Influence of glutamine infusion on ubiquitin, caspase-3, cathepsins L and B, and m-calpain expression in sheep with nutritionally induced metabolic acidosis1

Abstract

Provision of AA has shown success in attenuating proteolytic activity in monogastrics suffering from metabolic acidosis. However, it is unknown whether AA supplementation can provide any beneficial effects to ruminants with nutritionally induced metabolic acidosis. The objective of the current study was to examine the effects of glutamine infusion on various protein degradation components across several tissues in sheep with induced metabolic acidosis. Sheep were assigned to a randomized complete block design with 2 x 2 factorial arrangement of treatments (n = 6 sheep/treatment) consisting of a control or acidosis diet, and receiving a saline or L-glutamine infusion. Sheep were fed diets for 10 d and slaughtered on d 11. Liver, kidney, and muscle samples were collected at slaughter and examined for relative messenger RNA (mRNA) expression of ubiquitin, C8, E2, cathepsin L, cathepsin B, caspase-3, and m-calpain, as well as protein expression of ubiquitin. Relative mRNA expression of C8 (P = 0.02), E2 (P = 0.06), and ubiquitin (P = 0.07) was less in kidney in acidotic vs. control sheep. Additionally, mRNA expression of m-calpain in kidney was greater (P = 0.01) as a result of glutamine infusion. There were no significant alterations (P > 0.10) in mRNA of any component as a result of acidosis in the liver or muscle. This study demonstrates the inability of metabolic acidosis to increase expression of the ubiquitin-mediated proteolytic pathway in skeletal muscle; however, downregulation of renal mRNA expression of these components is apparent during the induction of metabolic acidosis.

Differential regulation of components of the apoptotic and ubiquitin-mediated proteolytic pathway in slow- and fast- twitch muscle type in lambs receiving increasing amounts of dietary non-structural carbohydrate

“…Further research is required to examine if this downregulation of mRNA expression is also reflected in protein abundance or phosphorylation state. Previous research investigating metabolic acidosis in growing sheep has suggested that the UPP is not affected by the disease state in these animals (Greenwood et al 2008(Greenwood et al , 2009; however, the current research demonstrates that a high NFC diet downregulates soleus muscle UPP, and Bad mRNA expression in both the soleus and EDL muscle. Though further research must be conducted to understand whether this regulation at the mRNA level is reflective of the resulting protein synthesis and activity, dietary interactions that could influence protein turnover must also be discussed.…”

“…3). The authors have previously demonstrated a lack of response in Caspase-3 mRNA expression after induction of a moderate metabolic acidosis using dietary HCl (Greenwood et al 2009), but have successfully induced downregulation of soleus muscle specific mRNA expression of Caspase-3 using an ammonium chloride induction model of metabolic acidosis (Greenwood et al 2010). However, Caspase-3 serves functions in other cellular activities, such as apoptosis (Jesenberger and Jentsch 2002) and this role should not be overlooked, as this process could be the more dominant regulator of Caspase-3 mRNA expression.…”

“…Ultimately, this proteolysis provides amino acids, specifically glutamine, for increased inter-organ nitrogen transport and H ' buffering (Taylor and Curthoys 2004). Although we have begun to characterize the UPP response in sheep as a result of increasing dietary anionic load (Greenwood et al 2008(Greenwood et al , 2009, whether ubiquitin mediated proteolysis increases as a result of NFC challenge in ruminants has not been characterized. In addition, previous monogastric research suggests a differential proteolytic response between fast-and slow-twitch muscle in septic rats, with increased proteolysis in fast-twitch muscle and relatively little change in proteolytic activity in slowtwitch muscle (Hobbler et al 1999).…”