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

ABSTRACT: Chronic exposure of pancreatic β-cells to elevated nutrient levels impairs their function and potentially induces apoptosis. Like in other cell types, AMPK is activated in β-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 β-cells. Then, we investigated the expression profile of AMPK pathways in β-cells following metabolic stresses. INS-1E β-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 β-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 β-cell, AMPK activation might be implicated in the pre-diabetic state, potentially as a protective mechanism.

SUBMITTER: Brun T

PROVIDER: S-EPMC7312098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK) has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids) effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP). Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes.

Project description:ObjectiveIn type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in β cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions.MethodsTranslating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment.ResultsTRAP-seq in a PDX1-deficiency model of β cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in β cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of β cell dysfunction, consistent with a maladaptive role for JUND in islets.ConclusionsA translation-centric approach uncovered JUND as a stress-responsive factor in β cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.

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

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