A lipidomic screen of palmitate-treated MIN6 β-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking

Boslem, Ebru; MacIntosh, Gustavo C.; Preston, Amanda M.; Bartley, Christopher R.; Busch, Anna K.; Fuller, Matthew D.; Laybutt, D. Ross; Meikle, Peter J.; Biden, Trevor J.

doi:10.1042/bj4390517

Cited by 34 publications

(61 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Trafficking assay ER-to-Golgi protein trafficking was assessed using the temperature-sensitive mutant vesicular stomatitis viral glycoprotein (VSVG) assay [21,22]. MIN6 cells seeded on coverslips were transfected with VSVG protein tagged with green fluorescent protein (GFP).…”

Section: Methodsmentioning

confidence: 99%

“…After addition of cycloheximide (20 μmol/l) to prevent novel VSVG synthesis, trafficking was commenced by transfer to the permissive temperature of 32°C. VSVG localisation was scored using confocal images according to the scale: 1, exclusively ER; 2, mostly ER with some Golgi; 3, equivalent ER and Golgi; 4, mostly Golgi with some ER; 5 exclusively Golgi [22].…”

Section: Methodsmentioning

confidence: 99%

“…A recent study showed that impaired Golgi-to-ER retrograde transport can induce beta cell ER stress [44]. Moreover, blockage of ER-to-Golgi protein trafficking causes accumulation of proteins in the ER and thus ER stress in beta cells chronically exposed to saturated fatty acids [21,22,45].…”

Section: Hypoxia Impairs Er-to-golgi Protein Trafficking In Beta Cellsmentioning

confidence: 99%

See 2 more Smart Citations

Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells

et al. 2016

View full text Add to dashboard Cite

Aims/hypothesis Hypoxia may contribute to beta cell failure in type 2 diabetes and islet transplantation. The adaptive unfolded protein response (UPR) is required for endoplasmic reticulum (ER) homeostasis. Here we investigated whether or not hypoxia regulates the UPR in beta cells and the role the adaptive UPR plays during hypoxic stress. Methods Mouse islets and MIN6 cells were exposed to various oxygen (O 2 ) tensions. DNA-damage inducible transcript 3 (DDIT3), hypoxia-inducible transcription factor (HIF)1α and HSPA5 were knocked down using small interfering (si)RNA; Hspa5 was also overexpressed. db/db mice were used. Results Hypoxia-response genes were upregulated in vivo in the islets of diabetic, but not prediabetic, db/db mice. In isolated mouse islets and MIN6 cells, O 2 deprivation (1-5% vs 20%; 4-24 h) markedly reduced the expression of adaptive UPR genes, including Hspa5, Hsp90b1, Fkbp11 and spliced Xbp1. Coatomer protein complex genes (Copa, Cope, Copg [also known as Copg1], Copz1 and Copz2) and ER-to-Golgi protein trafficking were also reduced, whereas apoptotic genes (Ddit3, Atf3 and Trb3 [also known as Trib3]), c-Jun N-terminal kinase (JNK) phosphorylation and cell death were increased. Inhibition of JNK, but not HIF1α, restored adaptive UPR gene expression and ER-to-Golgi protein trafficking while protecting against apoptotic genes and cell death following hypoxia. DDIT3 knockdown delayed the loss of the adaptive UPR and partially protected against hypoxia-induced cell death. The latter response was prevented by HSPA5 knockdown. Finally, Hspa5 overexpression significantly protected against hypoxia-induced cell death. Conclusions/interpretation Hypoxia inhibits the adaptive UPR in beta cells via JNK and DDIT3 activation, but independently of HIF1α. Downregulation of the adaptive UPR contributes to reduced ER-to-Golgi protein trafficking and increased beta cell death during hypoxic stress.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Cell death was determined, as previously described [28], using a Cell Death Detection ELISA (Roche Diagnostics, NSW, Australia).…”

Section: Methodsmentioning

confidence: 99%

“…Lalistat treatment MIN6 cells were pelleted in ice-cold PBS (two wells of a six-well plate per condition) and subjected to lipid MS as previously described [28,30].…”

Section: Lipidomic Profiling Using Ms Following 24 H Control Ormentioning

confidence: 99%

Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells

et al. 2013

View full text Add to dashboard Cite

Aims/hypothesis Lipolytic breakdown of endogenous lipid pools in pancreatic beta cells contributes to glucosestimulated insulin secretion (GSIS) and is thought to be mediated by acute activation of neutral lipases in the amplification pathway. Recently it has been shown in other cell types that endogenous lipid can be metabolised by autophagy, and this lipophagy is catalysed by lysosomal acid lipase (LAL). This study aimed to elucidate a role for LAL and lipophagy in pancreatic beta cells. Methods We employed pharmacological and/or genetic inhibition of autophagy and LAL in MIN6 cells and primary islets. Insulin secretion following inhibition was measured using RIA. Lipid accumulation was assessed by MS and confocal microscopy (to visualise lipid droplets) and autophagic flux was analysed by western blot. Results Insulin secretion was increased following chronic (≥8 h) inhibition of LAL. This was more pronounced with glucose than with non-nutrient stimuli and was accompanied by augmentation of neutral lipid species. Similarly, following inhibition of autophagy in MIN6 cells, the number of lipid droplets was increased and GSIS was potentiated. Inhibition of LAL or autophagy in primary islets also increased insulin secretion. This augmentation of GSIS following LAL or autophagy inhibition was dependent on the acute activation of neutral lipases. Conclusions/interpretation Our data suggest that lysosomal lipid degradation, using LAL and potentially lipophagy, contributes to neutral lipid turnover in beta cells. It also serves as a constitutive negative regulator of GSIS by depletion of substrate for the non-lysosomal neutral lipases that are activated acutely by glucose.

show abstract

The role of sphingolipids in endoplasmic reticulum stress

Park

2020

FEBS Letters

View full text Add to dashboard Cite

The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER-associated protein degradation and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long-chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis and cell-cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events.

show abstract

A lipidomic screen of palmitate-treated MIN6 β-cells links sphingolipid metabolites with endoplasmic reticulum (ER) stress and impaired protein trafficking

Cited by 34 publications

References 0 publications

Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells

Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells

Lysosomal acid lipase and lipophagy are constitutive negative regulators of glucose-stimulated insulin secretion from pancreatic beta cells

The role of sphingolipids in endoplasmic reticulum stress

Contact Info

Product

Resources

About