Leucine and mTORc1 act independently to regulate 2-deoxyglucose uptake in L6 myotubes

Qiong Yin,Tim Parr,Andrew J Murton,John M Brameld

doi:10.1007/s00726-020-02829-0

Abstract

Chronic mTORc1 hyperactivation via obesity-induced hyperleucinaemia has been implicated in the development of insulin resistance, yet the direct impact of leucine on insulin-stimulated glucose uptake in muscle cells remains unclear. To address this, differentiated L6 myotubes were subjected to various compounds designed to either inhibit mTORc1 activity (rapamycin), blunt leucine intracellular import (BCH), or activate mTORc1 signalling (3BDO), prior to the determination of the uptake of the glucose analogue, 2-deoxyglucose (2-DG), in response to 1 mM insulin. In separate experiments, L6 myotubes were subject to various media concentrations of leucine (0–0.8 mM) for 24 h before 2-DG uptake in response to insulin was assessed. Both rapamycin and BCH blunted 2-DG uptake, irrespective of insulin administration, and this occurred in parallel with a decline in mTOR, 4E-BP1, and p70S6K phosphorylation status, but little effect on AKT phosphorylation. In contrast, reducing leucine media concentrations suppressed 2-DG uptake, both under insulin- and non-insulin-stimulated conditions, but did not alter the phosphorylation state of AKT-mTORc1 components examined. Unexpectedly, 3BDO failed to stimulate mTORc1 signalling, but, nonetheless, caused a significant increase in 2-DG uptake under non-insulin-stimulated conditions. Both leucine and mTORc1 influence glucose uptake in muscle cells independent of insulin administration, and this likely occurs via distinct but overlapping mechanisms.

Highlights

The global incidence of diabetes continues to increase unabated, with 451 million adults in 2017 estimated to have the condition, a number that is expected to increase to 693 million by 2045 (Cho et al 2018)
In attempting to delineate the aetiology of type-2 diabetes mellitus (T2DM) which accounts for the majority of cases, it has been proposed that chronic increases in circulating concentrations of branched chain amino acids (BCAA) could contribute to the impaired ability of insulin to stimulate peripheral glucose disposal
Utilizing cultured cells, we circumvented the issues associated with the impact of leucine on distal tissues and systems, and by applying a multipronged approach to modulate leucine transport and availability, we demonstrated that the amino acid exerts a direct effect on glucose uptake in muscle cells, albeit the lack of a direct measure of intracellular free leucine concentrations is a limitation of the current study

Summary

Introduction

The global incidence of diabetes continues to increase unabated, with 451 million adults in 2017 estimated to have the condition, a number that is expected to increase to 693 million by 2045 (Cho et al 2018). In attempting to delineate the aetiology of type-2 diabetes mellitus (T2DM) which accounts for the majority of cases, it has been proposed that chronic increases in circulating concentrations of branched chain amino acids (BCAA) could contribute to the impaired ability of insulin to stimulate peripheral glucose disposal. Leucine is a potent stimulator of protein synthesis within muscle cells, a process achieved through activation of the kinase mammalian target of rapamycin (mTORc1), which, in turn, drives translation initiation as a consequence of the phosphorylation of downstream targets 4E-BP1 and p70S6K (see Murton et al 2008). In recent years, it has been suggested

Objectives

Methods

Results

Discussion

Conclusion