Loading

Commentary Open Access
Volume 3 | Issue 2 | DOI: https://doi.org/10.33696/diabetes.3.036

CERT-Dependent Ceramide Transport, A Crucial Process in Cells

  • 2Institut Hospitalo-Universitaire ICAN, Paris, France
  • 1Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, F-75006 Paris, France
+ Affiliations - Affiliations

*Corresponding Author

Eric Hajduch, eric.hajduch@crc.jussieu.fr

Received Date: March 24, 2021

Accepted Date: April 19, 2021

Commentary

In mammalian cells, ceramides are central lipids in sphingolipid metabolism and serve both as signaling lipids and as precursors for other bioactive sphingolipids, ranging from complex glycosphingolipids to “simpler” lipids such as ceramide-1-phosphate, sphingomyelin (SM), sphingosine and sphingosine-1-phosphate (S1P). Ceramides are largely distributed in cell membranes where they play an important structural role. In addition, ceramides play also key roles in intracellular signaling, and regulate growth, proliferation, cell migration, apoptosis, and differentiation [1].

Ceramides consist of a spingoid long chain base to which a fatty acid is attached via an amide bond. They can be generated either by hydrolysis of sphingomyelin by sphingomyelinases, degradation of complex sphingolipids localized in lysosomes, or produced de novo from saturated fatty acids, like palmitic acid, in the endoplasmic reticulum (ER) [2].

Synthesis of both SM and glucosylceramides (GlcCer) from ceramide occurs into the Golgi apparatus [2]. Ceramides are transported through a non-ATP dependent vesicular transport from the ER toward the cis-Golgi, where GlcCer synthase adds a glucose molecule to ceramides to give GlcCer [3]. Ceramides are also transported to the trans- Golgi to give SM after addition of a phosphocholine by the sphingomyelin synthase 1 (SMS1) [4]. This latter transport is ensured by a specific ceramide transporter called CERT (for ceramide transporter) in an ATP-dependent manner [5].

CERT, a 68 kDa protein, possess several domains to provide its precise function. The StAR-related lipid transfer (START) domain specifically extracts the ceramide molecule from the ER membrane to the Golgi membrane [6]. CERT also have a pleckstrin homology (PH) domain at its N-terminal side, which binds phosphatidylinositol 4-phosphate (PIP4), abundant phospholipid found in the trans-Golgi region (Figure 1) [7]. Two phenylalanine residues localized in the acidic tract (FFAT) domain allows CERT to bind the VAMP-associated protein (VAP) in the ER membrane [7]. FFAT domain can be phosphorylated on its serine 315 (S315), which improves ceramide transport from ER to Golgi, since CERT displays a higher affinity to VAP when S315 is phosphorylated (Figure 2) [7]. CERT possess also a serine-repeated motif that can be phosphorylated by protein kinase D (PKD) to decrease its binding with PIP4 and thus, reduces CERT ceramide transfer activity [7]. The serine-repeat motif (SRM), localized near the PH domain, carries several phosphorylation sites [7], and it has been shown that multiple phosphorylation of this site decreases CERT function, result of a conformational hindrance [7] .

Two CERT isoforms exist. CERTL  – also known as Goodpasture antigen-binding protein (GPBP) which is 26 amino acids residues longer than CERT/GPBPΔ26 [8]. The two forms can be found in most tissues, but CERTL is mainly expressed in tissues which can targeted by autoimmune response [8].

The importance of the CERT transporter is becoming increasingly evident for the regulation of intracellular concentration of ceramides and their transformation into other sphingolipid derivatives. Several studies have indeed shown that changes in expression / activity of CERT could modulate the action of ceramides on several physiological processes.

CERT and Insulin Resistance

Ceramide accumulation in cells (inducing lipotoxicity) is now well known to induce insulin resistance in muscle [9], in liver [10], in adipose tissue [11], and also pancreatic beta cells death [12].

A recent study showed that the increased ceramide concentrations in response to saturated FA was associated with their defective transport from the ER to the Golgi apparatus in muscle cells [13]. The authors demonstrated that CERT expression was decreased in the muscle cell line C2C12 after palmitate treatment to induce cell insulin resistance, muscle cells from diabetic mice and also in muscle from human diabetic patient, and that this drop in expression was the consequence of ceramide-dependent CERT cleavage by caspases 3 and 9. Interestingly, in vitro CERT overexpression in C2C12 myotubes treated with palmitate, or in vivo by electro transfer in the anterior tibial muscle of diabetic mice, decreased some ceramide species content (C16, C22, C24 and C24:1-ceramides), and improved insulin sensitivity in CERT-overexpressing muscle cells [13]. Overall, this study highlighted an important and original mechanism for regulating ceramide flow in muscle cells and to prevent their accumulation under lipotoxic conditions.

Interestingly, no decrease of CERT expression in a lipotoxic context was observed in liver [14]. Indeed, primary rat hepatocytes treated with palmitic acid for 16h showed an inhibition of the insulin signal following an increase in both ceramide content and CERT expression [14]. CERT expression returned to basal level 40 h after palmitic acid incubation while ceramide content remained elevated [14]. The authors hypothesized that ceramide transported from ER to Golgi could be converted to SM, which may be hydrolyzed latter on to form more ceramide molecules that intensified ceramide accumulation and lipotoxicity in cells. It is important to note that several studies demonstrated that another lipid species, diacylglycerol’s, play also an important and deleterious role in the loss of insulin sensitivity in liver in lipotoxic conditions [15].

Differences between the two articles regarding the action of ceramides on CERT expression could also be explained by the fact that, unlike what happens in muscle cells, ceramides do not really accumulate in hepatocytes [16]. Indeed, the upper study [14] and another one carried out in HepG2 liver cells showed that palmitate rapidly induced extracellular ceramide build-up in a dose- and time-dependent manner, suggesting that liver cells rapidly secreted the newly synthesized ceramide molecules [16]. As a result, it is possible that, unlike in muscle cells, ceramide concentrations remained relatively low in hepatocytes, at a level too small to induce caspase activation and to decrease CERT expression. Therefore, CERT expression remains unaffected by ceramides in hepatocytes.

Overall, these studies highlight the importance of a good ceramide transport in muscle to maintain a physiological insulin sensitivity. Maintaining a good CERT expression in cells appears to be crucial to prevent ceramide accumulation and cellular dysfunction.

CERT and Apoptosis

Some studies were interested in the role of ceramides and their transport in a context of aging. In females, advanced age induced changes in mitochondrial function and structure of oocytes and decreased their quality. Perez and collaborators observed that oocyte from old mice showed higher apoptosis levels after ceramide injection compared to apoptosis levels of ceramide-injected oocyte from young mice [17]. Interestingly, a decrease in CERT expression in oocytes from old mice compared to CERT expression in oocytes from young mice was observed, suggesting that a defect in ceramide metabolism/localization due to the lack of CERT expression could be responsible for the decreased developmental potential observed in old oocytes. This was confirmed when co-treatment of aged oocytes with ceramide and a cytoplasmic lipid carrier (l-carnitine) enhanced mitochondrial morphology and function, suggesting that the absence of ceramide transport surely induced the lipid accumulation and prevented its transformation into other lipid derivatives less toxic for the cells [17].

A recent study investigated the possible link between ceramide and mitochondrial apoptosis. Indeed, mitochondria play a central role in apoptotic cell death. Induction of mitochondrial apoptosis occurs upon outer membrane permeabilization, leading to the release of intermembrane proteins, such as the hemeprotein cytochrome c, and through the induction of caspase activation, ultimately causing cell apoptosis [18]. Ceramide are also known to promote cell apoptosis [1], and to study a possible involvement of this sphingolipid into mitochondrial apoptosis, the authors diverted CERTmediated ceramide transport to mitochondria by targeting CERT to the outer mitochondrial membrane (OMM) while retaining its ability to interact with VAP proteins in the ER [19]. To do so, they armed CERT with an OMM anchor and called it mitoCERT [19]. When HeLa cells were transfected by mitoCERT, an increased in cytosolic translocation of cytochrome c and caspase 9 activity was observed, leading to cell apoptosis [19]. Pre-treatment of cells with the CERT inhibitor HPA12 prevented cell death [19]. Overall, this study clearly demonstrates that transport of ceramide to mitochondria specifically induces apoptotic cell death.

All these results suggest again that CERT expression remains crucial in order to prevent ceramides to harm the cells. A dynamic between ceramide concentration and CERT expression is crucial to induce or to inhibit apoptosis in cells.

CERT and Cancer

Given the deleterious role of ceramides in the regulation of cell growth and apoptosis, a novel strategy to induce tumor cell apoptosis by modifying ceramide metabolism was looked at [20]. A study performed in three cancer cell lines (human alveolar adenocarcinoma A549; human colorectal carcinoma HCT116; human breast adenocarcinoma MDAMB- 231), observed that the inhibition of CERT activity through the use of the chemical inhibitor (HPA-12) or a CERT siRNA, led to a higher sensitivity of tumors to cancer drugs [21]. Indeed, in HCT116 cells, HPA-12 induced the increase of an actor of the three axes of the unfolded protein response (UPR), PERK [22], leading to a chronic activation of ER stress in cells, ultimately leading to cell apoptosis [22]. In another study performed in colorectal and breast cancer cells, the authors highlighted that CERT inhibition induced ceramide accumulation in cells, leading to enhanced lysosomal-autophagosome flux [23].

These independent studies demonstrated that CERT modulation could modify cell sensitivity to certain drugs through ceramide accumulation, certainly an interesting possibility to fight cancers and other pathologies.

CERT and Brain Related Diseases

Sphingomyelin, synthetized after transport of ceramide by CERT from the ER towards the Golgi, is the most abundant sphingolipid in cell membranes [24]. SM plays crucial roles in brain myelination, which is important for brain development [25]. This is why SM dysregulation may lead to cell dysfunctions, or even important diseases [26]. In Niemann-Pick disease, for example, SM accumulates in cell lysosomes, leading to several organs dysfunction like in the central nervous system [27].

A recent study identified a novel CERT variant from a patient with intellectual disability, where the serine at position 135 (S135) was substituted by a proline in the serine-repeat motif domain [28]. This domain is crucial to downregulate CERT activity [7]. Indeed, it can be phosphorylated several times on different serine/ threonine residues, and this multiple phosphorylation leads to a decrease of CERT activity [7]. The novel CERT variant cannot be phosphorylated and is abnormally active in this patient [28]. As consequence, the patient displays a strong intellectual retardation. MRI performed revealed a general cerebral atrophy [28], more specially at the frontal lobe responsible for reasoning, speaking and voluntary movement [29]. A hypoplasia of the corpus callosum, which ensures the communication between the two cerebral hemispheres, was also observed [28].

Alzheimer’s disease (AD), the most common neurodegenerative disorder, is characterized by extracellular deposits of amyloid β-peptides (Aβ), leading to a strong crippling disease [30]. Ceramides are associated with AD because they were shown to stabilize β-secretase, one of the key enzymes that cleaves the amyloid-precursor protein (APP) into the deleterious Aβ. Thus, ceramides contribute to Aβ accumulation in the nervous central system [30]. High ceramide content was observed into the cortex from patients with mild to moderate symptoms, suggesting that ceramide accumulation could occur in the early stage of the disease [31]. As consequence, ceramide content is more elevated in brains from AD patient compared to control patient [32].

CERTL expression was reported to binds the amyloidprecursor protein and to be reduced in the cortex in a mouse model of familial Alzheimer disease (5xFAD) [33]. In opposite, ceramide content was elevated in brains of Alzheimer patient [33]. Interestingly, a real interaction between CERTL and APP was observed using a co-immunoprecipitation system into primary neurons from 5xFAD brains. Addition of amyloid-β decreased cell viability but CERTL addition was able to restore this viability. In addition, brain cortex from 5xFAD mice revealed an increase in C16-ceramides, C18-ceramides, C20-ceramides, C22-ceramide, while CERTL expression was decreased [33].

CERTL  overexpression in 5xFAD mice through a viral vector did not affect behavior compared to mice who received a control virus [33]. However, a decrease of C16- ceramides into the cortex of 5xFAD mice overexpressing CERTL was observed, together with an increase of C16- SM, C18-SM, and C18:1-SM. Even if plaque numbers were not reduced in brain of mice overexpressing CERTL, the percentage of small plaque size was decreased in total brain, concomitantly with a decrease of amyloid-β in brain homogenate. In opposite, AD transgenic mice treated with the CERT inhibitor HPA-12 during 4 weeks displayed an increase of C16-ceramide, C20-ceramide, C22-ceramide and C24:1-ceramide in the cortex compared to control mice. As expected, amyloid-β was increased in brain homogenate.

All together, these data suggest that alteration of ceramide transport from ER to the Golgi, involving CERT, alters APP processing and leads to expand a critical phenomenon in Alzheimer disease.

New CERT Inhibitors

The development of HPA-12, a competitive inhibitor of CERT, helped a lot to understand the activity, function and regulation of CERT [34]. A recent study identified two others CERT inhibitors [35]. The authors used a fluorescence-based assay to measure the capacity of CERT to transfer labelled-ceramide to a liposome containing a lipid-quencher that can be tracked. This approach was combined with the Förster resonance energy transfer, technique allowing to see whether two light-sensitive molecules are close enough to carry out an energy transfer. 2000 different compounds were screened, and HPA-12 was used as a reference molecule. Following this protocol, four promising compound candidates were isolated [35]. Among them, only 2 were able to inhibit CERT and therefore decrease SM concentration, and increase ceramide content in cells. One, called Fluralaner, was already used as a veterinary drug. It targets the arthropod parasite channel and displayed an unknown target in mammals [35]. The other one, called Lomitapide, is a human drug that had already been shown to target the human microsomal triglyceride transfer protein. Lomitapide is used to treat familial hypercholesterolemia [36]. Both CERT inhibitors, already prescribed as medication in animals and human, could also be used as tools to study ceramide transfer.

Conclusion

Considering the growing importance of related studies around ceramides, they could even become a new biomarker for detecting cardiovascular disease [37], and studying the dynamics of flows between different sphingolipids remains crucial in several fields. All these studies show that the dysregulation of ceramide transfer from the ER to the Golgi apparatus, via the alteration of the activity / expression of CERT, presents serious consequences. These studies also highlight the importance of CERT as a major regulator of ceramide metabolism. Considering the number of cellular processes affected by a dysregulation of CERT, further studies will be necessary to get a better understanding of its implication in several pathologies and to consider the use of the different and new existing CERT inhibitors to fight the diseases.

Author Contributions Statement

Conceptualization, C.L.B. and E.H.; writing-original draft preparation, C.L.B.; writing-review and editing, C.L.B. and E.H. Both authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the “Société Francophone du diabète” and the “Fondation de France”.

References

1. Hannun YA, Obeid LM. Principles of bioactive lipid signalling: lessons from sphingolipids. Nature Reviews Molecular Cell Biology. 2008 Feb;9(2):139-50.

2. Mullen TD, Hannun YA, Obeid LM. Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochemical Journal. 2012 Feb 1;441(3):789-802.

3. Halter D, Neumann S, van Dijk SM, Wolthoorn J, De Maziere AM, Vieira OV, et al. Pre-and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis. The Journal of Cell biology. 2007 Oct 8;179(1):101-15.

4. Huitema K, van den Dikkenberg J, Brouwers JF, Holthuis JC. Identification of a family of animal sphingomyelin synthases. The EMBO Journal. 2004 Jan 14;23(1):33-44.

5. Yamaji T, Hanada K. Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins. Traffic. 2015 Feb;16(2):101-22.

6. Hanada K. Co-evolution of sphingomyelin and the ceramide transport protein CERT. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids. 2014 May 1;1841(5):704-19.

7. Kumagai K, Kawano M, Shinkai-Ouchi F, Nishijima M, Hanada K. Interorganelle trafficking of ceramide is regulated by phosphorylation-dependent cooperativity between the PH and START domains of CERT. Journal of Biological Chemistry. 2007 Jun 15;282(24):17758-66.

8. Raya A, Revert-Ros F, Martinez-Martinez P, Navarro S, Roselló E, Vieites B, et al. Goodpasture antigen-binding protein, the kinase that phosphorylates the goodpasture antigen, is an alternatively spliced variant implicated in autoimmune pathogenesis. Journal of Biological Chemistry. 2000 Dec 22;275(51):40392-9.

9. Bandet CL, Tan-Chen S, Bourron O, Le Stunff H, Hajduch E. Sphingolipid metabolism: new insight into ceramideinduced lipotoxicity in muscle cells. International Journal of Molecular Sciences. 2019 Jan;20(3):479.

10. Turpin SM, Nicholls HT, Willmes DM, Mourier A, Brodesser S, Wunderlich CM, et al. Obesity-induced CerS6-dependent C16: 0 ceramide production promotes weight gain and glucose intolerance. Cell Metabolism. 2014 Oct 7;20(4):678-86.

11. Li Y, Talbot CL, Chaurasia B. Ceramides in Adipose Tissue. Frontiers in Endocrinology. 2020;11.

12. Bellini L, Campana M, Mahfouz R, Carlier A, Véret J, Magnan C, et al. Targeting sphingolipid metabolism in the treatment of obesity/type 2 diabetes. Expert Opinion on Therapeutic Targets. 2015 Aug 3;19(8):1037-50.

13. Bandet CL, Mahfouz R, Véret J, Sotiropoulos A, Poirier M, Giussani P, et al. Ceramide transporter CERT is involved in muscle insulin signaling defects under lipotoxic conditions. Diabetes. 2018 Jul 1;67(7):1258-71.

14. Konstantynowicz-Nowicka K, Harasim E, Baranowski M, Chabowski A. New evidence for the role of ceramide in the development of hepatic insulin resistance. PLoS One. 2015 Jan 30;10(1):e0116858.

15. Erion DM, Shulman GI. Diacylglycerol-mediated insulin resistance. Nature Medicine. 2010 Apr;16(4):400-2.

16. Watt MJ, Barnett AC, Bruce CR, Schenk S, Horowitz JF, Hoy AJ. Regulation of plasma ceramide levels with fatty acid oversupply: evidence that the liver detects and secretes de novo synthesised ceramide. Diabetologia. 2012 Oct;55(10):2741-6.

17. Perez GI, Jurisicova A, Matikainen T, Moriyama T, Kim MR, Takai Y, et al. A central role for ceramide in the age-related acceleration of apoptosis in the female germline. The FASEB journal. 2005 May;19(7):1-23.

18. Abate M, Festa A, Falco M, Lombardi A, Luce A, Grimaldi A, et al. Mitochondria as playmakers of apoptosis, autophagy and senescence. InSeminars in cell & developmental biology 2020 Feb 1 (Vol. 98, pp. 139-153). Academic Press.

19. Jain A, Beutel O, Ebell K, Korneev S, Holthuis JC. Diverting CERT-mediated ceramide transport to mitochondria triggers Bax-dependent apoptosis. Journal of Cell Science. 2017 Jan 15;130(2):360-71.

20. Scheffer L, Rao Raghavendra P, Ma J, K Acharya J. Ceramide transfer protein and cancer. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 2011 Nov 1;11(9):904-10.

21. Swanton C, Marani M, Pardo O, Warne PH, Kelly G, Sahai E, et al. Regulators of mitotic arrest and ceramide metabolism are determinants of sensitivity to paclitaxel and other chemotherapeutic drugs. Cancer Cell. 2007 Jun 12;11(6):498-512.

22. Flamment M, Hajduch E, Ferré P, Foufelle F. New insights into ER stress-induced insulin resistance. Trends in Endocrinology & Metabolism. 2012 Aug 1;23(8):381-90.

23. Lee AJ, Roylance R, Sander J, Gorman P, Endesfelder D, Kschischo M, et al. CERT depletion predicts chemotherapy benefit and mediates cytotoxic and polyploid-specific cancer cell death through autophagy induction. The Journal of Pathology. 2012 Feb;226(3):482-94.

24. Gault CR, Obeid LM, Hannun YA. An overview of sphingolipid metabolism: from synthesis to breakdown. Sphingolipids as Signaling and Regulatory Molecules. 2010:1-23.

25. Schneider N, Hauser J, Oliveira M, Cazaubon E, Mottaz SC, Neill BV, et al. Sphingomyelin in brain and cognitive development: Preliminary Data. Eneuro. 2019 Jul 19.

26. Bienias K, Fiedorowicz A, Sadowska A, Prokopiuk S, Car H. Regulation of sphingomyelin metabolism. Pharmacological Reports. 2016 Jun 1;68(3):570-81.

27. Ferreira CR, Gahl WA. Lysosomal storage diseases. Translational Science of Rare Diseases. 2017 Jan 1;2(1-2):1-71.

28. Murakami H, Tamura N, Enomoto Y, Shimasaki K, Kurosawa K, Hanada K. Intellectual disability-associated gain-of-function mutations in CERT1 that encodes the ceramide transport protein CERT. PloS One. 2020 Dec 21;15(12):e0243980.

29. Chayer C, Freedman M. Frontal lobe functions. Current Neurology and Neuroscience reports. 2001 Nov;1(6):547-52.

30. Jazvinšćak Jembrek M, Hof PR, Šimić G. Ceramides in Alzheimer’s disease: key mediators of neuronal apoptosis induced by oxidative stress and Aβ accumulation. Oxidative Medicine and Cellular Longevity. 2015 Oct;2015.

31. Cutler RG, Kelly J, Storie K, Pedersen WA, Tammara A, Hatanpaa K, et al. Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer’s disease. Proceedings of the National Academy of Sciences. 2004 Feb 17;101(7):2070- 5.

32. Filippov V, Song MA, Zhang K, Vinters HV, Tung S, Kirsch WM, et al. Increased ceramide in brains with Alzheimer’s and other neurodegenerative diseases. Journal of Alzheimer’s Disease. 2012 Jan 1;29(3):537-47.

33. Crivelli SM, Luo Q, Stevens JA, Giovagnoni C, van Kruining D, Bode G, et al. CERT L reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease. Alzheimer’s Research & Therapy. 2021 Dec;13(1):1-21.

34. Yasuda S, Kitagawa H, Ueno M, Ishitani H, Fukasawa M, Nishijima M, et al. A novel inhibitor of ceramide trafficking from the endoplasmic reticulum to the site of sphingomyelin synthesis. Journal of Biological Chemistry. 2001 Nov 23;276(47):43994-4002.

35. Samaha D, Hamdo HH, Cong X, Schumacher F, Banhart S, Aglar Ö, et al. Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT). Chemistry (Weinheim an der Bergstrasse, Germany). 2020 Dec 15;26(70):16616.

36. Berberich AJ, Hegele RA. Lomitapide for the treatment of hypercholesterolemia. Expert Opinion on Pharmacotherapy. 2017 Aug 13;18(12):1261-8.

37. Summers SA. Could ceramides become the new cholesterol?. Cell Metabolism. 2018 Feb 6;27(2):276-80.

Author Information X