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Chronic metabolic acidosis plays a role in cachexia by enhancing total proteolysis in skeletal muscle. Glucocorticoid also triggers proteolysis and plays a permissive role in the effect of acidosis. The System A amino acid transporter SNAT2/SLC38A2 is ubiquitously expressed in mammalian cells including muscle, performing Na+‐dependent active import of neutral amino acids, and is strongly inhibited by low pH. Exposure of rat skeletal muscle cell line L6‐G8C5 to low pH rapidly inhibits SNAT2 transport activity and enhances total proteolysis rate. Pharmacological inhibition or silencing of SNAT2 also enhances proteolysis. This study tests the hypothesis that the glucocorticoid dexamethasone (DEX), like low pH, inhibits SNAT2 activity in L6‐G8C5 myotubes, thus contributing to total proteolysis. Incubation with 500 nM DEX for 4 h reduced the System A amino acid transport rate to half the rate in control cultures. This inhibition depended on glucocorticoid receptor‐mediated gene transcription, but SNAT2 mRNA levels were unaffected by DEX. In contrast, the SNAT2 protein assessed by immunoblotting was significantly depleted. The co‐inhibitory effects of DEX and low pH on System A transport activity were additive in stimulating total proteolysis. In keeping with this mechanism, DEX's inhibitory effect on SNAT2 transport activity was significantly blunted by the proteasome inhibitor MG132. Proof of principle was achieved in similar experiments using recombinant expression of a GFP‐tagged SNAT2 fusion protein in HEK293A cells. It is concluded that DEX acutely depletes the SNAT2 transporter protein, at least partly through proteasome‐dependent degradation of this functionally important transporter. [ABSTRACT FROM AUTHOR]

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca2+ through L-type voltage gated Ca2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation. [ABSTRACT FROM AUTHOR]

The branched-chain amino acids (BCAA) leucine, isoleucine and valine, are essential amino acids that play a critical role in cellular signalling and metabolism. They acutely stimulate insulin secretion and activate the regulatory serine/threonine kinase mammalian target of rapamycin complex 1 (mTORC1), a kinase that promotes increased β-cell mass and function. The effects of BCAA on cellular function are dependent on their active transport into the mammalian cells via amino acid transporters and thus the expression and activity of these transporters likely influence β-cell signalling and function. In this report, we show that the System-L transporters are required for BCAA uptake into clonal β-cell lines and pancreatic islets, and that these are essential for signalling to mTORC1. Further investigation revealed that the System-L amino acid transporter 1 (LAT1) is abundantly expressed in the islets, and that knockdown of LAT1 using siRNA inhibits mTORC1 signalling, leucine-stimulated insulin secretion and islet cell proliferation. In summary, we show that the LAT1 is required for regulating β-cell signalling and function in islets and thus may be a novel pharmacological/nutritional target for the treatment and prevention of type 2 diabetes. [ABSTRACT FROM AUTHOR]

Glucagon-like peptide 1 receptor (GLP1R) agonists, such as exendin-4, potentiate glucose-stimulated insulin secretion and are currently used in the management of type 2 diabetes. Interestingly, GLP1R agonists also have the ability to augment β-cell mass. In this report, we provide evidence that in the presence of glucose, exendin-4 stimulates rodent islet cell DNA replication via the activation of ribosomal protein S6 kinase 1 (S6K1) and that this is mediated by the protein kinase B (PKB)-dependent activation of mTOR complex 1 (mTORC1). We show that activation of this pathway is caused by the autocrine or paracrine activation of the IGF1 receptor (IGF1R), as siRNA-mediated knockdown of the IGF1R effectively blocked exendin-4-stimulated PKB and mTORC1 activation. In contrast, pharmacological inactivation of the epidermal growth factor receptor has no discernible effect on exendin-4-stimulated PKB or mTORC1 activation. Therefore, we conclude that GLP1R agonists stimulate β-cell proliferation via the PKB-dependent stimulation of mTORC1/S6K1 whose activation is mediated through the autocrine/paracrine activation of the IGF1R. This work provides a better understanding of the molecular basis of GLP1 agonist-induced β-cell proliferation which could potentially be exploited in the identification of novel drug targets that increase β-cell mass. [ABSTRACT FROM AUTHOR]

Inositol-requiring enzyme 1 alpha (IRE1α) is an endoplasmic reticulum (ER)-transmembrane endonuclease that is activated in response to ER stress as part of the unfolded protein response (UPR). Chronic activation of the UPR has been implicated in the pathogenesis of many common diseases including diabetes, cancer, and neurological pathologies such as Huntington's and Alzheimer's disease. 7-Hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde (4µ8C) is widely used as a specific inhibitor of IRE1α ribonuclease activity (IC 50 of 6.89 µM in cultured cells). However, in this paper, we demonstrate that 4µ8C acts as a potent reactive oxygen species (ROS) scavenger, both in a cell-free assay and in cultured cells, at concentrations lower than that widely used to inhibit IRE1α activity. In vitro we show that, 4µ8C effectively decreases xanthine/xanthine oxidase catalysed superoxide production with an IC 50 of 0.2 µM whereas in cultured endothelial and clonal pancreatic β-cells, 4µ8C inhibits angiotensin II-induced ROS production with IC 50 values of 1.92 and 0.29 µM, respectively. In light of this discovery, conclusions reached using 4µ8C as an inhibitor of IRE1α should be carefully evaluated. However, this unexpected off-target effect of 4µ8C may prove therapeutically advantageous for the treatment of pathologies that are thought to be caused by, or exacerbated by, both oxidative and ER stress such as endothelial dysfunction and/or diabetes., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In the present study, we demonstrate that, in pancreatic beta-cells, eIF2alpha (eukaryotic initiation factor 2alpha) phosphorylation in response to a decrease in glucose concentration is primarily mediated by the activation of PERK [PKR (protein kinase RNA activated)-like endoplasmic reticulum kinase]. We provide evidence that this increase in PERK activity is evoked by a decrease in the energy status of the cell via a potentially novel mechanism that is independent of IRE1 (inositol requiring enzyme 1) activation and the accumulation of unfolded nascent proteins within the endoplasmic reticulum. The inhibition of eIF2alpha phosphorylation in glucose-deprived cells by the overexpression of dominant-negative PERK or an N-terminal truncation mutant of GADD34 (growth-arrest and DNA-damage-inducible protein 34) leads to a 53% increase in the rate of total protein synthesis. Polysome analysis revealed that this coincides with an increase in the amplitude but not the number of ribosomes per mRNA, indicating that eIF2alpha dephosphorylation mobilizes hitherto untranslated mRNAs on to polysomes. In summary, we show that PERK is activated at low glucose concentrations in response to a decrease in energy status and that this plays an important role in glucose-regulated protein synthesis in pancreatic beta-cells.

In pancreatic beta-cells, following an acute (within 1 h) increase in glucose concentration, there are rapid changes in the expression of a large subset of proteins. The change in the expression of many of these proteins is mediated by a post-transcriptional mechanism through either increases or decreases in the rate of translation from pre-existing transcripts. These proteins, whose synthesis is rapidly up- or down-regulated in response to glucose, are likely important in mounting the correct response to changes in plasma glucose concentrations. However, the vast majority of these proteins remain unidentified. Therefore, in order to identify these proteins, we analysed changes in the levels of mRNAs associated with polysomes (i.e. actively translating mRNAs) isolated from mouse insulinoma 6 cells incubated at either 0.5 or 20 mM glucose for 1 h. Changes in the levels of polysomal mRNAs in response to glucose were analysed using affymetrix oligonucleotide microarrays (translational profiling). This work revealed that, in response to a change in glucose concentration, the abundance of 313 transcripts associated with polysomes changed by more than 1.5-fold, of which the abundance of 37 changed by more than twofold. The majority of these transcripts encoded proteins associated with metabolism or gene expression. More detailed analysis showed that a number of mRNAs encoding proteins associated with the induction of oxidative stress, including thioredoxin-2 and thioredoxin-interacting protein were rapidly redistributed onto heavier polysomes at high glucose concentration, indicating an increase in their expression. At low glucose concentration, when the general rate of protein synthesis is low, a number of mRNAs encoding integrated stress response proteins, including ATF4 and CHOP10, associate with heavier polysomes, indicating that their expression is up-regulated. In conclusion, translational profiling has revealed that, at either low or at high glucose concentration, beta-cells rapidly increase the synthesis of a specific subset of proteins that are likely important in maintaining beta-cell integrity and survival during conditions of nutritional stress.

Glucose acutely stimulates proinsulin synthesis in pancreatic beta-cells through a poorly understood post-transcriptional mechanism. In the present study, we demonstrate in pancreatic beta-cells that glucose stimulates the recruitment of ribosome-associated proinsulin mRNA, located in the cytoplasm, to the ER (endoplasmic reticulum), the site of proinsulin synthesis, and that this plays an important role in glucose-stimulated proinsulin synthesis. Interestingly, glucose has greater stimulatory effect on the recruitment of proinsulin mRNA to the ER compared with other mRNAs encoding secretory proteins. This, as far as we are aware, is the first example whereby mRNAs encoding secretory proteins are selectively recruited to the ER and provides a novel regulatory mechanism for secretory protein synthesis. Contrary to previous reports, and importantly in understanding the mechanism by which glucose stimulates proinsulin synthesis, we demonstrate that there is no large pool of 'free' proinsulin mRNA in the cytoplasm and that glucose does not increase the rate of de novo initiation on the proinsulin mRNA. However, we show that glucose does stimulate the rate of ribosome recruitment on to ribosome-associated proinsulin mRNA. In conclusion, our results provide evidence that the selective recruitment of proinsulin mRNA to the ER, together with increases in the rate of initiation are important mediators of glucose-stimulated proinsulin synthesis in pancreatic beta-cells.