Project description:Myostatin (MSTN) is a negative regulator of myogenesis in vertebrates. Depletion of mstn resulted in elevated muscle growth in several animal species. However, the report on the complete ablation of mstn in teleost fish has not yet become available. In this study, two independent mstnb-deficient mutant lines in zebrafish were generated with the TALENs technique. In the mstnb-deficient zebrafish, enhanced muscle growth with muscle fiber hyperplasia was achieved. Beginning at the adult stage (80 days postfertilization), the mstnb-deficient zebrafish exhibited increased circumferences and body weights compared with the wild-type sibling control fish. Although the overall total lipid/body weight ratios remained similar between the mstnb-deficient zebrafish and the control fish, the distribution of lipids was altered. The size of the visceral adipose tissues became smaller while more lipids accumulated in skeletal muscle in the mstnb-deficient zebrafish than in the wild-type control fish. Based on the transcriptional expression profiles, our results revealed that lipid metabolism, including lipolysis and lipogenesis processes, was highly activated in the mstnb-deficient zebrafish, which indicated the transition of energy metabolism from protein-dependent to lipid-dependent in mstnb-deficient zebrafish. Our mstnb-deficient model could be valuable in understanding not only the growth trait regulation in teleosts but also the mechanisms of teleost energy metabolism.
Project description:LPP2 is one of three enzymes in the lipid phosphate phosphatase family (LPP1-3) that dephosphorylate extracellular and intracellular bioactive lipid phosphates and pyrophosphates. LPP2 increases cell growth and LPP2 expression is elevated in a variety of malignancies, implying that LPP2 is a pro-tumorigenic factor. Methods: LPP2 expression in human breast tumors and normal breast tissue was measured by qPCR. To understand the role of LPP2, we knocked out its expression in multiple cell lines using CRISPR/Cas9. Cell proliferation and migration were compared between wild type and LPP2 knockout cells. Cell cycle was measured by flow cytometry, and cell cycle proteins were determined by western blotting. Effects of LPP2 on tumor growth were investigated using syngeneic and xenograft mouse breast cancer models. Results: LPP2 mRNA levels were higher in ER/PR positive, ER/HER2 positive, and triple negative human breast tumors, relative to normal breast tissue. Higher levels of LPP2 in breast tumors, hepatocellular carcinoma, pancreatic adenocarcinoma, and melanomas were prognostic of poorer survival. LPP2 mRNA expression is also increased in Hs-578T, MDA-MB-231, MCF7 and MDA-MB-468 breast cancer cell lines, relative to non-malignant Hs-578Bst, MCF10A and MCF-12A cells. LPP2 knockout in breast cancer cells decreased cell growth by inhibiting G1/S transition, whereas, increasing LPP2 levels in Hs-578Bst and MCF10A cells promoted proliferation. The effects of LPP2 on cell cycle were associated with changes in cyclin A2, cyclin B1, and cell cycle inhibitors, p27 or p21. The level of c-Myc was downregulated by knocking out LPP2, and it was partly restored by re-expressing LPP2. The positive correlation between the expression of LPP2 and c-Myc exists in multiple cancer cell lines including breast, lung, upper aerodigestive tract and urinary tract cancer. LPP2 knockout in MDA-MB-231 or 4T1 cells suppressed tumor formation in mouse breast cancer models, and decreased the in vivo expression of Ki67 and c-Myc of the cancer cells. Conclusion: Targeting LPP2 could provide a new strategy for decreasing c-Myc expression and tumor growth.
Project description:Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein associated with several human pathologies, including obesity, depression, and autism. Recently, significantly enlarged white adipose tissue, hepatic lipid accumulation, and decreased muscle capacity were reported in Negr1-deficient mice. However, the mechanism behind these phenotypes was not clear. In the present study, we found NEGR1 to interact with cluster of differentiation 36 (CD36), the major fatty acid translocase in the plasma membrane. Binding assays with a soluble form of NEGR1 and in situ proximal ligation assays indicated that NEGR1-CD36 interaction occurs at the outer leaflet of the cell membrane. Furthermore, we show that NEGR1 overexpression induced CD36 protein destabilization in vitro. Both mRNA and protein levels of CD36 were significantly elevated in the white adipose tissue and liver tissues of Negr1-/- mice. Accordingly, fatty acid uptake rate increased in NEGR1-deficient primary adipocytes. Finally, we demonstrated that Negr1-/- mouse embryonic fibroblasts showed elevated reactive oxygen species levels and decreased adenosine monophosphate-activated protein kinase activation compared with control mouse embryonic fibroblasts. Based on these results, we propose that NEGR1 regulates cellular fat content by controlling the expression of CD36.
Project description:Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14KO cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth.
Project description:Glomerulosclerosis and tubulointerstitial fibrosis are pathological features of chronic kidney disease. Transforming growth factor β (TGFβ) is a key player in the development of fibrosis. However, of the three known TGFβ isoforms, only TGFβ1 has an established role in fibrosis, and the pathophysiological relevance of TGFβ2 and TGFβ3 is unknown. Because Tgfb3 deficiency in mice results in early postnatal lethality, we analyzed the kidney phenotype of heterozygous Tgfb3-knockout mice (Tgfb3+/-) and compared it with that of matched wild-type mice. Four-month-old Tgfb3+/- mice exhibited incipient renal fibrosis with epithelial-mesenchymal transition, in addition to glomerular basement membrane thickening and podocyte foot process effacement associated with albuminuria. Also evident was insulin resistance and oxidative stress at the renal level, together with aberrant renal lipid metabolism and mitochondrial function. Omics analysis revealed toxic species, such as diacylglycerides and ceramides, and dysregulated mitochondrial metabolism in Tgfb3+/- mice. Kidneys of Tgfb3+/- mice showed morphological alterations of mitochondria and overactivation of non-canonical MAPK ERK1/2 and JNK cascades. Our study indicates that renal TGFβ3 might have antifibrotic and renoprotective properties, opposing or counteracting the activity of TGFβ1. This article has an associated First Person interview with the first author of the paper.
Project description:Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1(-/-) primary glia upon incubation with fatty acids was reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis.
Project description:The molecular mechanisms by which extracellular guidance cues regulate axonal morphology are not fully understood. Recent findings suggest that increased activity of the protein kinase Akt promotes dendritic branching and elongation in hippocampal neurons. We tested whether expression of constitutively active Akt (CA-Akt) in primary sensory neurons would promote axonal branching and whether targeting CA-Akt to lipid rafts, common sites of Akt function, would differentially regulate axonal morphology. Biolistic transduction of sensory neurons induced a rapid expression of CA-Akt, resulting in increased axonal branching, cell hypertrophy, and growth cone expansion. Additionally, we found that targeting of CA-Akt to lipid rafts significantly potentiated growth cone expansion compared with expression of CA-Akt throughout the neuron. Because lipid rafts are concentrated within the growth cone, this finding suggests that signaling of expansion is likely regulated locally. We found that CA-Akt-mediated growth cone expansion, but not axonal branching, was attenuated by coexpression of dominant-negative Rac1. In contrast, blockade of mammalian target of rapamycin (mTOR) prevented axonal branching and hypertrophy in response to CA-Akt, but not growth cone expansion. These data indicate that Akt activity can regulate growth cone expansion via localized Rac1 signaling and regulate axonal branching and soma size via activation of mTOR.
Project description:Lipid incorporation from endoplasmic reticulum (ER) to lipid droplet (LD) is important in controlling LD growth and intracellular lipid homeostasis. However, the molecular link mediating ER and LD cross talk remains elusive. Here, we identified Rab18 as an important Rab guanosine triphosphatase in controlling LD growth and maturation. Rab18 deficiency resulted in a drastically reduced number of mature LDs and decreased lipid storage, and was accompanied by increased ER stress. Rab3GAP1/2, the GEF of Rab18, promoted LD growth by activating and targeting Rab18 to LDs. LD-associated Rab18 bound specifically to the ER-associated NAG-RINT1-ZW10 (NRZ) tethering complex and their associated SNAREs (Syntaxin18, Use1, BNIP1), resulting in the recruitment of ER to LD and the formation of direct ER-LD contact. Cells with defects in the NRZ/SNARE complex function showed reduced LD growth and lipid storage. Overall, our data reveal that the Rab18-NRZ-SNARE complex is critical protein machinery for tethering ER-LD and establishing ER-LD contact to promote LD growth.
Project description:BackgroundCommon genetic variation in close proximity to the ILRUN gene are significantly associated with coronary artery disease as well as with plasma lipid traits. We recently demonstrated that hepatic inflammation and lipid regulator with ubiquitin-associated domain-like and NBR1-like domains (ILRUN) regulates lipoprotein metabolism in vivo in mice. However, whether ILRUN, which is expressed in vascular cells, directly impacts atherogenesis remains unclear. We sought to determine the role of ILRUN in atherosclerosis development in mice.MethodsFor our study, we generated global Ilrun-deficient (IlrunKO) male and female mice on 2 hyperlipidemic backgrounds: low density lipoprotein receptor knockout (LdlrKO) and apolipoprotein E knockout (ApoeKO; double knockout [DKO]).ResultsCompared with littermate control mice (single LdlrKO or ApoeKO), deletion of Ilrun in DKO mice resulted in significantly attenuated both early and advanced atherosclerotic lesion development, as well as reduced necrotic area. DKO mice also had significantly decreased plasma cholesterol levels, primarily attributable to non-HDL (high-density lipoprotein) cholesterol. Hepatic-specific reconstitution of ILRUN in DKO mice on the ApoeKO background normalized plasma lipids, but atherosclerotic lesion area and necrotic area remained reduced in DKO mice. Further analysis showed that loss of Ilrun increased efferocytosis receptor MerTK expression in macrophages, enhanced in vitro efferocytosis, and significantly improved in situ efferocytosis in advanced lesions.ConclusionsOur results support ILRUN as an important novel regulator of atherogenesis that promotes lesion progression and necrosis. It influences atherosclerosis through both plasma lipid-dependent and lipid-independent mechanisms. These findings support ILRUN as the likely causal gene responsible for genetic association of variants with coronary artery disease at this locus and suggest that suppression of ILRUN activity might be expected to reduce atherosclerosis.
Project description:UnlabelledFat-specific protein 27 (Fsp27) is a lipid droplet-associated protein that promotes lipid droplet (LD) growth and triglyceride (TG) storage in white adipocytes. Fsp27 is also highly expressed in the steatotic liver and contributes to TG accumulation. In this study we discovered that the liver produces Fsp27β, an alternative Fsp27 isoform, which contains 10 additional amino acids at the N-terminus of the original Fsp27 (Fsp27α). White adipose tissue (WAT) and the liver specifically expressed Fsp27α and Fsp27β transcripts, respectively, which were driven by distinct promoters. The Fsp27β promoter was activated by the liver-enriched transcription factor cyclic-AMP-responsive-element-binding protein H (CREBH) but not by peroxisome proliferator-activated receptor gamma (PPARγ), which activated the Fsp27α promoter. Enforced expression of the constitutively active CREBH strongly induced Fsp27β and the human ortholog CIDEC2 in mouse hepatocytes and HepG2 cells, respectively. In contrast, loss of CREBH decreased hepatic Fsp27β in fasted mice, suggesting that CREBH plays a critical role in Fsp27β expression in the liver. Similar to Fsp27α, Fsp27β localized on the surface of lipid droplets and suppressed lipolysis. Consequently, enforced expression of Fsp27β or CREBH promoted lipid droplet enlargement and TG accumulation in the liver.ConclusionThe CREBH-Fsp27β axis is important for regulating lipid droplet dynamics and TG storage in the liver.