Quantitative comparison of presenilin protein expression reveals greater activity of PS2-γ-secretase.

γ-secretase processing of amyloid precursor protein (APP) has long been of interest in the pathological progression of Alzheimer's disease (AD) due to its role in the generation of amyloid-ß. The catalytic component of the enzyme is the presenilins of which there are two homologues, Presenilin-1 (PS1) and Presenilin-2 (PS2). The field has focussed on the PS1 form of this enzyme, as it is typically considered the more active at APP processing. However, much of this work has been completed without appropriate consideration of the specific levels of protein expression of PS1 and PS2. We propose that expression is an important factor in PS1- and PS2-γ-secretase activity, and that when this is considered, PS1 does not have greater activity than PS2. We developed and validated tools for quantitative assessment of PS1 and PS2 protein expression levels to enable the direct comparison of PS in exogenous and endogenous expression systems, in HEK-293 PS1 and/or PS2 knockout cells. We show that exogenous expression of Myc-PS1-NTF is 5.5-times higher than Myc-PS2-NTF. Quantitating endogenous PS protein levels, using a novel PS1/2 fusion standard we developed, showed similar results. When the marked difference in PS1 and PS2 protein levels is considered, we show that compared to PS1-γ-secretase, PS2-γ-secretase has equal or more activity on APP and Notch1. This study has implications for understanding the PS1- and PS2-specific contributions to substrate processing, and their potential influence in AD pathogenesis.

Therapeutic blockade of ER stress and inflammation prevents NASH and progression to HCC.

The incidence of hepatocellular carcinoma (HCC) is rapidly rising largely because of increased obesity leading to nonalcoholic steatohepatitis (NASH), a known HCC risk factor. There are no approved treatments to treat NASH. Here, we first used single-nucleus RNA sequencing to characterize a mouse model that mimics human NASH-driven HCC, the MUP-uPA mouse fed a high-fat diet. Activation of endoplasmic reticulum (ER) stress and inflammation was observed in a subset of hepatocytes that was enriched in mice that progress to HCC. We next treated MUP-uPA mice with the ER stress inhibitor BGP-15 and soluble gp130Fc, a drug that blocks inflammation by preventing interleukin-6 trans-signaling. Both drugs have progressed to phase 2/3 human clinical trials for other indications. We show that this combined therapy reversed NASH and reduced NASH-driven HCC. Our data suggest that these drugs could provide a potential therapy for NASH progression to HCC.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential.

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

TWEAK/Fn14 Signalling Regulates the Tissue Microenvironment in Chronic Pancreatitis.

Chronic pancreatitis increases the risk of developing pancreatic cancer through the upregulation of pathways favouring proliferation, fibrosis, and sustained inflammation. We established in previous studies that the ligand tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) signals through its cognate receptor fibroblast growth factor-inducible 14 (Fn14) to regulate these underlying cellular processes in the chronic liver injury niche. However, the role of the TWEAK/Fn14 signalling pathway in pancreatic disease is entirely unknown. An analysis of publicly available datasets demonstrated that the TWEAK receptor Fn14 is upregulated in pancreatitis and pancreatic adenocarcinoma, with single cell RNA sequencing revealing pancreatic ductal cells as the main Fn14 producers. We then used choline-deficient, ethionine-supplemented (CDE) diet feeding of wildtype C57BL/6J and Fn14 knockout littermates to (a) confirm CDE treatment as a suitable model of chronic pancreatitis and (b) to investigate the role of the TWEAK/Fn14 signalling pathway in pancreatic ductal proliferation, as well as fibrotic and inflammatory cell dynamics. Our time course data obtained at three days, three months, and six months of CDE treatment reveal that a lack of TWEAK/Fn14 signalling significantly inhibits the establishment and progression of the tissue microenvironment in CDE-induced chronic pancreatitis, thus proposing the TWEAK/Fn14 pathway as a novel therapeutic target.

Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution.

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model.

Maraviroc Prevents HCC Development by Suppressing Macrophages and the Liver Progenitor Cell Response in a Murine Chronic Liver Disease Model.

Maraviroc (MVC), a CCR5 antagonist, reduces liver fibrosis, injury and tumour burden in mice fed a hepatocarcinogenic diet, suggesting it has potential as a cancer therapeutic. We investigated the effect of MVC on liver progenitor cells (LPCs) and macrophages as both have a role in hepatocarcinogenesis. Mice were fed the hepatocarcinogenic choline-deficient, ethionine-supplemented diet (CDE) ± MVC, and immunohistochemistry, RNA and protein expression were used to determine LPC and macrophage abundance, migration and related molecular mechanisms. MVC reduced LPC numbers in CDE mice by 54%, with a smaller reduction seen in macrophages. Transcript and protein abundance of LPC-associated markers correlated with this reduction. The CDE diet activated phosphorylation of AKT and STAT3 and was inhibited by MVC. LPCs did not express Ccr5 in our model; in contrast, macrophages expressed high levels of this receptor, suggesting the effect of MVC is mediated by targeting macrophages. MVC reduced CD45+ cells and macrophage migration in liver and blocked the CDE-induced transition of liver macrophages from an M1- to M2-tumour-associated macrophage (TAM) phenotype. These findings suggest MVC has potential as a re-purposed therapeutic agent for treating chronic liver diseases where M2-TAM and LPC numbers are increased, and the incidence of HCC is enhanced.

Previous liver regeneration induces fibro-protective mechanisms during thioacetamide-induced chronic liver injury.

Chronic liver injury is characterised by continuous or repeated epithelial cell loss and inflammation. Hepatic wound healing involves matrix deposition through activated hepatic stellate cells (HSCs) and the expansion of closely associated Ductular Reactions and liver progenitor cells (LPCs), which are thought to give rise to new epithelial cells. In this study, we used the murine thioacetamide (TAA) model to reliably mimic these injury and regeneration dynamics and assess the impact of a recovery phase on subsequent liver injury and fibrosis. Age-matched naïve or 6-week TAA-treated/4-week recovered mice (C57BL/6 J, n = 5-9) were administered TAA for six weeks (C57BL/6 J, n = 5-9). Sera and liver tissues were harvested at key time points to assess liver injury biochemically, by real-time PCR for fibrotic mediators, Sirius Red staining and hydroxyproline assessment for collagen deposition as well as immunofluorescence for inflammatory, HSC and LPC markers. In addition, primary HSCs and the HSC cell line LX-2 were co-cultured with the well-characterised LPC line BMOL and analysed for potential changes in expression of fibrogenic mediators. Our data demonstrate that recovery from a previous TAA insult, with LPCs still present on day 0 of the second treatment, led to a reduced TAA-induced disease progression with less severe fibrosis than in naïve TAA-treated animals. Importantly, primary activated HSCs significantly reduced pro-fibrogenic gene expression when co-cultured with LPCs. Taken together, previous TAA injury established a fibro-protective molecular and cellular microenvironment. Our proof-of principle HSC/LPC co-culture data demonstrate that LPCs communicate with HSCs to regulate fibrogenesis, highlighting a key role for LPCs as regulatory cells during chronic liver disease.

Nitric Oxide and Redox State Measurements in Pancreatic Beta Cells.

The role of oxidative stress in the pathogenesis of type 2 diabetes (T2D), especially pancreatic ß-cell dysfunction and death, has become apparent in the last two decades. Peroxidase- and catalase-based antioxidant mechanisms are particularly weak in ß-cells and can be easily overwhelmed by excessive production of reactive oxygen and nitrogen species in the course of pathological processes. Recent research has attempted to define in detail the mechanistic aspects of oxidative stress-induced ß-cell dysfunction. Here, we describe the procedures for the measurement of various parameters important to assess oxidative stress in pancreatic ß-cells. Detailed protocols for determination of nitric oxide (NO) production, the glutathione redox status, and general oxidative status in ß-cells are presented in this chapter.

Effects of vitamin D on primary human skeletal muscle cell proliferation, differentiation, protein synthesis and bioenergetics.

The active form of Vitamin D (1,25(OH)2D), has been suggested to have a regulatory role in skeletal muscle function and metabolism, however, the effects and mechanisms of vitamin D (VitD) action in this tissue remain to be fully established. In this study, we have used primary human skeletal muscle myoblast (HSMM) cells that display typical characteristics of human skeletal muscle function and protein levels, to investigate the effects of the active form of VitD on proliferation, differentiation, protein synthesis and bioenergetics. Myoblast cells were treated with 100 nM of VitD for 24 h, 48 h, 72 h and five days (cells were differentiated into myotubes) and then analyses were performed. We report that VitD inhibits myoblast proliferation and enhances differentiation by altering the expression of myogenic regulatory factors. In addition, we found that protein synthesis signaling improved in myotubes after VitD treatment in the presence of insulin. We also report an increase in oxygen consumption rate after 24 h of treatment in myoblasts and after 5 days of treatment in myotubes after VitD exposure. VitD significantly impacted HSMM myogenesis, as well as protein synthesis in the presence of insulin.

Oxidative stress pathways in pancreatic ß-cells and insulin-sensitive cells and tissues: importance to cell metabolism, function, and dysfunction.

It is now accepted that nutrient abundance in the blood, especially glucose, leads to the generation of reactive oxygen species (ROS), ultimately leading to increased oxidative stress in a variety of tissues. In the absence of an appropriate compensatory response from antioxidant mechanisms, the cell, or indeed the tissue, becomes overwhelmed by oxidative stress, leading to the activation of intracellular stress-associated pathways. Activation of the same or similar pathways also appears to play a role in mediating insulin resistance, impaired insulin secretion, and late diabetic complications. The ability of antioxidants to protect against the oxidative stress induced by hyperglycemia and elevated free fatty acid (FFA) levels in vitro suggests a causative role of oxidative stress in mediating the latter clinical conditions. In this review, we describe common biochemical processes associated with oxidative stress driven by hyperglycemia and/or elevated FFA and the resulting clinical outcomes: ß-cell dysfunction and peripheral tissue insulin resistance.

Mechanisms of vitamin D action in skeletal muscle.

Vitamin D receptor expression and associated function have been reported in various muscle models, including C2C12, L6 cell lines and primary human skeletal muscle cells. It is believed that 1,25-hydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D, has a direct regulatory role in skeletal muscle function, where it participates in myogenesis, cell proliferation, differentiation, regulation of protein synthesis and mitochondrial metabolism through activation of various cellular signalling cascades, including the mitogen-activated protein kinase pathway(s). It has also been suggested that 1,25(OH)2D3 and its associated receptor have genomic targets, resulting in regulation of gene expression, as well as non-genomic functions that can alter cellular behaviour through binding and modification of targets not directly associated with transcriptional regulation. The molecular mechanisms of vitamin D signalling, however, have not been fully clarified. Vitamin D inadequacy or deficiency is associated with muscle fibre atrophy, increased risk of chronic musculoskeletal pain, sarcopenia and associated falls, and may also decrease RMR. The main purpose of the present review is to describe the molecular role of vitamin D in skeletal muscle tissue function and metabolism, specifically in relation to proliferation, differentiation and protein synthesis processes. In addition, the present review also includes discussion of possible genomic and non-genomic pathways of vitamin D action.

The A allele of the UCP2 -866G/A polymorphism changes UCP2 promoter activity in HUVECs treated with high glucose.

The mitochondrial uncoupling protein 2 (UCP2) decreases reactive oxygen species (ROS) formation by mitochondria. Our group previously showed that the UCP2 -866A allele was associated with risk of diabetic retinopathy (DR), which is caused by hyperglycemia-induced oxidative stress. To date, it is still unclear if the -866A allele directly affects UCP2 expression in endothelial cells. Thus, we investigated the effect of the A allele on UCP2 promoter activity in HUVECs treated with high glucose (HG) or hydrogen peroxide (H2O2). To quantify UCP2 promoter activity, HUVECs were transfected with pGL3 plasmids containing the UCP2 promoter and the firefly luciferase coding sequence. Experimental groups were: (1) pGL3-866G-transfected cells and (2) pGL3-866A cells, both under normal (4 mM) or HG (25 mM) concentrations for 24 h and 48 h or incubated with H2O2 (0.1 mM) for 1 h. UCP2 promoter activity was monitored by Luminescent Dual-luciferase Assay. HG induced an upregulation of UCP2 promoter activity in PGL3-866G cells after 24 h of treatment (P = 0.027), but not after 48 h. Compared to pGL3-866G cells, pGL3-866A cells seems to have reduced UCP2 promoter activity following 24 h and 48 h of normal glucose treatment (P = 0.087 and P = 0.022). After HG treatment, pGL3-866A cells had more marked UCP2 downregulation (24 h: - 3.2-folds, P < 0.001; and 48 h: - 2.5-folds, P < 0.001 vs. G cells). Both pGL3-866G and pGL3-866A cells treated with H2O2 showed a ≅ 4-fold increase in UCP2 promoter activity (both P < 0.001). The -866A allele modifies UCP2 promoter activity in HUVECs under HG treatment but not in the H2O2 condition.

Glutamine deprivation induces metabolic adaptations associated with beta cell dysfunction and exacerbate lipotoxicity.

Studies have reported that plasma glutamine is reduced in type 2 diabetes (T2D) patients. Glutamine supplementation improves glycaemic control, however the mechanisms are unclear. Here, we evaluated in vitro the pancreatic beta cell bioenergetic and insulin secretory responses to various levels of glutamine availability, or treatment in the presence of an inhibitor of intracellular glutamine metabolism. The impact of glutamine deprivation to the pathological events induced by the saturated fatty acid palmitate was also investigated. Glutamine deprivation induced a reduction in mitochondrial respiration and increase in glucose uptake and utilization. This phenotype was accompanied by impairment in beta cell function, as demonstrated by diminished insulin production and secretion, and activation of the unfolded protein response pathway. Palmitate led to insulin secretory dysfunction, loss of viability and apoptosis. Importantly, glutamine deprivation significantly exacerbated these phenotypes, suggesting that low glutamine levels could participate in the process of beta cell dysfunction in T2D.

Renal effects of exendin-4 in an animal model of brain death.

Organ transplantation is the gold standard therapy for the majority of patients with terminal organ failure. However, it is still a limited treatment especially due to the low number of brain death (BD) donors in relation to the number of waiting list recipients. Strategies to increase the quantity and quality of donor organs have been studied, and the administration of exendin-4 (Ex-4) to the donor may be a promising approach. Male Wistar rats were randomized into 3 groups: (1) control, without central nervous system injury; (2) BD induced experimentally, and (3) BD induced experimentally + Ex-4 administered immediately after BD induction. After BD induction, animals were monitored for 6 h before blood collection and kidney biopsy. Kidney function was assessed by biochemical quantification of plasma kidney markers. Gene and protein expressions of inflammation- and stress-related genes were evaluated by RT-qPCR and immunoblot analysis. Animals treated with Ex-4 had lower creatinine and urea levels compared with controls. BD induced oxidative stress in kidney tissue through increased expression of Ucp2, Sod2 and Inos, and Ex-4 administration reduced the expression of these genes. Ex-4 also induced increased expression of the anti-apoptotic Bcl2 gene. Nlrp3 and Tnf expressions were up-regulated in the BD group compared with controls, but Ex-4 treatment had no effect on these genes. Our findings suggest that Ex-4 administration in BD rats reduces BD-induced kidney damage by decreasing the expression of oxidative stress genes and increasing the expression of Bcl2.

Method Protocols for Metabolic and Functional Analysis of the BRIN-BD11 ß-Cell Line: A Preclinical Model for Type 2 Diabetes.

In type 2 diabetes, prolonged dysregulation of signalling and ß-cell metabolic control leads to ß-cell dysfunction, and is increasingly associated with abnormal metabolic states which disrupt normal cellular physiology. Utilization of appropriate ß-cell models enables a systematic approach to understand the impact of perturbations to the biological system. The BRIN-BD11 ß-cell line is a useful, pre-clinical cell model for ß-cell dysfunction associated with type 2 diabetes, among other metabolic disorders. The present chapter describes detection and analysis of ß-cell dysfunction with respect to changes in bioenergetics and metabolism, generation of intracellular reactive oxygen species, and acute and chronic insulin secretion in the BRIN-BD11 cell line.

Lupin seed hydrolysate promotes G-protein-coupled receptor, intracellular Ca2+ and enhanced glycolytic metabolism-mediated insulin secretion from BRIN-BD11 pancreatic beta cells.

Lupin seed proteins have been reported to exhibit hypoglycaemic effects in animals and humans following oral administration, however little is known about its mechanism of action. This study investigated the signalling pathway(s) responsible for the insulinotropic effect of the hydrolysate obtained from lupin (Lupinus angustifolius L.) seed extracts utilizing BRIN-BD11 ß-cells. The extract was treated with digestive enzymes to give a hydrolysate rich in biomolecules ≤7 kDa. Cells exhibited hydrolysate induced dose-dependent stimulation of insulin secretion and enhanced intracellular Ca2+ and glucose metabolism. The stimulatory effect of the hydrolysate was potentiated by depolarizing concentrations of KCl and was blocked by inhibitors of the ATP sensitive K+ channel, Gαq protein, phospholipase C (PLC) and protein kinase C (PKC). These findings reveal a novel mechanism for lupin hydrolysate stimulated insulin secretion via Gαq mediated signal transduction (Gαq/PLC/PKC) in the ß-cells. Thus, lupin hydrolysates may have potential for nutraceutical treatment in type 2 diabetes.

Pleiotropic Effects of GLP-1 and Analogs on Cell Signaling, Metabolism, and Function.

The incretin hormone Glucagon-Like Peptide-1 (GLP-1) is best known for its "incretin effect" in restoring glucose homeostasis in diabetics, however, it is now apparent that it has a broader range of physiological effects in the body. Both in vitro and in vivo studies have demonstrated that GLP-1 mimetics alleviate endoplasmic reticulum stress, regulate autophagy, promote metabolic reprogramming, stimulate anti-inflammatory signaling, alter gene expression, and influence neuroprotective pathways. A substantial body of evidence has accumulated with respect to how GLP-1 and its analogs act to restore and maintain normal cellular functions. These findings have prompted several clinical trials which have reported GLP-1 analogs improve cardiac function, restore lung function and reduce mortality in patients with obstructive lung disease, influence blood pressure and lipid storage, and even prevent synaptic loss and neurodegeneration. Mechanistically, GLP-1 elicits its effects via acute elevation in cAMP levels, and subsequent protein kinase(s) activation, pathways well-defined in pancreatic ß-cells which stimulate insulin secretion in conjunction with elevated Ca2+ and ATP. More recently, new studies have shed light on additional downstream pathways stimulated by chronic GLP-1 exposure, findings which have direct relevance to our understanding of the potential therapeutic effects of longer lasting analogs recently developed for clinical use. In this review, we provide a comprehensive description of the diverse roles for GLP-1 across multiple tissues, describe downstream pathways stimulated by acute and chronic exposure, and discuss novel pleiotropic applications of GLP-1 mimetics in the treatment of human disease.

Oleoyl-lysophosphatidylinositol enhances glucagon-like peptide-1 secretion from enteroendocrine L-cells through GPR119.

The gastrointestinal tract is increasingly viewed as critical in controlling glucose metabolism, because of its role in secreting multiple glucoregulatory hormones, such as glucagon like peptide-1 (GLP-1). Here we investigate the molecular pathways behind the GLP-1- and insulin-secreting capabilities of a novel GPR119 agonist, Oleoyl-lysophosphatidylinositol (Oleoyl-LPI). Oleoyl-LPI is the only LPI species able to potently stimulate the release of GLP-1 in vitro, from murine and human L-cells, and ex-vivo from murine colonic primary cell preparations. Here we show that Oleoyl-LPI mediates GLP-1 secretion through GPR119 as this activity is ablated in cells lacking GPR119 and in colonic primary cell preparation from GPR119-/- mice. Similarly, Oleoyl-LPI-mediated insulin secretion is impaired in islets isolated from GPR119-/- mice. On the other hand, GLP-1 secretion is not impaired in cells lacking GPR55 in vitro or in colonic primary cell preparation from GPR55-/- mice. We therefore conclude that GPR119 is the Oleoyl-LPI receptor, upstream of ERK1/2 and cAMP/PKA/CREB pathways, where primarily ERK1/2 is required for GLP-1 secretion, while CREB activation appears dispensable.

Insulin and IGF-1 receptor autocrine loops are not required for Exendin-4 induced changes to pancreatic ß-cell bioenergetic parameters and metabolism in BRIN-BD11 cells.

Pharmacological long lasting Glucagon-like peptide-1 (GLP-1) analogues, such as Exendin-4, have become widely used diabetes therapies. Chronic GLP-1R stimulation has been linked to ß-cell protection and these pro-survival actions of GLP-1 are dependent on the activation of the mammalian target of rapamycin (mTOR) leading to accumulation of Hypoxia inducible factor 1 alpha (HIF-1α). Recent studies from our lab indicate that prolonged GLP-1R stimulation promotes metabolic reprograming of ß-cells towards a highly glycolytic phenotype and activation of the mTOR/HIF-1α pathway was required for this action. We hypothesised that GLP-1 induced metabolic changes depend on the activation of mTOR and HIF-1α, in a cascade that occurs after triggering of a potential Insulin-like growth factor 1 receptor (IGF-1R) or the Insulin receptor (IR) autocrine loops. Loss of function of these receptors, through the use of small interfering RNA, or neutralizing antibodies directed towards their products, was undertaken in conjunction with functional assays. Neither of these strategies mitigated the effect of GLP-1 on glucose uptake, protein expression or bioenergetic flux. Our data indicates that activation of IGF-1R and/or the IR autocrine loops resulting in ß-cell protection and function, involve mechanisms independent to the enhanced metabolic effects resulting from sustained GLP-1R activation.

GLP-1 receptor signalling promotes ß-cell glucose metabolism via mTOR-dependent HIF-1α activation.

Glucagon-like peptide-1 (GLP-1) promotes insulin secretion from pancreatic ß-cells in a glucose dependent manner. Several pathways mediate this action by rapid, kinase phosphorylation-dependent, but gene expression-independent mechanisms. Since GLP-1-induced insulin secretion requires glucose metabolism, we aimed to address the hypothesis that GLP-1 receptor (GLP-1R) signalling can modulate glucose uptake and utilization in ß-cells. We have assessed various metabolic parameters after short and long exposure of clonal BRIN-BD11 ß-cells and rodent islets to the GLP-1R agonist Exendin-4 (50 nM). Here we report for the first time that prolonged stimulation of the GLP-1R for 18 hours promotes metabolic reprogramming of ß-cells. This is evidenced by up-regulation of glycolytic enzyme expression, increased rates of glucose uptake and consumption, as well as augmented ATP content, insulin secretion and glycolytic flux after removal of Exendin-4. In our model, depletion of Hypoxia-Inducible Factor 1 alpha (HIF-1α) impaired the effects of Exendin-4 on glucose metabolism, while pharmacological inhibition of Phosphoinositide 3-kinase (PI3K) or mTOR completely abolished such effects. Considering the central role of glucose catabolism for stimulus-secretion coupling in ß-cells, our findings suggest that chronic GLP-1 actions on insulin secretion include elevated ß-cell glucose metabolism. Moreover, our data reveal novel aspects of GLP-1 stimulated insulin secretion involving de novo gene expression.