Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Mar 25;291(13):6989-7003.
doi: 10.1074/jbc.M115.691212. Epub 2016 Feb 7.

Ceramide Synthase 5 Is Essential to Maintain C16:0-Ceramide Pools and Contributes to the Development of Diet-induced Obesity

Dominic Gosejacob  1 , Philipp S Jäger  1 , Katharina Vom Dorp  2 , Martin Frejno  1 , Anne C Carstensen  1 , Monika Köhnke  1 , Joachim Degen  1 , Peter Dörmann  2 , Michael Hoch  3
Affiliations

Ceramide Synthase 5 Is Essential to Maintain C16:0-Ceramide Pools and Contributes to the Development of Diet-induced Obesity

Dominic Gosejacob et al. J Biol Chem. .

Abstract

Ceramides are bioactive sphingolipids, which are composed of sphingoid bases carrying acyl chains of various lengths. Ceramides are synthesized by a family of six ceramide synthases (CerS) in mammals, which produce ceramides with differentN-linked acyl chains. Increased ceramide levels are known to contribute to the development of obesity and insulin resistance. Recently, it has been demonstrated that the ceramide acylation pattern is of particular importance for an organism to maintain energy homeostasis. However, which of theCerSfamily members are involved in this process is not yet completely known. Using newly developedCerS5knock-out mice, we show here thatCerS5is essential to maintain cellular C16:0sphingolipid pools in lung, spleen, muscle, liver, and white adipose tissue. Glycerophospholipid levels inCerS5-deficient mice were not altered. We found a strong impact of CerS5-dependent ceramide synthesis in white adipose tissue after high fat diet feeding. In skeletal muscle, liver, and spleen, C16:0-ceramide levels were altered independent of feeding conditions. The loss ofCerS5is associated with reduced weight gain and improved systemic health, including maintenance of glucose homeostasis and reduced white adipose tissue inflammation after high fat diet challenge. Our findings indicate that reduction of endogenous C16:0-ceramide by genetic inhibition ofCerS5is sufficient to ameliorate obesity and its comorbidities.

Keywords: adipose tissue metabolism; ceramide; ceramide synthase; diabetes; gene knockout; insulin resistance; lipid metabolism; metabolism; obesity; sphingolipid.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
CerS are central enzymes in the sphingolipid metabolism pathway. A, schematic representation of sphingolipid synthesis with CerS enzymes at a central point, mediating N-acyl chain specificity. Sptlc, serine palmitoyltransferase; Kdsr, 3-ketodihydrosphingosine reductase; Degs, sphingolipid δ(4)-desaturase. B, N-acyl chain specificity of mammalian CerS enzymes adapted from Refs. .
FIGURE 2.
FIGURE 2.
Generation and verification of the CerS5 KO mouse model. A, gene targeting strategy. Schematic illustration of the genomic organization of the WT CerS5 locus, the recombined locus, including a frt site flanked pgk-Neo cassette for positive ES cell selection and the recombined locus after FLP-mediated deletion of the frt site flanked pgk-Neo cassette. B, Southern blot demonstrating recombination of the CerS5 gene locus and deletion of the frt site flanked pgk-Neo cassette with a probe against lacZ after genomic digestion with the enzyme SacI, resulting in no signal for the WT locus, a 9.5-kb band for the CerS5lacZ-neo allele, and a 7.6-kb band for the CerS5lacZ allele. C, CerS5 mRNA levels in WT (CerS5+/+), heterozygous (CerS5+/lacZ), and KO (CerS5lacZ/lacZ) lung tissue. D, detection of CerS5 protein using custom-made antibodies against the C terminus of CerS5 in MEFs generated from WT (CerS5+/+), heterozygous (CerS5+/lacZ), and KO (CerS5lacZ/lacZ) mice.
FIGURE 3.
FIGURE 3.
CerS5 is predominantly expressed in testis, eWAT, lung, spleen, and thymus. CerS5 protein expression was analyzed by Western blotting. CerS5 is predominantly expressed in eWAT, lung, and testis. CerS5 is modestly expressed in thymus, spleen, and subcutaneous WAT (scWAT). CerS5 is only expressed at very low levels in liver, muscle, and interscapular brown adipose tissue (iBAT).
FIGURE 4.
FIGURE 4.
Sphingolipids with C16:0N-acyl chains are reduced in lung tissue of CerS5 KO mice. A, ceramide levels (with d18:1 as long chain base) were quantified in lung tissue of adult CerS5 KO mice by mass spectrometry. B–D, analysis of sphingolipids containing C16:0-acyl chains in lung tissue (n = 5 ± S.D. per group) (p values calculated using an unpaired Student's t test). E, click ceramide synthase assay using alkyne-sphinganine as substrate and C16:0-CoA to demonstrate loss of CerS5 activity in lung tissue (n = 3 ± S.D. per group) (p values calculated with an unpaired Student's t test). F, NBD-ceramide synthase activity assay using NBD-sphinganine as substrate in combination with various acyl-CoAs (chain length indicated on the x axis of the graph, n = 4 ± S.D.). G, total phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS) levels. H, individual PC levels in lung tissues (n = 4–5 ± S.D. per group) (p values calculated using an unpaired Student's t test). I, mass spectrometric analysis of most abundant sphingolipids and glycerophospholipids demonstrating specific decrease of sphingolipids carrying C16:0-acyl chains.
FIGURE 5.
FIGURE 5.
High fat diet-dependent and -independent alterations of C16:0-ceramide and sphingomyelin in tissues. Analysis of ceramide (A, C, E, and G) and sphingomyelin (B, D, F, and H) in muscle, liver, eWAT, and serum (n = 3–5 mice per condition, values indicate mean ± S.E.). The inset in G shows S1P concentrations in serum. p values were calculated by ANOVA, and experimental groups were compared using Tukey post hoc tests. #, LFD versus HFD in WT mice; ¶, WT versus KO on LFD; *, WT versus KO on HFD.
FIGURE 6.
FIGURE 6.
CerS5 deficiency suppresses diet-induced obesity. A, growth curve of CerS5 KO and WT mice after consumption of LFD or HFD (n = 15–27/group ±S.E.). B, longitudinal growth of Cer5 KO and WT mice on LFD and HFD. C, analysis of eWAT mass after HFD and LFD feeding (n = 12–26/group). D, eWAT cell area (n = 5–8/group). E, serum Leptin concentrations (n = 8–10/group). F, relative concentration of hepatic lipids determined by TLC analysis and densitometric quantification (n = 4/group ± S.E.). G and H, representative H&E staining of WAT (scale bar, 100 μm) and liver (scale bar, 50 μm), respectively (p values were calculated by ANOVA, and experimental groups were compared using Tukey post hoc tests).
FIGURE 7.
FIGURE 7.
Improved glucose tolerance and insulin sensitivity after HFD exposure in CerS5 KO compared with WT mice. A, glucose tolerance test (GTT) from WT and CerS5 KO mice. (n = 9; 6; 14 and 11 mice for WT LFD, KO LFD, WT HFD, and KO HFD, respectively. Data represent mean ± S.E.). B, insulin tolerance test (ITT) from WT and CerS5 KO mice (n = 10; 6; 12 and 11 mice for WT LFD, KO LFD, WT HFD, and KO HFD, respectively ± S.E.). C and D, area under the curve (AUC) of insulin and glucose tolerance tests. E, serum insulin concentration (n = 7; 8; 8 and 9 mice for WT LFD, KO LFD, WT HFD, and KO HFD, respectively). F, starvation glucose levels (n = 9; 6; 14 and 11 mice for WT LFD, KO LFD, WT HFD, and KO HFD, respectively) (p values were calculated by ANOVA and experimental groups were compared using Tukey post hoc tests).
FIGURE 8.
FIGURE 8.
CerS5 deficiency is associated with improved adipose tissue health after HFD feeding. A, analysis of phosphorylation of acetyl-CoA carboxylase at serine 79 (ACC) and AKT at serine 473 (AKT) and LC3 lipidation as marker for autophagy induction (unlipidated (LC3I) and lipidated (LC3II) LC3) in eWAT by Western blotting (WT n = 5, KO n = 5) Quantification of phosphorylation status, expression levels, and LC3 lipidation is depicted in the graph next to the blot. B, analysis of expression and phosphorylation of AMP-activated protein kinase α (AMPKα) at threonine 172and AMP-activated protein kinase β (AMPKβ) at serine 108. C, protein expression of the procaspase-1 p45 (Casp1) in eWAT after HFD challenge. D, F4/80 staining of eWAT under LFD and HFD (scale bar, 50 μm). E, expression levels of central players in eWAT function after HFD (WT n = 8, KO n = 6). F, hepatic expression levels of genes involved in lipid homeostasis after HFD (WT n = 10, KO n = 12). G, acylcarnitine profile of serum from mice fed a HFD (WT and KO n = 6) (data represent mean ± S.E. p values calculated using an unpaired Student's t test).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mizutani Y., Kihara A., and Igarashi Y. (2005) Mammalian Lass6 and its related family members regulate synthesis of specific ceramides. Biochem. J. 390, 263–271 - PMC - PubMed
    1. Jennemann R., Rabionet M., Gorgas K., Epstein S., Dalpke A., Rothermel U., Bayerle A., van der Hoeven F., Imgrund S., Kirsch J., Nickel W., Willecke K., Riezman H., Gröne H.-J., and Sandhoff R. (2012) Loss of ceramide synthase 3 causes lethal skin barrier disruption. Hum. Mol. Genet. 21, 586–608 - PubMed
    1. Imgrund S., Hartmann D., Farwanah H., Eckhardt M., Sandhoff R., Degen J., Gieselmann V., Sandhoff K., and Willecke K. (2009) Adult ceramide synthase 2 (CERS2)-deficient mice exhibit myelin sheath defects, cerebellar degeneration, and hepatocarcinomas. J. Biol. Chem. 284, 33549–33560 - PMC - PubMed
    1. Ginkel C., Hartmann D., vom Dorp K., Zlomuzica A., Farwanah H., Eckhardt M., Sandhoff R., Degen J., Rabionet M., Dere E., Dörmann P., Sandhoff K., and Willecke K. (2012) Ablation of neuronal ceramide synthase 1 in mice decreases ganglioside levels and expression of myelin-associated glycoprotein in oligodendrocytes. J. Biol. Chem. 287, 41888–41902 - PMC - PubMed
    1. Pewzner-Jung Y., Park H., Laviad E. L., Silva L. C., Lahiri S., Stiban J., Erez-Roman R., Brügger B., Sachsenheimer T., Wieland F., Prieto M., Merrill A. H. Jr., and Futerman A. H. (2010) A critical role for ceramide synthase 2 in liver homeostasis: I. alterations in lipid metabolic pathways. J. Biol. Chem. 285, 10902–10910 - PMC - PubMed

Publication types

MeSH terms