Woo-Jae Park

Ceramide synthases as potential targets for therapeutic intervention in human diseases

Ceramide is located at a key hub in the sphingolipid metabolic pathway and also acts as an important cellular signaling molecule. Ceramide contains one acyl chain which is attached to a sphingoid long chain base via an amide bond, with the acyl chain varying in length and degree of saturation. The identification of a family of six mammalian ceramide synthases (CerS) that synthesize ceramide with distinct acyl chains, has led to significant advances in our understanding of ceramide biology, including further delineation of the role of ceramide in various pathophysiologies in both mice and humans. Since ceramides, and the complex sphingolipids generated from ceramide, are implicated in disease, the CerS might potentially be novel targets for therapeutic intervention in the diseases in which the ceramide acyl chain length is altered. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Ablation of very long acyl chain sphingolipids causes hepatic insulin resistance in mice due to altered detergent‐resistant membranes

Sphingolipids are important structural components of cell membranes and act as critical regulators of cell function by modulating intracellular signaling pathways. Specific sphingolipids, such as ceramide, glucosylceramide, and ganglioside GM3, have been implicated in various aspects of insulin resistance, because they have been shown to modify several steps in the insulin signaling pathway, such as phosphorylation of either protein kinase B (Akt) or of the insulin receptor. We now explore the role of the ceramide acyl chain length in insulin signaling by using a ceramide synthase 2 (CerS2) null mouse, which is unable to synthesize very long acyl chain (C22‐C24) ceramides. CerS2 null mice exhibited glucose intolerance despite normal insulin secretion from the pancreas. Both insulin receptor and Akt phosphorylation were abrogated in liver, but not in adipose tissue or in skeletal muscle. The lack of insulin receptor phosphorylation in liver correlated with its inability to translocate into detergent‐resistant membranes (DRMs). Moreover, DRMs in CerS2 null mice displayed properties significantly different from those in wild‐type mice, suggesting that the altered sphingolipid acyl chain length directly affects insulin receptor translocation and subsequent signaling. Conclusion: We conclude that the sphingolipid acyl chain composition of liver regulates insulin signaling by modifying insulin receptor translocation into membrane microdomains. (HEPATOLOGY 2013)

Sortilin deficiency improves the metabolic phenotype and reduces hepatic steatosis of mice subjected to diet-induced obesity

BACKGROUND & AIMS Sortilin traffics newly synthesized molecules from the trans-Golgi apparatus along secretory pathways to endosomes, lysosomes or to the cell surface. Sortilin trafficking of acid sphingomyelinase (aSMase) may regulate ceramide levels, a major modulator of insulin signalling. We therefore tested whether sortilin deficiency reduces hepatic and adipose tissue aSMase activity, improving insulin sensitivity in diet-induced obesity (DIO). METHODS DIO in C57BL/6 (WT) and sortilin(-/-) mice was induced by high-fat diet feeding for 10 weeks. RESULTS Sortilin(-/-) mice gained less body weight and less visceral fat, despite similar food intake compared to WT type mice and had enhanced glucose uptake in insulin tolerance tests, which was further corroborated by enhanced hepatic pAkt expression. Sortilin deficiency led to attenuated hepatic steatosis, reduced expression of genes involved in lipogenesis, ceramide synthesis and inflammatory cytokine production and reduced activity of ceramide synthase 5/6 (CerS5/6). Sortilin(-/-) mice had reduced hepatic aSMase activity under both steady-state and DIO. Likewise, sortilin(-/-) hepatocytes displayed hypersensitivity to insulin, due to enhanced insulin receptor downstream signalling. In adipose tissue, sortilin(-/-) mice exhibited lower expression of inflammatory cytokines and lower expression and activity of CerS5/6. As in liver, adipose tissue displayed increased insulin signalling, accompanied by attenuated aSMase activity. CONCLUSIONS Sortilin deficiency induces a beneficial metabolic phenotype in liver and adipose tissue upon DIO, mediated in part by reduced aSMase activity.

Protection of a Ceramide Synthase 2 Null Mouse from Drug-induced Liver Injury ROLE OF GAP JUNCTION DYSFUNCTION AND CONNEXIN 32 MISLOCALIZATION

Background: Ceramide synthase 2 null mice cannot synthesize very long acyl chain ceramides and display severe hepatopathy. Results: Ceramide synthase 2 null mice are protected from drug- and chemical-induced liver injury and display impaired gap junction function. Conclusion: Altering sphingolipid levels modulates gap junction function. Significance: Sphingolipids may play a key role in regulating drug-induced liver injury. Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl4, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.

The effect of altered sphingolipid acyl chain length on various disease models.

Abstract Sphingolipids have emerged as an important lipid mediator in intracellular signalling and metabolism. Ceramide, which is central to sphingolipid metabolism, is generated either via a de novo pathway, by attaching fatty acyl CoA to a long-chain base, or via a salvage pathway, by degrading pre-existing sphingolipids. As a ‘sphingolipid rheostat’ has been proposed, the balance between ceramide and sphingosine-1-phosphate has been the object of considerable attention. Ceramide has recently been reported to have a different function depending on its acyl chain length: six ceramide synthases (CerS) determine the specific ceramide acyl chain length in mammals. All CerS-deficient mice generated to date show that sphingolipids with defined acyl chain lengths play distinct pathophysiological roles in disease models. This review describes recent advances in understanding the associations of CerS with various diseases and includes clinical case reports.

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on β-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.

Development of pheochromocytoma in ceramide synthase 2 null mice

Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in ∼15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the ceramide synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes ceramides with very-long (C22-C24) chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullary tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ∼13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulation of ceroid in the X-zone was observed as early as 2 months of age and as a consequence, older mice displayed elevated levels of lysosomal cathepsins, reduced proteasome activity and reduced activity of mitochondrial complex IV by 6 months of age. Together, these findings implicate an additional pathway that can lead to PCC formation, which involves alterations in the sphingolipid acyl chain length. Analysis of the role of sphingolipids in PCC may lead to further understanding of the mechanism by which PCC develops, and might implicate the sphingolipid pathway as a possible novel therapeutic target for this rare tumor.

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate-induced colitis in mice due to increased intestinal permeability

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long‐chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very‐long acyl chain ceramides with concomitant increase of long chain bases and C16‐ceramides, were more susceptible to dextran sodium sulphate‐induced colitis, and their survival rate was markedly decreased compared with that of wild‐type littermates. Using mixed bone‐marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule‐A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco‐2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2‐knockdown via CRISPR‐Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long‐chain bases and C16‐ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC‐dextran levels, indicating that altered SLs including deficiency of very‐long‐chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.

Bortezomib alleviates drug-induced liver injury by regulating CYP2E1 gene transcription

Acute liver failure, i.e., the fatal deterioration of liver function, is the most common indication that emergency liver transplantation is necessary. Moreover, in the USA, drug-induced liver injury (DILI), including acetaminophen (APAP)-induced hepatotoxicity, is the main cause of acute liver failure. Matching a donor for liver transplantation is extremely difficult, and thus the development of a novel therapy for DILI is urgently needed. Following recent approval by the FDA of the proteasomal inhibitor bortezomib, its therapeutic effects on various human diseases, including solid and hematologic malignancies, have been validated. However, the specific action of proteasomal inhibition in cases of DILI had not been elucidated prior to this study. To examine the effects of proteasomal inhibition in DILI experimentally, male C56Bl/6 mice were injected with 1 mg bortezomib/kg before APAP treatment. Bortezomib not only alleviated APAP-induced hepatotoxicity in a time- and dose-dependent manner, it also alleviated CCl4- and thioacetamide-induced hepatotoxicity. We also noted that bortezomib significantly reduced cytochrome P450 2E1 (CYP2E1) expression and activity in the liver, which was accompanied by the induction of endoplasmic reticulum (ER) stress. In addition, bortezomib decreased hepatocyte nuclear factor-1α-induced promoter activation of CYP2E1 in Hep3B cells. By contrast, another proteasome inhibitor, MG132, did not cause ER stress and did not markedly affect CYP2E1 enzyme activity. Liver injury induced by APAP was aggravated by MG132, possibly via elevation of connexin 32 expression. This study suggests that proteasome inhibition has different effects in cases of DILI depending on the specific inhibitor being used. Furthermore, results from the mouse model indicated that bortezomib, but not MG132, was effective in alleviating DILI. ER stress induced by proteasome inhibition has previously been shown to exert various effects on DILI patients, and thus each available proteasomal inhibitor should be evaluated individually in order to determine its potential for clinical application.

P283 SORTILIN DEFICIENCY IMPROVES HEPATIC AND ADIPOSE TISSUE INSULIN RESISTANCE AND INFLAMMATION IN DIET-INDUCED OBESITY

Background and Aims: Sortilin is a trafficking molecule directing newly synthesized molecules from the trans-Golgi network to secretory pathways, endosomes, lysosomes or cell surface. Several of the molecules trafficked by sortilin, such as acidic sphingomyelinase (ASM) and sphingolipid activating proteins (SAPs), regulate the synthesis of ceramide, a major modulator of insulin signaling. We hypothesized that in sortilin mice, reduced hepatic ASM may improve insulin sensitivity and reduce steatosis in a diet-induced obesity (DIO) model. Methods: DIO and insulin resistance were induced by feeding high fat diet for 10 weeks to WT and sortilin mice. Results: Sortilin mice had significantly reduced body weight and visceral fat, despite similar food intake. Sortilin mice had better insulin tolerance test and displayed increased insulin signaling in both liver and adipose tissue as demonstrated by increased Akt phosphorylation. In accordance with the proposed role of sortilin in ASM trafficking, ASM activity in both liver and adipose tissue of sortilin mice was significantly reduced. Sortilin mice had almost no steatosis and a three-fold reduction in total hepatic triglyceride levels compared to WT mice. In addition, expression of proinflammatory cytokines was reduce in both livers and adipose tissue of sortilin. Looking at ceramide synthesis enzymes, we observed a significant reduction in ceramide synthase 6, which synthesizes long chain ceramides, in both liver and adipose tissue of sortilin. Conclusions: Sortilin deficiency induces a beneficial metabolic phenotype in DIO, with respect to both liver and adipose tissue, which may be mediated in part by reduced ASM activity and reduced ceramide levels.