Signalling pathways and combinatory effects of insulin and amino acids in isolated rat hepatocytes

Abstract

Liver metabolism is influenced by hormones and nutrients. Amino acids such as glutamine or leucine induce an anabolic response, which resembles that of insulin in muscle and adipose tissue. In this work, the signalling pathways and the effects of insulin were compared to those of glutamine and leucine in isolated hepatocytes from normal and streptozotocin-diabetic rats. Glutamine increased cell volume and induced an anabolic response characterized by an activation of acetyl-CoA carboxylase (ACC), glycogen synthase (GS) and p70 ribosomal S6 kinase (p70S6K), the key enzymes in fatty acid, glycogen and protein synthesis, respectively. The effects of glutamine were independent of insulin and did not share its signalling components. Leucine, which is poorly metabolized by the liver and does not modify cell volume, activated ACC and p70S6K, and exerted a synergistic effect on the glutamine-induced activation of ACC and p70S6K.These amino acids did not affect insulin signalling. Insulin alone had no anabolic effect in hepatocytes, despite the activation of protein kinase B. Nevertheless, it enhanced the activation of ACC and p70S6K induced by leucine. However, insulin injected intravenously activated rat liver p70S6K. In hepatocytes from streptozotocin-diabetic animals, the metabolic responses to the amino acids and insulin were similar to those in normal hepatocytes. We conclude that glutamine, insulin and leucine exert different effects that are mediated by different signalling pathways, although their effects are combinatory. The anabolic effect of insulin in hepatocytes was strictly dependent on the permissive action of leucine.Keywords: insulin; amino acids; p70S6K; acetyl-CoA carboxylase (ACC); diabetic rat.In recent years, experimental evidence demonstrating the involvement of certain amino acids in the control of metabolism and signal transduction has accumulated (reviewed in [1]). A cross-talk between nutrients and cell signalling was first reported in studies of the regulation of hepatic autophagy, a process responsible for starvationinduced proteolysis [2]. Autophagy is inhibited by amino acids even in the absence of insulin. The mechanism involves phosphorylation of ribosomal protein S6 [3,4], which is implicated in the control of translation of certain mRNAs (reviewed in [5,6]). An amino-acid-dependent activation of the ribosomal protein S6 kinase (p70S6K), the Ser/Thr protein kinase responsible for S6-phosphorylation, was first reported in hepatocytes [7], and confirmed in several tissues and cultured cells where branched-chain amino acids like leucine play a predominant role in p70S6K activation [8][9][10][11][12][13][14]. Moreover, the branched-chain amino acids amplify the effect of insulin in activating p70S6K (reviewed in [15]).Apart from their inhibition of proteolysis through p70S6K activation, amino acids also control fatty acid and glycogen metabolism. In rat hepatocytes, Na + -cotransported amino acids like glutamine, alanine or proline stimulate lipogenesis by activation of acetyl-CoA carboxyla...

Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR

“…The absence of BCATm from the liver may prevent "first-pass" losses of leucine and may thereby facilitate postprandial rises in plasma leucine concentration that would allow leucine to operate as a nutrient signal. The inability of liver to metabolize leucine is relevant to the hypothesis tested in this communication, because it has been demonstrated by several groups that leucine can regulate mTOR signaling in freshly isolated hepatocytes (6,36,37). It is unclear how leucine signaling to mTOR could be accomplished in hepatocytes if leucine metabolism was required to activate mTOR.…”

Involvement of Integrins and Src in Insulin Signaling toward Autophagic Proteolysis in Rat Liver

“…Proteolysis inhibition by swelling induced by either hypoosmolarity (15) or insulin (this study) in perfused rat liver is insensitive to rapamycin, indicating that mTOR is not involved. Moreover, in isolated hepatocytes hypoosmotic swelling and insulin do not increase phosphorylation of S6, a downstream target of mTOR involved in proteolysis inhibition by certain amino acids (34,48). However, both hypoosmolarity and insulin were reported to amplify amino acid-induced S6 phosphorylation in isolated hepatocytes (34,48), suggesting some synergy that obviously does not require cell adherence.…”

“…Moreover, in isolated hepatocytes hypoosmotic swelling and insulin do not increase phosphorylation of S6, a downstream target of mTOR involved in proteolysis inhibition by certain amino acids (34,48). However, both hypoosmolarity and insulin were reported to amplify amino acid-induced S6 phosphorylation in isolated hepatocytes (34,48), suggesting some synergy that obviously does not require cell adherence. In the present study, only 100 M leucine were added to the perfusion medium to prevent reutilization of [ 3 H]leucine for protein synthesis.…”

“…Insensitive to Rapamycin-mTOR plays an important role in the regulation of protein metabolism by nutrient availability (33)(34)(35)(36). Proteolysis inhibition induced by hypoosmotic swelling, however, is insensitive to rapamycin (15).…”

mTOR-Dependent Cell Survival Mechanisms

“…In starved cells, AMPK tightly binds ULK1, and this interaction is enhanced by rapamycin and disrupted by Rheb overexpression (Behrends et al 2010;. In addition, AMPK can also inhibit mTORC1 by phosphorylating and activating TSC2, and directly by phosphorylating the RAPTOR subunit, which inhibits mTORC1 activity (Krause et al 2002;Inoki et al 2003;Gwinn et al 2008). Interestingly, mTORC1 can be reactivated after prolonged starvation by the autolysosomal products generated by autophagy, indicating that a minimal level of mTORC1 activity is required for survival (Liang et al 2007;Matsui et al 2007;Herrero-Martin et al 2009).…”