Effect of acute and chronic alcohol treatment and their superimposition on lysosomal, cytoplasmic, and proteosomal protease activities in rat skeletal muscle in vivo

Abstract

Alcohol can be considered as a nutritional toxin when ingested in excess amounts and leads to skeletal muscle myopathy. We hypothesized that altered protease activities contribute to this phenomenon, and that differential effects on protease activities may occur when: (1) rats at different stages in their development are administered alcohol in vivo; (2) acute ethanol treatment is superimposed on chronic alcohol-feeding in vivo; and (3) muscles are exposed to alcohol and acetaldehyde in vivo and in vitro. In acute studies, rats weighing approximately 0.1 kg (designated immature) or approximately 0.25 kg (designated mature) body weight (BW) were dosed acutely with alcohol (75 mmol/kg BW; intraperitoneal [IP], 2.5 hours prior to killing) or identically treated with 0.15 mol/L NaCl as controls. In chronic studies, rats (approximately 0.1 kg BW) were fed between 1 to 6 weeks, with 35% of dietary energy as ethanol, controls were identically treated with isocaloric glucose. Other studies included administration of cyanamide (aldehyde dehydrogenase inhibitor) in vivo or addition of alcohol and acetaldehyde to muscle preparations in vitro. At the end of the treatments, cytoplasmic (alanyl-, arginyl-, leucyl-, prolyl-, tripeptidyl-aminopeptidase and dipeptidyl aminopeptidase IV), lysosomal (cathepsins B, D, H, and L, dipeptidyl aminopeptidase I and II), proteasomal (chymotrypsin-, trypsin-like, and peptidylglutamyl peptide hydrolase activities) and Ca(2+)-activated (micro- and milli-calpain and calpastatin) activities were assayed. (1) Acute alcohol dosage in mature rats reduced the activities of alanyl-, arginyl- and leucyl aminopeptidase (cytoplasmic), dipeptidyl aminopeptidase II (lysosomal), and the chymotrypsin- and trypsin-like activities (proteosomal). No significant effects were observed in similarly treated immature rats. (2) Alcohol feeding in immature rats did not alter the activities of any of the enzymes assayed at 6 weeks. (3) In immature rats, activities of cathepsins B and D were not overtly affected at either 3, 7, 14, 28, or 42 days. (4) Superimposing acute (2.5 hours) on chronic (4 weeks feeding of immature rats) ethanol treatment (ie, chronic + acute) reduced the activities of cytoplasmic proline aminopeptidase and the chymotrypsin- and trypsin-like activities of the proteasome. (5) Cathepsin D activities were reduced in muscle homogenates upon addition of alcohol and acetaldehyde in vitro. (6) Cyanamide pretreatment in combination with alcohol dosage in immature rats did not significantly alter any protease activities. The data suggests that mature rats are more sensitive to the effects of acute alcohol on muscle proteases. Protease activities may be affected by acetaldehyde or alcohol levels as indicated by in vitro experiments. The reduction in muscle protease activities in chronic + acute alcohol superimposition may reflect the effect of acute alcohol dosage alone. Overall, there was no evidence for increased protease activity in any of the experimental situations.

Skeletal muscle protein synthesis and degradation exhibit sexual dimorphism after chronic alcohol consumption but not acute intoxication

“…In one study, the fractional rate of myofibrillar breakdown as estimated by the difference between muscle protein content and protein synthesis was reported to be decreased by chronic alcohol consumption (31). However, the tissue proteasome activity was not significantly altered after 6 wk of alcohol feeding (14). Historically, the ubiquitination of proteins in the N end-rule pathway by the ubiquitin-conjugating enzyme E2 14k and the ubiquitin ligase E3␣ was considered the primary regulatory pathway for muscle loss in wasting states (23,35).…”

Alcohol abuse and glycoconjugate metabolism

“…For example, an acute ethanol exposure suppresses the LPS-induced clustering of Toll-like receptor (TLR4) and CD14, whereas chronic ethanol exposure exhibits a greater amount of expression of CD14, following LPS exposure [78]. LPS induces cytosolic phospholipase A 2 activation (which is linked to mucin synthesis impairment) [79] and TNF-a1 protein production (which affects proteolysis) [80]. Therefore chronic ethanol exposure throughout LPS induced www.fhc.viamedica.pl cytokine production impairs glycoconjugate synthesis to a higher degree than acute exposure.…”

Acute and chronic effects of alcohol exposure on skeletal muscle c-myc, p53, and Bcl-2 mRNA expression

“…However, a number of groups have resolved this by cyanamide predosing, which inhibits acetaldehyde dehydrogenase and markedly raises endogenous acetaldehyde (30,31). Recently, we have adopted such a protocol to investigate the putative effects of acetaldehyde in alcohol-related pathology (2,26,33,42,46,59). Finally, our previous studies on c-myc mRNA in alcohol-exposed muscle were carried out without reference to other regulator genes involved in apoptosis (41).…”