohol, although chronic alcohol intake does decrease the association of RagA with 23

620

Raptor (presumably decreasing mTORC1 activation) in the rat gastrocnemius (62). Collectively,

621

current evidence indicates acute alcohol intoxication prevents the maximal anabolic response to

622

feeding and the amino acid leucine, and may contribute to the development of alcoholic

623

myopathy.

624 625 626

Muscle contraction An anabolic response in skeletal muscle is also generated by fiber contraction (i.e.,

627

exercise). Until recently little attention had been paid to the potential ability of alcohol to impair

628

this stimulus or the therapeutic potential of muscle contraction in the treatment of muscle disease

629

in individuals with AUD. Induction of muscle contractions using electrical stimulation is currently

630

being investigated as a clinical therapy to reduce illness- and immobilization-induced muscle

631

atrophy (88). In mice, acute alcohol intoxication (3 g/kg) immediately prior to a bout of maximal

632

muscle contractions prevented the increase in protein synthesis and S6K1/4E-BP1

633

phosphorylation for at least 4 hr (140). While the suppressive effect of alcohol waned over time,

634

contraction-stimulated increases in protein synthesis and mTORC1 were still partially suppressed

635

up to 12 hr after alcohol. Such data suggest that inducing muscle contraction in individuals with

636

elevated BALs may be ineffective as a treatment for alcoholic myopathy and demonstrate that

637

alcohol-induced anabolic resistance is a generalizable muscle response.

638

Alcohol administered prior to an anabolic agent clearly impairs stimulation of protein

639

synthesis/mTORC1 signaling. However, whether alcohol also reduced rates of synthesis which

640

had already been increased by an anabolic agent remained unexplored despite its direct societal

641

relevance (e.g. alcohol is commonly consumed in the evening following a meal or exercise). This

642

concept has now been addressed in both humans and mice although unfortunately protocols and

643

reported results differ considerably. For example, alcohol administered in mice 2 h after muscle

644

contraction completely reversed the contraction-induced increase in protein synthesis (141). It is

645

noteworthy that this decrease in protein synthesis was not due to suppressed mTORC1 signaling, 24

646

but was associated with inhibition of translation elongation by alcohol intoxication. In humans,

647

alcohol was ingested after a high-intensity exercise session which included both endurance and

648

resistance exercise (108). Unfortunately, the lack of appropriate control groups (i.e., exercise

649

alone and alcohol alone) precluded direct comparison with the aforementioned mouse study, but

650

the data did show alcohol suppressed the combined stimulatory effects of exercise and protein

651

supplementation on muscle S6K1 Thr389-phosphorylation. In contradistinction, alcohol did not

652

suppress myofibrillar fractional synthetic rates compared to the unexercised control group.

653

Finally, chronic alcohol feeding did not prevent the normal mTOR-dependent increase in protein

654

synthesis or hypertrophic response of muscle whereby overload was produced by synergistic

655

ablation (142). Additional work is needed to reconcile the apparently divergent responses

656

observed between mice and humans, and to assess the importance of the prevailing BAL in

657

modulating contraction and hypertrophic signaling.

658 659

Limitations and opportunities

660

While multiple studies have characterized the existence of alcohol-induced anabolic

661

resistance, the physiological importance of growth factor-, nutrient- and contraction induced

662

resistance is not known nor is the relative contribution of each to the erosion of LBM in AUD. The

663

underlying mechanism(s) for each remain to be fully elucidated. Anabolic resistance caused by

664

alcohol intoxication represents a major barrier to the successful treatment of alcoholic myopathy

665

as it prevents the full repertoire of responses necessary for the normal accretion of muscle

666

protein. As both growth factor and amino acid signaling is required for maximal mTORC1

667

stimulation, mechanistic studies investigating the potential effects of alcohol on lysosomal

668

regulation as well as on the activity of the proteins (i.e., Rags, v-ATPase, Ragulator, Rheb-GTP)

669

which are integral in each signaling process would be of value. Finally, the majority of work in this

670

area has been performed using a model of acute intoxication, but it unclear whether chronic

671

alcohol consumption produces a similar level of anabolic resistance. 25

672 673

VI.

INTERACTION OF ALCOHOL AND CATABOLIC STATES The final section reviews studies on the interaction of alcohol and various catabolic

674

stressors on skeletal muscle protein balance, primarily protein synthesis. The most thoroughly

675

investigated pairing is that of alcohol and acquired immunodeficiency syndrome (AIDS) (95), due

676

in part to the disproportionately high percentage of human immunodeficiency virus (HIV)-infected

677

individuals with AUD (53, 124). Several studies have combined simian immunodeficiency virus

678

(SIV) infection, a widely accepted model of HIV, and chronic alcohol ingestion in rhesus monkeys.

679

During the early asymptomatic period of SIV infection alcohol feeding increases the

680

proinflammatory environment of muscle [e.g., increased tumor necrosis factor (TNF)-α and

681

interleukin (IL)-6 mRNA), but does not alter muscle protein synthesis or degradation (97).

682

However, as the SIV disease progressed in the presence of alcohol, a greater decrease in limb

683

muscle mass was detected (96). These changes appeared independent of alterations in IL-6,

684

IGF-I or myostatin mRNA in the affected muscle, although the upregulation of TNFα mRNA

685

expression was exaggerated. In vitro-determined 20S proteasome activity of muscle from alcohol-

686

fed animals with late-stage SIV infection was increased, compared to SIV infection alone (84). A

687

similar exacerbation of muscle wasting was reported in HIV-1 transgenic rats fed an alcohol-

688

containing diet (10). Finally, while anti-retroviral therapy has decreased morbidity and mortality

689

from AIDS, several of the HIV-1 protease inhibitors have been reported to impair basal protein

690

synthesis and accentuate alcohol-induced changes in skeletal muscle (39, 40, 46). For example,

691

the co-incubation of myocytes with both alcohol and the HIV protease inhibitor Indinavir led to a

692

greater reduction in mTOR activity and cap-dependent initiation than produced by either condition

693

alone (39). Collectively, these studies demonstrate an adverse interaction between alcohol, HIV

694

and/or selective drugs used to treat the disease, but definitive mechanistic studies are largely

695

lacking.

696 697

Despite the extensive literature on sepsis-induced changes in muscle protein balance per se (24, 132) and the recognized detrimental interaction of alcohol + sepsis on other organ 26

698

systems (e.g., lung, liver and intestine) (13, 52, 161), information is lacking on the effect of

699

alcohol on muscle protein metabolism produced by bacterial infection. Related to this topic is a

700

report where E. coli endotoxin injected 24-h post-acute alcohol intoxication exaggerated the

701

increase in muscle IL-6 mRNA and protein as well as the late-phase cytokine high-mobility group

702

protein-1 (28) which, because of their proinflammatory properties, might be expected to impair

703

protein synthesis and/or increase proteolysis (27, 145). This report is limited by the lack of

704

accompanying data on muscle protein balance. As chronic alcohol consumption decreases

705

survival following bacterial infection (161), this would appear to be a fruitful area for future

706

research.

707

Epidemiological studies report that excessive alcohol consumption can increase or

708

decrease the incidence of certain types of cancer in humans. Such studies will not be reviewed,

709

but we will instead focus on the potential interaction of alcohol and cancer cachexia. Chronic

710

alcohol consumption exacerbates the loss of body weight in melanoma-bearing mice compared to

711

tumor-bearing control-fed mice. This response was associated with a reduction in carcass and

712

gonadal fat mass, but also with a decrease in urinary 3-MH excretion (e.g., decreased muscle

713

proteolysis) (100). These results could not be explained by changes in food consumption or

714

energy production, but suggest that impaired protein synthetic processes was the principal cause

715

for the loss of body mass. As alcohol consumption is a recognized risk factor for a variety of

716

cancers (87), research in this area has as high translational relevance.

717

Muscle disuse can occur in isolation (e.g., extended bed rest and immobilization) or

718

concomitant with general catabolic illness; hence, alcohol may modulate either the disuse-

719

induced loss of muscle or the accretion of muscle during the reloading phase. In the sole study in

720

this category, oral gavage of alcohol for three days exaggerated the mTOR-independent

721

decrease in muscle protein synthesis produced by unilateral hindlimb immobilization (151).

722

Additionally, alcohol ingestion during the atrophic immobilization period exaggerated the disuse-

723

induced increases in atrogin-1 and MuRF1 (implying increased proteolysis), and accordingly the 27

724

proteasome inhibitor Velcade prevented the exaggerated loss of muscle mass in alcohol-fed rats.

725

Further, when administered during the reloading phase, alcohol suppressed mTORC1 signaling

726

and increased atrogene expression in association with AMPK activation (151). Collectively, these

727

data suggest alcohol has the potential to negatively interact with other catabolic stressors to

728

induce derangements in protein metabolism not observed in response to the individual conditions.

729

An erosion of LBM commonly occurs as part of the aging process (i.e., sarcopenia)

730

resulting in age-related muscle dysfunction (11). As the relative age of the population in the US

731

and other developed countries continues to increase, the prevalence of AUD among the elderly

732

represents an increasing medical concern (139). Similar to heart disease, low- to moderate-level

733

alcohol consumption does not appear to contribute to age-induced loss of muscle, but sarcopenia

734

is exacerbated when alcohol consumption is sustained and excessive (89, 148). When adult (4

735

months) and aged (18 months) rats were placed on an alcohol-containing diet for 20 wks, the

736

latter demonstrated greater decreases in LBM and gastrocnemius weight (62). The ability of

737

alcohol to accentuate the age-induced decrease in muscle mass resulted primarily from a

738

reduction in protein synthesis (both sarcoplasmic and myofibrillar), but not activation of the UPP.

739

Alcohol-fed aged rats had enhanced dephosphorylation of 4E-BP1 suggesting the underlying

740

mechanism might be mTOR-mediated. Additionally, alcohol-fed aged rats showed enhanced

741

binding of Deptor (a negative regulator) with mTOR, which was associated with activation of

742

AMPK signaling (e.g., increased LKB1 and AMPK phosphorylation) and total REDD1 protein (62).

743

Finally, the accentuation of the age-induced impairment in protein synthesis and mTORC1

744

signaling was not associated with an exaggerated cytokine (TNFα, IL-6) response in muscle, but

745

did correlate with the reduction in muscle IGF-I.

746 747 748 749

Limitations and opportunities With the exception of the work on alcohol and SIV infection, research on the interaction of alcohol either prior to, or during the recovery phase of different catabolic stresses represents 28

750

singular studies for which the underlying etiology has not been defined and the fidelity of the

751

preclinical models employed not critically evaluated. Moreover, many of the studies have been

752

conducted in young growing animals in which there is a net accretion of muscle protein over time.

753

Regardless, the translational importance of such studies is evident as the population ages and

754

there are more individuals living with one or more chronic catabolic illnesses (aging, chronic

755

obstructive pulmonary disease, heart failure) or convalescing from traumatic injury (surgery, falls,

756

disuse).

757 758 759

VI.

SUMMARY The effects of alcohol, like all drugs, can be beneficial or detrimental based on the dose

760

and duration of exposure. Low- to moderate doses of alcohol have little to no effect, either directly

761

or indirectly, on muscle protein balance. Whereas acute alcohol intoxication and chronic alcohol

762

abuse decrease basal muscle protein synthesis via what appears to be a largely mTOR-

763

dependent mechanism. Of potentially greater importance is the ability of alcohol to induce a

764

resistance to a variety of anabolic stimuli all of which converge on mTORC1 to influence muscle

765

protein homeostasis. It is unclear whether this diminished responsiveness to growth factors,

766

nutrients and muscle contraction is mediated via a common mechanism or, more likely, via

767

defects in multiple pathways which intersect and result in mTORC1 inhibition. In addition, alcohol

768

has the potential to interact with and exacerbate the derangements in muscle protein balance

769

produced by other catabolic conditions. As protein synthesis and degradation are coordinately

770

controlled, what appears to be the relative selective alcohol-induced decrease of protein

771

synthesis in muscle is somewhat unexpected. With methodological advances, future studies

772

should be able to illuminate more subtle or nuanced effects of alcohol on the synthesis and

773

degradation of individual proteins. Such studies will yield insight into the mechanism of action for

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alcohol and how such changes negatively impact muscle structure and function potentially

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contributing to the increased morbidity associated with alcohol use disorders. 29

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ACKNOWLEDGEMENTS

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We thank our many collaborators who helped to make our work possible, especially Drs. L.

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Jefferson, S. Kimball, C. Lynch and the late T Vary. We apologize to those investigators whose

780

work was not cited due to space limitations.

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GRANTS

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This work was supported in part by NIAAA grant R37 AA011290 (CHL) and F32 AA23422 (JLS).

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DISCLOSURES

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No conflicts, financial or otherwise, are declared by the authors.

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39

1215

FIGURE LEGEND

1216 1217

Figure 1. Pathways modulating skeletal muscle protein balance. Signals from various anabolic

1218

stimuli enter the cell via membrane transporters (i.e. amino acids), or receptors (i.e. myostatin,

1219

hormones). Amino acids induce mTOR translocation and activation at the lysosomal surface via

1220

the Rag family of proteins and the GTP loading of Rheb. In contrast, TGFβs, GDFs and growth

1221

factors converge at Akt to either activate mTOR at the lysosome via TSC1/TBC complex

1222

phosphorylation and subsequent Rheb GTP-loading, or enhance proteasome activity (UPP) and

1223

muscle degradation. Upon lysosomal translocation and activation, mTOR phosphorylates several

1224

downstream targets including 4E-BP1, S6K1 and ULK-1. Phosphorylation of 4E-BP1 and S6K1

1225

enhances translation initiation and protein elongation, while ULK-1 regulates autophagosome

1226

formation and autophagy.

1227

40

Amino acids

Muscle contraction

TGFβs, GDFs (myostatin)

Growth factors (Inslin, IGF-1)

? IRS1 Smad2/3

PI3K

Akt

FOXOs

DGKζ Atrogin1 MuRF1

GATOR2 PA GATOR1 GTP

V-ATPase

TSC/TBC complex

TSC/TBC complex

RagA/B mTORC1 Regulator complex RagC/D (active)

Rheb

UPP mTORC1 (active)

GTP

Late endosome / lysosome

GDP

eIF2

4E-BP1

GTP

Late endosome / lysosome

4E-BP1 (active)

eIF4E

Rheb

S6K1

ULK-1

FIP2000 Atg13

eIF4E

eIF2B subunits

eIF4G eIF4A

beclin1 Vps34 PDCD4

eIF4B

rpS6

eEF2K LC3-II

GTP

LC3-I

eIF4F complex

eIF2 Met-tRNAi

eEF2

Autophagosome formation

Elongation

Autophagy

48S ribosomal subunit

Translation initiation

Endocrinology Journal E-00006-15: Dr. Jin Figure 01 ScEYEnce Studios 02/25/15

Protein Synthesis

LC3