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Pancreatic 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. RNA-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. A 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. Our results identify a novel ERK/hnRNPK/DDX3X pathway that influences β cell survival and is activated under conditions associated with T2D. • A polyC motif is identified in the 3′UTRs of post-transcriptionally regulated genes. • hnRNPK mediates the post-transcriptional induction of JUND during metabolic stress. • hnRNPK is phosphorylated in β cells during metabolic stress. • MEK/ERK signaling is necessary and sufficient to activate hnRNPK/JUND. • DDX3X interacts with hnRNPK and regulates translation of JUND mRNA. [ABSTRACT FROM AUTHOR]

In 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. Translating 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. TRAP-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. A translation-centric approach uncovered JUND as a stress-responsive factor in β cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress. • TRAP-seq uncovers genes with altered ribosome occupancy during PDX1 deficiency. • JUND is post-transcriptionally upregulated in β cells during metabolic stress. • Depletion of JUND protects β cells from oxidative stress and apoptosis. • JUND regulates a set of pro-oxidant and pro-inflammatory genes in β cells. • JUND target genes are commonly dysregulated in models of β cell dysfunction. [ABSTRACT FROM AUTHOR]

Objectives Chronic hyperlipidemia and hyperglycemia are characteristic features of type 2 diabetes (T2DM) that are thought to cause or contribute to β-cell dysfunction by “glucolipotoxicity.” Previously we have shown that acute treatment of pancreatic islets with fatty acids (FA) decreases acetylcholine-potentiated insulin secretion. This acetylcholine response is mediated by M3 muscarinic receptors, which play a key role in regulating β-cell function. Here we examine whether chronic FA exposure also inhibits acetylcholine-potentiated insulin secretion using mouse and human islets. Methods Islets were cultured for 3 or 4 days at different glucose concentration with 0.5 mM palmitic acid (PA) or a 2:1 mixture of PA and oleic acid (OA) at 1% albumin (PA/BSA molar ratio 3.3). Afterwards, the response to glucose and acetylcholine were studied in perifusion experiments. Results FA-induced impairment of insulin secretion and Ca 2+ signaling depended strongly on the glucose concentrations of the culture medium. PA and OA in combination reduced acetylcholine potentiation of insulin secretion more than PA alone, both in mouse and human islets, with no evidence of a protective role of OA. In contrast, lipotoxicity was not observed with islets cultured for 3 days in medium containing less than 1 mM glucose and a mixture of glutamine and leucine (7 mM each). High glucose and FAs reduced endoplasmic reticulum (ER) Ca 2+ storage capacity; however, preserving ER Ca 2+ by blocking the IP3 receptor with xestospongin C did not protect islets from glucolipotoxic effects on insulin secretion. In contrast, an inhibitor of casein kinase 2 (CK2) protected the glucose dependent acetylcholine potentiation of insulin secretion in mouse and human islets against glucolipotoxicity. Conclusions These results show that chronic FA treatment decreases acetylcholine potentiation of insulin secretion and that this effect is strictly glucose dependent and might involve CK2 phosphorylation of β-cell M3 muscarinic receptors. [ABSTRACT FROM AUTHOR]

Objectives Hyperglycemia and elevated blood lipids are the presumed precipitating causes of β-cell damage in T2DM as the result of a process termed “glucolipotoxicity”. Here, we tested whether glucolipotoxic pathophysiology is caused by defective bioenergetics using islets in culture. Methods Insulin secretion, respiration, ATP generation, fatty acid (FA) metabolite profiles and gene expression were determined in isolated islets treated under glucolipotoxic culture conditions. Results Over time, chronic exposure of mouse islets to FAs with glucose leads to bioenergetic failure and reduced insulin secretion upon stimulation with glucose or amino acids. Islets exposed to glucolipotoxic conditions displayed biphasic changes of the oxygen consumption rate (OCR): an initial increase in baseline and Vmax of OCR after 3 days, followed by decreased baseline and glucose stimulated OCR after 5 days. These changes were associated with lower islet ATP levels, impaired glucose-induced ATP generation, a trend for reduced mitochondrial DNA content and reduced expression of mitochondrial transcription factor A (Tfam). We discovered the accumulation of carnitine esters of hydroxylated long chain FAs, in particular 3-hydroxytetradecenoyl-carnitine. Conclusions As long chain 3-hydroxylated FA metabolites are known to uncouple heart and brain mitochondria [53–55] , we propose that under glucolipotoxic condition, unsaturated hydroxylated long-chain FAs accumulate, uncouple and ultimately inhibit β-cell respiration. This leads to the slow deterioration of mitochondrial function progressing to bioenergetics β-cell failure. [ABSTRACT FROM AUTHOR]

Insulin secretion by pancreatic β-cells is stimulated by glucose, amino acids, and other metabolic fuels. Glutamate dehydrogenase (GDH) has been shown to play a regulatory role in this process. The importance of GDH was underscored by features of hyperinsulinemia/hyperammonemia syndrome, where a dominant mutation causes the loss of inhibition by GTP and ATP. Here we report the effects of green tea polyphenols on GDH and insulin secretion. Of the four compounds tested, epigallocatechin gallate (EGCG) and epicatechin gallate were found to inhibit GDH with nanomolar ED50 values and were therefore found to be as potent as the physiologically important inhibitor GTP. Furthermore, we have demonstrated that EGCG inhibits BCH-stimulated insulin secretion, a process that is mediated by GDH, under conditions where GDH is no longer inhibited by high energy metabolites. EGCG does not affect glucose-stimulated insulin secretion under high energy conditions where GDH is probably fully inhibited. We have further shown that these compounds act in an allosteric manner independent of their antioxidant activity and that the β-cell stimulatory effects are directly correlated with glutamine oxidation. These results demonstrate that EGCG, much like the activator of GDH (BCH), can facilitate dissecting the complex regulation of insulin secretion by pharmacologically modulating the effects of GDH. [ABSTRACT FROM AUTHOR]

Background Mutations in C9orf72 and SOD1 account for most of the familial and late-onset sporadic ALS cases. Mutations in FUS can be identified in around 5% of FALS and 1% of overall sporadic ALS cases. Methods We screened a large cohort of 15 very early onset (onset age <36 years) and 10 middle aged (36–53 years) sporadic ALS patients to determine the frequency of mutations in C9orf72 , SOD1 and FUS in this distinct patient cohort. Results In the cohort of the very early onset patients, we identified 6 subjects (40.0%) carrying a FUS mutation. Genetic testing of the patients’ parents was possible in 5 families and revealed that the mutation in these individuals arose de novo . Two of the identified mutations had not been described before (c.1484delG; c.1504delG). Three patients carried the already in younger ALS patients described FUS mutation p.P525L. No mutations were found in SOD1 , C9orf72 . Five of the six identified patients presented with rapidly progressive bulbar symptoms. Conclusion Our study identifies FUS mutations as the most frequent genetic cause in early-onset ALS and suggests that de novo FUS mutations may be more common for sporadic ALS cases than recognized so far. Genetic testing of the FUS gene seems worthwhile in early onset ALS patients showing predominant bulbar symptoms and an aggressive disease course. [ABSTRACT FROM AUTHOR]