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.

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 ß-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.

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury.

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.

Hepatic triglyceride accumulation via endoplasmic reticulum stress-induced SREBP-1 activation is regulated by ceramide synthases.

The endoplasmic reticulum (ER) is not only important for protein synthesis and folding but is also crucial for lipid synthesis and metabolism. In the current study, we demonstrate an important role of ceramide synthases (CerS) in ER stress and NAFLD progression. Ceramide is important in sphingolipid metabolism, and its acyl chain length is determined by a family of six CerS in mammals. CerS2 generates C22-C24 ceramides, and CerS5 or CerS6 produces C16 ceramide. To gain insight into the role of CerS in NAFLD, we used a high-fat diet (HFD)-induced NAFLD mouse model. Decreased levels of CerS2 and increased levels of CerS6 were observed in the steatotic livers of mice fed a HFD. In vitro experiments with Hep3B cells indicated the protective role of CerS2 and the detrimental role of CerS6 in the ER stress response induced by palmitate treatment. In particular, CerS6 overexpression increased sterol regulatory element-binding protein-1 (SREBP-1) cleavage with decreased levels of INSIG-1, leading to increased lipogenesis. Blocking ER stress abrogated the detrimental effects of CerS6 on palmitate-induced SREBP-1 cleavage. In accordance with the protective role of CerS2 in the palmitate-induced ER stress response, CerS2 knockdown enhanced ER stress and SREBP-1 cleavage, and CerS2 heterozygote livers exhibited a stronger ER stress response and higher triglyceride levels following HFD. Finally, treatment with a low dose of bortezomib increased hepatic CerS2 expression and protected the development of NAFLD following HFD. These results indicate that CerS and its derivatives impact hepatic ER stress and lipogenesis differently and might be therapeutic targets for NAFLD.

Proteasome inhibition protects against diet-induced gallstone formation through modulation of cholesterol and bile acid homeostasis.

Gallstone disease is one of the most prevalent and costly gastrointestinal disorders worldwide. Gallstones are formed in the biliary system by cholesterol secretions in bile, which result from excess cholesterol, a deficiency in bile salts or a combination of the two. The present study examined the effects of proteasome inhibition on gallstone formation using the proteasome inhibitors bortezomib (BT) and carfilzomib (CF). C57BL/6J mice were fed a lithogenic diet to generate gallstones and injected with BT or CF for 12 weeks. After 12 weeks of the lithogenic diet, 8 out of the 10 mice in the control group had developed gallstones, whereas none of the mice who received proteasome inhibitors had developed gallstones. Notably, the expression of genes associated with cholesterol synthesis (sterol regulatory element­binding protein­2 and 3­hydroxy­3­methylglutaryl­CoA reductase), cholesterol secretion [ATP­binding cassette subfamily G member 5 (ABCG5) and ABCG8] and bile acid synthesis [cytochrome P450 family 7 subfamily A member 1 (Cyp7a1), Cyp7b1, Cyp27a1 and Cyp8b1] was reduced in the livers of mice injected with BT or CF. Cyp7a1 encodes cholesterol 7α­hydroxylase, the rate­limiting enzyme in the synthesis of bile acid from cholesterol. The present study therefore measured the expression levels of transcription factors that are known to inhibit Cyp7a1 expression, namely farnesoid X receptor (FXR), pregnane X receptor (PXR) and small heterodimer partner (SHP). Although FXR, PXR and SHP expression was predicted to increase in the presence of proteasome inhibitors, the expression levels were actually reduced; thus, it was concluded that they were not involved in the proteasome inhibition­induced regulation of Cyp7a1. Further investigation of the mitogen­activated protein kinase and protein kinase A (PKA) signaling pathways in human hepatoma cells revealed that proteasome inhibition­induced c­Jun N­terminal kinase (JNK) phosphorylation reduced CYP7A1 and CYP27A1 expression. In addition, reduced PKA phosphorylation as a result of proteasome inhibition regulated ABCG5 and ABCG8 expression. In conclusion, these findings suggest that proteasome inhibition regulates cholesterol and biliary metabolism via the JNK and PKA pathways, and is a promising therapeutic strategy to prevent gallstone disease.

Hepatic inflammatory cytokine production can be regulated by modulating sphingomyelinase and ceramide synthase 6.

Chronic inflammation is associated with the pathogenesis of type 2 diabetes and diabetic complications, and palmitate has been nominated as a candidate for the molecular link between these disorders. Recently, a crucial role of ceramide in inflammation and metabolic diseases has been reported. Therefore, in this study, we investigated whether ceramide formation is involved in palmitate­induced hepatic inflammation in vitro and in vivo. Ceramide can be generated either by the de novo pathway or by sphingomyelin degradation, and six different ceramide synthases (CerS) determine the specific acyl chain length of ceramide in mammals. We examined the roles of CerS and sphingomyelinases (SMases) in the secretion of inflammatory cytokines, such as tumour necrosis factor (TNF)­α, interleukin (IL)­1ß, and IL­6 in Hep3B cells. Among the six CerS, CerS6 overexpression uniquely elevated TNF­α secretion via p38 mitogen­activated protein kinase (MAPK) activation. In addition, the treatment of CerS6 overexpressing cells with palmitate synergistically increased cytokine secretion. However, neither palmitate treatment nor CerS6 overexpression altered lipopolysaccharide (LPS)-induced cytokine secretion. Instead, the activation of acidic (A)­SMase was involved in LPS­induced cytokine secretion via the MAPK/NF­κB pathway. Finally, the suppression of ceramide generation via A­SMase inhibition or de novo ceramide synthesis decreased high­fat diet­induced hepatic cytokine production in vivo. On the whole, our results revealed that CerS6 played a role in TNF­α secretion, and palmitate augmented inflammatory responses in pathophysiological conditions in which CerS6 is overexpressed. In addition, A­SMase activation was shown to be involved in LPS­induced inflammatory processes, suggesting that the modulation of CerS6 and A­SMase may be a therapeutic target for controlling hepatic inflammation.

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 factor­1α-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.