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Pancreatic ?-cells respond to fuel pressure with an early metabolic switch.

ABSTRACT: Pancreatic ?-cells become irreversibly damaged by long-term exposure to excessive glucose concentrations and lose their ability to carry out glucose stimulated insulin secretion (GSIS) upon damage. The ?-cells are not able to control glucose uptake and they are therefore left vulnerable for endogenous toxicity from metabolites produced in excess amounts upon increased glucose availability. In order to handle excess fuel, the ?-cells possess specific metabolic pathways, but little is known about these pathways. We present a study of ?-cell metabolism under increased fuel pressure using a stable isotope resolved NMR approach to investigate early metabolic events leading up to ?-cell dysfunction. The approach is based on a recently described combination of 13C metabolomics combined with signal enhanced NMR via dissolution dynamic nuclear polarization (dDNP). Glucose-responsive INS-1 ?-cells were incubated with increasing concentrations of [U-13C] glucose under conditions where GSIS was not affected (2-8 h). We find that pyruvate and DHAP were the metabolites that responded most strongly to increasing fuel pressure. The two major divergence pathways for fuel excess, the glycerolipid/fatty acid metabolism and the polyol pathway, were found not only to operate at unchanged rate but also with similar quantity.

SUBMITTER: Malinowski RM

PROVIDER: S-EPMC7508987 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Pancreatic β-cells respond to fuel pressure with an early metabolic switch.

Malinowski Ronja M RM Ghiasi Seyed M SM Mandrup-Poulsen Thomas T Meier Sebastian S Lerche Mathilde H MH Ardenkjær-Larsen Jan H JH Jensen Pernille R PR

Scientific reports 20200922 1

Pancreatic β-cells become irreversibly damaged by long-term exposure to excessive glucose concentrations and lose their ability to carry out glucose stimulated insulin secretion (GSIS) upon damage. The β-cells are not able to control glucose uptake and they are therefore left vulnerable for endogenous toxicity from metabolites produced in excess amounts upon increased glucose availability. In order to handle excess fuel, the β-cells possess specific metabolic pathways, but little is known about ...[more]

PMID: 32963286

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Project description:Glucose metabolism promotes insulin secretion in β-cells via metabolic coupling factors that are incompletely defined. Moreover, chronically elevated glucose causes β-cell dysfunction, but little is known about how cells handle excess fuels to avoid toxicity. Here we sought to determine which among the candidate pathways and coupling factors best correlates with glucose-stimulated insulin secretion (GSIS), define the fate of glucose in the β-cell, and identify pathways possibly involved in excess-fuel detoxification. We exposed isolated rat islets for 1 h to increasing glucose concentrations and measured various pathways and metabolites. Glucose oxidation, oxygen consumption, and ATP production correlated well with GSIS and saturated at 16 mm glucose. However, glucose utilization, glycerol release, triglyceride and glycogen contents, free fatty acid (FFA) content and release, and cholesterol and cholesterol esters increased linearly up to 25 mm glucose. Besides being oxidized, glucose was mainly metabolized via glycerol production and release and lipid synthesis (particularly FFA, triglycerides, and cholesterol), whereas glycogen production was comparatively low. Using targeted metabolomics in INS-1(832/13) cells, we found that several metabolites correlated well with GSIS, in particular some Krebs cycle intermediates, malonyl-CoA, and lower ADP levels. Glucose dose-dependently increased the dihydroxyacetone phosphate/glycerol 3-phosphate ratio in INS-1(832/13) cells, indicating a more oxidized state of NAD in the cytosol upon glucose stimulation. Overall, the data support a role for accelerated oxidative mitochondrial metabolism, anaplerosis, and malonyl-CoA/lipid signaling in β-cell metabolic signaling and suggest that a decrease in ADP levels is important in GSIS. The results also suggest that excess-fuel detoxification pathways in β-cells possibly comprise glycerol and FFA formation and release extracellularly and the diversion of glucose carbons to triglycerides and cholesterol esters.

Project description:ObjectivePancreatic β cell failure plays a central role in the development of type 2 diabetes (T2D). While the transcription factors shaping the β cell gene expression program have received much attention, the post-transcriptional controls that are activated in β cells during stress are largely unknown. We recently identified JUND as a pro-oxidant transcription factor that is post-transcriptionally upregulated in β cells during metabolic stress. Here we seek to uncover the mechanisms underlying this maladaptive response to metabolic stress.MethodsRNA-protein and protein-protein interactions were measured using RNA immunoprecipitation and co-immunoprecipitation, respectively, in Min6 cells and mouse islets. Phos-tag analyses were used to assess hnRNPK phosphorylation in primary mouse and human islets and Min6 cells. Translating ribosome affinity purification (TRAP) followed by RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of CRISPR-Cas9 to Min6 cells. Apoptosis was measured in primary islets using a cell-permeable dye with a fluorescence readout of activated cleaved caspase-3 and-7.ResultsA de novo motif analysis was performed on a subset of genes previously found to be regulated at the level of ribosome binding during PDX1-deficiency, which identified a poly-cytosine (polyC) motif in the 3'UTR of the transcript encoding JUND. The polyC-binding protein hnRNPK bound to the mRNA encoding JUND, leading us to hypothesize that hnRNPK regulates JUND expression during glucolipotoxicity. Indeed, loss of hnRNPK blocked the post-transcriptional upregulation of JUND during metabolic stress. hnRNPK was phosphorylated in mouse and human islets during glucolipotoxicity and in islets of diabetic db/db mice. The MEK/ERK signaling pathway was both necessary and sufficient for the phosphorylation of hnRNPK, upregulation of JUND levels, and induction of pro-oxidant and pro-inflammatory genes. Further, we identified the RNA helicase DDX3X as a new binding partner for hnRNPK that is required for efficient translation of JUND mRNA. Loss of hnRNPK reduced DDX3X binding to translation machinery, suggesting that these factors cooperate to regulate translation in β cells.ConclusionsOur results identify a novel ERK/hnRNPK/DDX3X pathway that influences β cell survival and is activated under conditions associated with T2D.

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Pancreatic ?-cells respond to fuel pressure with an early metabolic switch.

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Pancreatic β-cells respond to fuel pressure with an early metabolic switch.

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