Glucolipotoxicity promotes the capacity of the glycerolipid/NEFA cycle supporting the secretory response of pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to high glucose and fatty acids has been proposed to induce glucolipotoxicity. However, contradictory results suggest adaptations of the beta cells, which might be instrumental for partial preservation of the secretory response. In this context, we delineated the expression pattern of genes related to lipid pathways along with fat storage/mobilisation during glucose-stimulated insulin secretion. METHODS: Insulin-secreting cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mmol/l) without or with BSA-complexed 0.4 mmol/l palmitate and oleate. Then, transcriptomic analyses of lipid pathways were performed in human islets by RNA-Seq and in INS-1E cells and rat islets by quantitative RT-PCR. Storage of fat was assessed in INS-1E cells by electron microscopy and Bodipy staining, which was also used for measuring lipid mobilisation rate. The secretory response was monitored during acute 15 mmol/l glucose stimulation using online luminescence assay for INS-1E cells and by radioimmunoassay for rat islets. RESULTS: In human islets, chronic exposure to palmitate and oleate modified expression of a panel of genes involved in lipid handling. Culture at 25 mmol/l glucose upregulated genes encoding for enzymes of the glycerolipid/NEFA cycle and downregulated receptors implicated in fatty acid signalling. Similar results were obtained in INS-1E cells, indicating enhanced capacity of the glycerolipid/NEFA cycle under glucotoxic conditions. Exposure to unsaturated C18:1 fatty acid favoured intracellular lipid accumulation in a glucose-dependent way, an effect also observed with saturated C16:0 fatty acid when combined with the panlipase inhibitor Orlistat. After the glucolipotoxic culture, intracellular fat mobilisation was required for acute glucose-stimulated secretion, particularly in oleate-treated cells under glucotoxic culture conditions. The lipid mobilisation rate was governed chiefly by the levels of stored fat as a direct consequence of the culture conditions rather than energetic demands, except in palmitate-loaded cells. CONCLUSIONS/INTERPRETATION: Glucolipotoxic conditions promote the capacity of the glycerolipid/NEFA cycle thereby preserving part of the secretory response. The cycle of fat storage/mobilisation emerges as a mechanism helping the beta cell to cope with glucotoxic conditions.

Biosensors Monitor Ligand-Selective Effects at Kappa Opioid Receptors.

The kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to ß-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor-biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.

Lipid-Induced Adaptations of the Pancreatic Beta-Cell to Glucotoxic Conditions Sustain Insulin Secretion.

Over the last decades, lipotoxicity and glucotoxicity emerged as established mechanisms participating in the pathophysiology of obesity-related type 2 diabetes in general, and in the loss of ß-cell function in particular. However, these terms hold various potential biological processes, and it is not clear what precisely they refer to and to what extent they might be clinically relevant. In this review, we discuss the basis and the last advances of research regarding the role of free fatty acids, their metabolic intracellular pathways, and receptor-mediated signaling related to glucose-stimulated insulin secretion, as well as lipid-induced ß-cell dysfunction. We also describe the role of chronically elevated glucose, namely, glucotoxicity, which promotes failure and dedifferentiation of the ß cell. Glucolipotoxicity combines deleterious effects of exposures to both high glucose and free fatty acids, supposedly provoking synergistic defects on the ß cell. Nevertheless, recent studies have highlighted the glycerolipid/free fatty acid cycle as a protective pathway mediating active storage and recruitment of lipids. Finally, we discuss the putative correspondence of the loss of functional ß cells in type 2 diabetes with a natural, although accelerated, aging process.

AMPK Profiling in Rodent and Human Pancreatic Beta-Cells under Nutrient-Rich Metabolic Stress.

Chronic exposure of pancreatic ß-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in ß-cells under conditions of nutrient deprivation, while little is known on AMPK responses to metabolic stresses. Here, we first reviewed recent studies on the role of AMPK activation in ß-cells. Then, we investigated the expression profile of AMPK pathways in ß-cells following metabolic stresses. INS-1E ß-cells and human islets were exposed for 3 days to glucose (5.5-25 mM), palmitate or oleate (0.4 mM), and fructose (5.5 mM). Following these treatments, we analyzed transcript levels of INS-1E ß-cells by qRT-PCR and of human islets by RNA-Seq; with a special focus on AMPK-associated genes, such as the AMPK catalytic subunits α1 (Prkaa1) and α2 (Prkaa2). AMPKα and pAMPKα were also evaluated at the protein level by immunoblotting. Chronic exposure to the different metabolic stresses, known to alter glucose-stimulated insulin secretion, did not change AMPK expression, either in insulinoma cells or in human islets. Expression profile of the six AMPK subunits was marginally modified by the different diabetogenic conditions. However, the expression of some upstream kinases and downstream AMPK targets, including K-ATP channel subunits, exhibited stress-specific signatures. Interestingly, at the protein level, chronic fructose treatment favored fasting-like phenotype in human islets, as witnessed by AMPK activation. Collectively, previously published and present data indicate that, in the ß-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.

Chronic fructose renders pancreatic ß-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.

Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic ß-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human ß-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the ß-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in ß-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve ß-cells potentiated insulin secretion stimulated by intermediate glucose, mimicking the fructose treatment. Conversely, the P2Y1 antagonist and Panx1 inhibitor reversed the effects of fructose, as confirmed using Panx1-null islets and by the clearance of extracellular ATP by apyrase. These results reveal an important function of ATP signaling in pancreatic ß-cells mediating fructose-induced hyper-responsiveness.

Role of fatty acids in the pathogenesis of ß-cell failure and Type-2 diabetes.

Pancreatic ß-cells are glucose sensors in charge of regulated insulin delivery to the organism, achieving glucose homeostasis and overall energy storage. The latter function promotes obesity when nutrient intake chronically exceeds daily expenditure. In case of ß-cell failure, such weight gain may pave the way for the development of Type-2 diabetes. However, the causal link between excessive body fat mass and potential degradation of ß-cells remains largely unknown and debated. Over the last decades, intensive research has been conducted on the role of lipids in the pathogenesis of ß-cells, also referred to as lipotoxicity. Among various lipid species, the usual suspects are essentially the non-esterified fatty acids (NEFA), in particular the saturated ones such as palmitate. This review describes the fundamentals and the latest advances of research on the role of fatty acids in ß-cells. This includes intracellular pathways and receptor-mediated signaling, both participating in regulated glucose-stimulated insulin secretion as well as being implicated in ß-cell dysfunction. The discussion extends to the contribution of high glucose exposure, or glucotoxicity, to ß-cell defects. Combining glucotoxicity and lipotoxicity results in the synergistic and more deleterious glucolipotoxicity effect. In recent years, alternative roles for intracellular lipids have been uncovered, pointing to a protective function in case of nutrient overload. This requires dynamic storage of NEFA as neutral lipid droplets within the ß-cell, along with active glycerolipid/NEFA cycle allowing subsequent recruitment of lipid species supporting glucose-stimulated insulin secretion. Overall, the latest studies have revealed the two faces of the same coin.

Subcellular location defines GPCR signal transduction.

Intracellular G protein-coupled receptors (GPCRs) can be activated by permeant ligands, which contributes to agonist selectivity. Opioid receptors (ORs) provide a notable example, where opioid drugs rapidly activate ORs in the Golgi apparatus. Our knowledge on intracellular GPCR function remains incomplete, and it is unknown whether OR signaling in plasma membrane (PM) and Golgi apparatus differs. Here, we assess the recruitment of signal transducers to mu- and delta-ORs in both compartments. We find that Golgi ORs couple to Gαi/o probes and are phosphorylated but, unlike PM receptors, do not recruit ß-arrestin or a specific Gα probe. Molecular dynamics simulations with OR-transducer complexes in bilayers mimicking PM or Golgi composition reveal that the lipid environment promotes the location-selective coupling. We then show that delta-ORs in PM and Golgi have distinct effects on transcription and protein phosphorylation. The study reveals that the subcellular location defines the signaling effects of opioid drugs.

Palmitate and oleate modify membrane fluidity and kinase activities of INS-1E ß-cells alongside altered metabolism-secretion coupling.

Chronic exposure to elevated levels of glucose and free fatty acids impairs beta-cell function, leading to insulin secretion defects and eventually beta-cell failure. Using a semi-high throughput approach applied to INS-1E beta-cells, we tested multiple conditions of chronic exposure to basal, intermediate and high glucose, combined with saturated versus mono- and polyunsaturated fatty acids in order to assess cell integrity, lipid metabolism, mitochondrial function, glucose-stimulated calcium rise and secretory kinetics. INS-1E beta-cells were cultured for 3 days at different glucose concentrations (5.5, 11.1, 25 mM) without or with BSA-complexed 0.4 mM saturated (C16:0 palmitate), monounsaturated (C18:1 oleate) or polyunsaturated (C18:2 linoleate, C18:3 linolenate) fatty acids, resulting in 0.1-0.5 µM unbound fatty acids. Accumulation of triglycerides in cells exposed to fatty acids was glucose-dependent, oleate inducing the strongest lipid storage and protecting against glucose-induced cytotoxicity. The combined chronic exposure to both high glucose and either palmitate or oleate altered mitochondrial function as well as glucose-induced calcium rise. This pattern did not directly translate at the secretory level since palmitate and oleate exhibited distinct effects on the first and the second phases of glucose-stimulated exocytosis. Both fatty acids changed the activity of kinases, such as the MODY-associated BLK. Additionally, chronic exposure to fatty acids modified membrane physicochemical properties by increasing membrane fluidity, oleate exhibiting larger effects compared to palmitate. Chronic fatty acids differentially and specifically exacerbated some of the glucotoxic effects, without promoting cytotoxicity on their own. Each of the tested fatty acids functionally modified INS-1E beta-cell, oleate inducing the strongest effects.