Down-regulation of the ubiquitin–proteasome proteolysis system by amino acids and insulin involves the adenosine monophosphate-activated protein kinase and mammalian target of rapamycin pathways in rat hepatocytes

Abstract

The purpose of this work was to examine whether changes in dietary protein levels could elicit differential responses of tissue proteolysis and the pathway involved in this response. In rats fed with a high protein diet (55%) for 14 days, the liver was the main organ where adaptations occurred, characterized by an increased protein pool and a strong, meal-induced inhibition of the protein breakdown rate when compared to the normal protein diet (14%). This was associated with a decrease in the key-proteins involved in expression of the ubiquitin-proteasome and autophagy pathway gene and a reduction in the level of hepatic ubiquitinated protein. In hepatocytes, we demonstrated that the increase in amino acid (AA) levels was sufficient to down-regulate the ubiquitin proteasome pathway, but this inhibition was more potent in the presence of insulin. Interestingly, AICAR, an adenosine monophosphate-activated protein kinase (AMPK) activator, reversed the inhibition of protein ubiquination induced by insulin at high AA concentrations. Rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, reversed the inhibition of protein ubiquination induced by a rise in insulin levels with both high and low AA concentrations. Moreover, in both low and high AA concentrations in the presence of insulin, AICAR decreased the mTOR phosphorylation, and in the presence of both AICAR and rapamycin, AICAR reversed the effects of rapamycin. These results demonstrate that the inhibition of AMPK and the activation of mTOR transduction pathways, are required for the down-regulation of protein ubiquitination in response to high amino acid and insulin concentrations.

Knockdown of PRAS40 inhibits insulin action via proteasome-mediated degradation of IRS1 in primary human skeletal muscle cells

“…4c). As PRAS40-KD inhibits mTORC1 signalling, and inhibition of mTORC1 activity has been linked to increased protein degradation by increased activity of the ubiquitinproteasome system [30,31], we next examined whether mRNA expression of the muscle-specific E3-ligases FBXO32 and MURF1, which are increased in mice heterozygous for mTOR [30], is affected by PRAS40-KD. As shown in Fig.…”

“…One may speculate that the effects of PRAS40-KD on the proteasome result from inhibition of mTORC1 activity. In primary rat hepatocytes, short-term pharmacological inhibition of mTOR was found to reverse the inhibition of the ubiquitin-proteasome pathway by insulin and amino acids [31]. Furthermore, skeletal muscle of mTOR-heterozygous mice displays elevated levels of the E3 ligases FBXO32 and MURF1 [30].…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…However, the ubiquitin-proteasome system likely confounds this investigation, as this pathway can also be inhibited by insulin and amino acids. 653 In addition, multiple disease states have been associated with an altered activity of the ubiquitin-proteasome system, further complicating the use of the amino acid exchange rate as a marker of autophagy. 654 When analyzing basal autophagic level in vivo using GFP-LC3 transgenic mice, 126 one pitfall is that GFP-LC3 expression is driven by the cytomegalovirus enhancer and β-actin (CAG) promoter, so that the intensity of the GFP signal may not always represent the actual autophagic activity, but rather the CAG promoter activity in individual cells.…”

Dietary restriction in moderately obese rats improves body size and glucose handling without the renal and hepatic alterations observed with a high-protein diet

“…HP diets can have a profound impact on the functioning of the liver because of its central role in amino acid and protein metabolism (Chotechuang et al 2011), with demonstrated reductions in serum levels of glycine and cysteine (Cys), 2 amino acids used for homocysteine (Hcy) clearance (Holecek and Kovarik 2011;Ohuchi et al 2009). Elevated serum Hcy levels have been observed with long-term HP feeding in some studies (Wakefield et al 2011;Xiao et al 2013), but not in others (Noakes et al 2005), and this is of interest because the serum Hcy level is being considered as an independent risk factor for many cardiovascular diseases (Refsum et al 1998).…”

“…Elevated serum Hcy levels have been observed with long-term HP feeding in some studies (Wakefield et al 2011;Xiao et al 2013), but not in others (Noakes et al 2005), and this is of interest because the serum Hcy level is being considered as an independent risk factor for many cardiovascular diseases (Refsum et al 1998). Liver enlargement in response to HP diets has been observed in rats (Chotechuang et al 2011) and mice (Hammond and Janes 1998), but no adverse effects on liver function were observed in overweight and obese human subjects consuming HP diets for 1 year (Li et al 2010). Studies in mice and humans have shown prevention and reversal of hepatic steatosis (Bortolotti et al 2009;Garcia-Caraballo et al 2013) and reduction in inflammation with HP diets (Garcia-Caraballo et al 2013).…”