siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis

Grimard, Vinciane; Massier, Julia; Richter, Doris; Schwudke, Dominik; Kalaidzidis, Yannis; Fava, Eugenio; Hermetter, Albin; Thiele, Christoph

doi:10.1194/jlr.m800168-jlr200

Cited by 15 publications

(8 citation statements)

References 60 publications

(61 reference statements)

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“…Lipids were recovered by a two‐step methanol/chloroform extraction as described (53). Settings that ensure accurate and robust quantification have been established in our previous work regarding sample amount, extraction procedure and mass spectrometric measurements (34,53,54). Basically, 900 000 cells (harvested following 4, 24 and 48 h of incubation) were taken up in 180 μL of 155 m m ammonia carbonate and 100 pmol of each lipid standard was added (PE‐O 20:0/‐O 20:0; PC‐O 18:0/‐O18:0; SM 35:1; Cer 35:1; GlcCer 30:1) and then extracted with 900 μL chloroform/methanol (10:1), while the water phase was further extracted with chloroform/methanol (2:1).…”

Section: Methodsmentioning

confidence: 99%

Glycosphingolipid Requirements for Endosome‐to‐Golgi Transport of Shiga Toxin

Raa

Grimmer

Schwudke

et al. 2009

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Shiga toxin binds to globotriaosylceramide (Gb3) receptors on the target cell surface. To enter the cytosol, Shiga toxin is dependent on endocytic uptake, retrograde transport to the Golgi apparatus and further to the endoplasmic reticulum before translocation of the enzymatically active moiety to the cytosol. Here, we have investigated the importance of newly synthesized glycosphingolipids for the uptake and intracellular transport of Shiga toxin in HEp-2 cells. Inhibition of glycosphingolipid synthesis by treatment with either PDMP or Fumonisin B 1 for 24-48 h strongly reduced the transport of Gb3-bound Shiga toxin from endosomes to the Golgi apparatus. This was associated with a change in localization of sorting nexins 1 and 2, and accompanied by a protection against the toxin. In contrast, there was no effect on transport or toxicity of the plant toxin ricin. High-resolution mass spectrometry revealed a 2-fold reduction in Gb3 at conditions giving a 10-fold inhibition of Shiga toxin transport to the Golgi. Furthermore, mass spectrometry showed that the treatment with PDMP (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol) and Fumonisin B 1 among other changes of the lipidome, affected the relative content of the different glycosphingolipid species. The largest depletion was observed for the hexosylceramide species with the N-amidated fatty acid 16:0, whereas hexosylceramide species with 24:1 were less affected. Quantitative lipid profiling with mass spectrometry demonstrated that PDMP did not influence the content of sphingomyelins, phospholipids and plasmalogens. In contrast, Fumonisin B 1 affected the amount and composition of sphingomyelin and glycolipids and altered the profiles of phospholipids and plasmalogens.

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Section: Methodsmentioning

confidence: 99%

Glycosphingolipid Requirements for Endosome‐to‐Golgi Transport of Shiga Toxin

Raa

Grimmer

Schwudke

et al. 2009

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“…This is of special concern when performing screens with mammalian cells, given the high number of genes with multiple paralogs and the complexity of the mammalian lipidome. In an attempt to perform a large‐scale screening of protein kinases involved in lipid homeostasis in mammalian cells, Grimard and others [47] perform a siRNA knockdown of 600 human kinases combined with TLC analysis of lipids. Despite the limitations of the approach in terms of sensitivity of lipid profiling, the screen yielded 91 hits with a lipid phenotype, eventually characterizing JNK2 as a new regulator of triglyceride homeostasis.…”

Section: Lipids Homeostasis and Function – Studies And Different Apprmentioning

confidence: 99%

Yeast as a model system for studying lipid homeostasis and function

Santos

Riezman

2012

FEBS Letters

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a b s t r a c tLipids are essential eukaryotic cellular constituents. Lipid metabolism has a strong impact on cell physiology, and despite good progress in this area, many important basic questions remain unanswered concerning the functional diversity of lipid species and on the mechanisms that cells employ to sense and adjust their lipid composition. Combining convenient experimental tractability, a large degree of conservation of metabolic pathways with other eukaryotes and the relative simplicity of its genome, proteome and lipidome, yeast represents the most advantageous model organism for studying lipid homeostasis and function. In this review we will focus on the importance of yeast as a model organism and some of the innovative advantages for the lipid research field.

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“…Small interfering RNA (siRNA) screening revealed that JNK2 is a regulator of LD homeostasis in HeLa cells ( 33 ). Furthermore, we recently showed that JNK2 is required for insulin-induced LD formation in human adipocytes ( 23 ).…”

Section: Methodsmentioning

confidence: 99%

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes

Ito¹,

Nagasawa²,

Omae³

et al. 2013

Journal of Lipid Research

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cular disease, nonalcoholic steatohepatitis, nephropathy, and several types of cancer ( 1-3 ). WAT mass is determined by the number and size of adipocytes ( 4, 5 ) regulated by cell differentiation, apoptosis, and lipid droplet (LD) formation (6)(7)(8). Insulin is known to inhibit apoptosis ( 9, 10 ) and increase LD formation ( 11,12 ) in adipocytes. Hyperinsulinemia is associated with weight gain in humans ( 13 ). Insulin signaling in adipocytes is critical for the development of obesity ( 14,15 ). Therefore, it has been suggested that insulin is one of the determinants involved in increasing the WAT mass.Our previous study showed that insulin decreases cell death-inducing DNA fragmentation factor-␣ -like effector (CIDE)A expression and increases CIDEC expression, both of which belong to the CIDE family and play critical roles in apoptosis ( 16,17 ) and LD formation ( 18-21 ), and the differential regulation of these genes is related, at least in part, to insulin-induced anti-apoptosis and LD formation in human adipocytes ( 22 ). Our recent study also showed that insulin regulates CIDEA and CIDEC expression via phosphatidylinositol 3-kinase (PI3K), and regulates expression of each protein via Akt1/2-and c-Jun N-terminal kinase (JNK)2-dependent pathways downstream of PI3K, respectively ( 23 ). These results suggest that insulin regulation of CIDEA and CIDEC contribute separately via different signaling pathways to insulin-induced antiapoptosis and LD formation, leading to increases in the number and size of adipocytes. Adipocyte size is determined by LD formation regulated by glucose uptake, de novo fatty acid synthesis, triacylglycerol synthesis, and lipolysis ( 24,25 ). Previous studies suggested that regulation of CIDEC expression contributes to LD maintenance by modulating lipolysis. However, regulation of other genes Press, March 19, 2013 DOI 10.1194 A novel JNK2/SREBP-1c pathway involved in insulininduced fatty acid synthesis in human adipocytes Abbreviations: ACC1, acetyl-CoA carboxylase 1; ACLY, ATP citrate lyase; CIDE, cell death-inducing DNA fragmentation factor-␣ -like effector; DAPI, 4 ′ ,6 ′ -diamidino-2-phenylindole; Dex, dexamethasone; FAS, fatty acid synthase; IPA, Ingenuity Pathway Analysis; JNK, c-Jun N-terminal kinase; LD, lipid droplet; n-SREBP-1c , nuclear form of sterol regulatory element binding protein-1c; PI3K, phosphatidylinositol 3-kinase; pre-SREBP-1c, precursor form of sterol regulatory element binding protein-1c; siJNK, JNK siRNA; siRNA, small interfering RNA; siSREBP-1, SREBP-1 siRNA; SREBP, sterol regulatory element binding protein; WAT, white adipose tissue. Published, JLR Papers in

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siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis

Cited by 15 publications

References 60 publications

Glycosphingolipid Requirements for Endosome‐to‐Golgi Transport of Shiga Toxin

Glycosphingolipid Requirements for Endosome‐to‐Golgi Transport of Shiga Toxin

Yeast as a model system for studying lipid homeostasis and function

A novel JNK2/SREBP-1c pathway involved in insulin-induced fatty acid synthesis in human adipocytes

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