Background & Aims Ceramide, a sphingolipid metabolite, affects T-cell signaling, induces apoptosis of cancer cells, and slows tumor growth in mice. However, it has not been used as a chemotherapeutic agent because of its cell impermeability and precipitation in aqueous solution. We developed a nanoliposome-loaded C6-ceremide (LipC6) to overcome this limitation and investigated its effects in mice with liver tumors. Methods Immune competent C57BL/6 mice received intraperitoneal injections of carbon tetrachloride and intra-splenic injections of oncogenic hepatocytes. As a result, tumors resembling human hepatocellular carcinomas developed in a fibrotic liver setting. After tumors formed, mice were given an injection of LipC6 or vehicle via tail vein every other day for 2 weeks. This was followed by administration, also via tail vein, of tumor antigen-specific (TAS) CD8⁺ T cells isolated from the spleens of line 416 mice, and subsequent immunization by intraperitoneal injection of tumor antigen-expressing B6/WT-19 cells. Tumor growth was monitored with magnetic resonance imaging. Tumor apoptosis, proliferation, and AKT expression were analyzed using immunohistochemistry and immunoblots. Cytokine production, phenotype, and function of TAS CD8⁺ T cells and tumor-associated macrophages (TAMs) were studied with flow cytometry, real-time polymerase chain reaction (PCR), and ELISA. Reactive oxygen species (ROS) in TAMs and bone marrow-derived macrophages, induced by colony stimulating factor 2 (GMCSF or CSF2) or colony stimulating factor 1 (MCSF or CSF1), were detected using a luminescent assay. Results Injection of LipC6 slowed tumor growth by reducing tumor cell proliferation and phosphorylation of AKT, and increasing tumor cell apoptosis, compared with vehicle. Tumors grew more slowly in mice given the combination of LipC6 injection and TAS CD8⁺ T cells followed by immunization compared with mice given vehicle, LipC6, the T cells, or immunization alone. LipC6 injection also reduced numbers of TAMs and their production of ROS. LipC6 induced TAMs to differentiate into an M1 phenotype, which reduced immune suppression and increased activity of CD8⁺ T cells. These results were validated by experiments with bone marrow-derived macrophages induced by GMCSF or MCSF. Conclusions In mice with liver tumors, injection of LipC6 reduces the number of TAMs and the ability of TAMs to suppress the anti-tumor immune response. LipC6 also increases the anti-tumor effects of TAS CD8⁺ T cells. LipC6 might therefore increase the efficacy of immune therapy in patients with hepatocellular carcinoma.

Lipid perturbations contribute to detrimental outcomes in obesity. We previously demonstrated that nervonic acid, a C24:1 ω‐9 fatty acid, predominantly acylated to sphingolipids, including ceramides, are selectively reduced in a mouse model of obesity. It is currently unknown if deficiency of nervonic acid‐sphingolipid metabolites contribute to complications of obesity. Mice were fed a standard diet, a high fat diet, or these diets supplemented isocalorically with nervonic acid. The primary objective was to determine if dietary nervonic acid content alters the metabolic phenotype in mice fed a high fat diet. Furthermore, we investigated if nervonic acid alters markers of impaired fatty acid oxidation in the liver. We observed that a nervonic acid‐enriched isocaloric diet reduced weight gain and adiposity in mice fed a high fat diet. The nervonic acid enrichment led to increased C24:1‐ceramides and improved several metabolic parameters including blood glucose levels, and insulin and glucose tolerance. Mechanistically, nervonic acid supplementation increased PPARα and PGC1α expression and improved the acylcarnitine profile in liver. These alterations indicate improved energy metabolism through increased β‐oxidation of fatty acids. Taken together, increasing dietary nervonic acid improves metabolic parameters in mice fed a high fat diet. Strategies that prevent deficiency of, or restore, nervonic acid may represent an effective strategy to treat obesity and obesity‐related complications.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase–dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.

Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of immature myeloid cells in the blood and bone marrow. The 5-year survival rate is approximately 25%, and recent therapeutic developments have yielded little survival benefit. Therefore, there is an urgent need to identify novel therapeutic targets. We previously demonstrated that acid ceramidase (ASAH1, referred to as AC) is upregulated in AML and high AC activity correlates with poor patient survival. Here, we characterized a novel AC inhibitor, SACLAC, that significantly reduced the viability of AML cells with an EC50 of approximately 3 μmol/L across 30 human AML cell lines. Treatment of AML cell lines with SACLAC effectively blocked AC activity and induced a decrease in sphingosine 1-phosphate and a 2.5-fold increase in total ceramide levels. Mechanistically, we showed that SACLAC treatment led to reduced levels of splicing factor SF3B1 and alternative MCL-1 mRNA splicing in multiple human AML cell lines. This increased proapoptotic MCL-1S levels and contributed to SACLAC-induced apoptosis in AML cells. The apoptotic effects of SACLAC were attenuated by SF3B1 or MCL-1 overexpression and by selective knockdown of MCL-1S. Furthermore, AC knockdown and exogenous C16-ceramide supplementation induced similar changes in SF3B1 level and MCL-1S/L ratio. Finally, we demonstrated that SACLAC treatment leads to a 37% to 75% reduction in leukemic burden in two human AML xenograft mouse models. Implications: These data further emphasize AC as a therapeutic target in AML and define SACLAC as a potent inhibitor to be further optimized for future clinical development.

In non-alcoholic steatohepatitis (NASH), many lines of investigation have reported a dysregulation in lipid homeostasis, leading to intrahepatic lipid accumulation. Recently, the role of dysfunctional sphingolipid metabolism has also been proposed. Human and animal models of NASH have been associated with elevated levels of long chain ceramides and pro-apoptotic sphingolipid metabolites, implicated in regulating fatty acid oxidation and inflammation. Importantly, inhibition of de novo ceramide biosynthesis or knock-down of ceramide synthases reverse some of the pathology of NASH. In contrast, cell permeable, short chain ceramides have shown anti-inflammatory actions in multiple models of inflammatory disease. Here, we investigated non-apoptotic doses of a liposome containing short chain C6-Ceramide (Lip-C6) administered to human hepatic stellate cells (hHSC), a key effector of hepatic fibrogenesis, and an animal model characterized by inflammation and elevated liver fat content. On the basis of the results from unbiased liver transcriptomic studies from non-alcoholic fatty liver disease patients, we chose to focus on adenosine monophosphate activated kinase (AMPK) and nuclear factor-erythroid 2-related factor (Nrf2) signaling pathways, which showed an abnormal profile. Lip-C6 administration inhibited hHSC proliferation while improving anti-oxidant protection and energy homeostasis, as indicated by upregulation of Nrf2, activation of AMPK and an increase in ATP. To confirm these in vitro data, we investigated the effect of a single tail-vein injection of Lip-C6 in the methionine-choline deficient (MCD) diet mouse model. Lip-C6, but not control liposomes, upregulated phospho-AMPK, without inducing liver toxicity, apoptosis, or exacerbating inflammatory signaling pathways. Alluding to mechanism, mass spectrometry lipidomics showed that Lip-C6-treatment reversed the imbalance in hepatic phosphatidylcholines and diacylglycerides species induced by the MCD-fed diet. These results reveal that short-term Lip-C6 administration reverses energy/metabolic depletion and increases protective anti-oxidant signaling pathways, possibly by restoring homeostatic lipid function in a model of liver inflammation with fat accumulation.

Therapies for head and neck squamous cell carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of methyl pyruvate rescues cells from CNL + CQ–dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.