Diabetic retinopathy is a ceramidopathy reversible by anti-ceramide immunotherapy.

Diabetic retinopathy is a microvascular disease that causes blindness. Using acid sphingomyelinase knockout mice, we reported that ceramide generation is critical for diabetic retinopathy development. Here, in patients with proliferative diabetic retinopathy, we identify vitreous ceramide imbalance with pathologic long-chain C16-ceramides increasing and protective very long-chain C26-ceramides decreasing. C16-ceramides generate pro-inflammatory/pro-apoptotic ceramide-rich platforms on endothelial surfaces. To geo-localize ceramide-rich platforms, we invented a three-dimensional confocal assay and showed that retinopathy-producing cytokines TNFα and IL-1ß induce ceramide-rich platform formation on retinal endothelial cells within seconds, with volumes increasing 2-logs, yielding apoptotic death. Anti-ceramide antibodies abolish these events. Furthermore, intravitreal and systemic anti-ceramide antibodies protect from diabetic retinopathy in standardized rodent ischemia reperfusion and streptozotocin models. These data support (1) retinal endothelial ceramide as a diabetic retinopathy treatment target, (2) early-stage therapy of non-proliferative diabetic retinopathy to prevent progression, and (3) systemic diabetic retinopathy treatment; and they characterize diabetic retinopathy as a "ceramidopathy" reversible by anti-ceramide immunotherapy.

Untargeted and temporal analysis of retinal lipidome in bacterial endophthalmitis.

Bacterial endophthalmitis is a blinding infectious disease typically acquired during ocular surgery. We previously reported significant alterations in retinal metabolism during Staphylococcus (S) aureus endophthalmitis. However, the changes in retinal lipid composition during endophthalmitis are unknown. Here, using a mouse model of S. aureus endophthalmitis and an untargeted lipidomic approach, we comprehensively analyzed temporal alterations in total lipids and oxylipin in retina. Our data showed a time-dependent increase in the levels of lipid classes, sphingolipids, glycerolipids, sterols, and non-esterified fatty acids, whereas levels of phospholipids decreased. Among lipid subclasses, phosphatidylcholine decreased over time. The oxylipin analysis revealed increased prostaglandin-E2, hydroxyeicosatetraenoic acids, docosahexaenoic acid, eicosapentaenoic acid, and α-linolenic acid. In-vitro studies using mouse bone marrow-derived macrophages showed increased lipid droplets and lipid-peroxide formation in response to S. aureus infection. Collectively, these findings suggest that S. aureus-infection alters the retinal lipid profile, which may contribute to the pathogenesis of bacterial endophthalmitis.

Dietary fish oil enriched in very-long-chain polyunsaturated fatty acid reduces cardiometabolic risk factors and improves retinal function.

Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.

Androgen-mediated Perturbation of the Hepatic Circadian System Through Epigenetic Modulation Promotes NAFLD in PCOS Mice.

In women, excess androgen causes polycystic ovary syndrome (PCOS), a common fertility disorder with comorbid metabolic dysfunctions including diabetes, obesity, and nonalcoholic fatty liver disease. Using a PCOS mouse model, this study shows that chronic high androgen levels cause hepatic steatosis while hepatocyte-specific androgen receptor (AR)-knockout rescues this phenotype. Moreover, through RNA-sequencing and metabolomic studies, we have identified key metabolic genes and pathways affected by hyperandrogenism. Our studies reveal that a large number of metabolic genes are directly regulated by androgens through AR binding to androgen response element sequences on the promoter region of these genes. Interestingly, a number of circadian genes are also differentially regulated by androgens. In vivo and in vitro studies using a circadian reporter [Period2::Luciferase (Per2::LUC)] mouse model demonstrate that androgens can directly disrupt the hepatic timing system, which is a key regulator of liver metabolism. Consequently, studies show that androgens decrease H3K27me3, a gene silencing mark on the promoter of core clock genes, by inhibiting the expression of histone methyltransferase, Ezh2, while inducing the expression of the histone demethylase, JMJD3, which is responsible for adding and removing the H3K27me3 mark, respectively. Finally, we report that under hyperandrogenic conditions, some of the same circadian/metabolic genes that are upregulated in the mouse liver are also elevated in nonhuman primate livers. In summary, these studies not only provide an overall understanding of how hyperandrogenism associated with PCOS affects liver gene expression and metabolism but also offer insight into the underlying mechanisms leading to hepatic steatosis in PCOS.

BSCL2/Seipin deficiency in hearts causes cardiac energy deficit and dysfunction via inducing excessive lipid catabolism.

BACKGROUND: Heart failure (HF) is one of the leading causes of death worldwide and is associated with cardiac metabolic perturbations. Human Type 2 Berardinelli-Seip Congenital Lipodystrophy (BSCL2) disease is caused by mutations in the BSCL2 gene. Global lipodystrophic Bscl2-/- mice exhibit hypertrophic cardiomyopathy with reduced cardiac steatosis. Whether BSCL2 plays a direct role in regulating cardiac substrate metabolism and/or contractile function remains unknown. METHODS: We generated mice with cardiomyocyte-specific deletion of Bscl2 (Bscl2cKO ) and studied their cardiac substrate utilisation, bioenergetics, lipidomics and contractile function under baseline or after either a treatment regimen using fatty acid oxidation (FAO) inhibitor trimetazidine (TMZ) or a prevention regimen with high-fat diet (HFD) feeding. Mice with partial ATGL deletion and cardiac-specific deletion of Bscl2 were also generated followed by cardiac phenotyping. RESULTS: Different from hypertrophic cardiomyopathy in Bscl2-/- mice, mice with cardiac-specific deletion of Bscl2 developed systolic dysfunction with dilation. Myocardial BSCL2 deletion led to elevated ATGL expression and FAO along with reduced cardiac lipid contents. Cardiac dysfunction in Bscl2cKO mice was independent of mitochondrial dysfunction and oxidative stress, but associated with decreased metabolic reserve and ATP levels. Importantly, cardiac dysfunction in Bscl2cKO mice could be partially reversed by FAO inhibitor TMZ, or prevented by genetic abolishment of one ATGL allele or HFD feeding. Lipidomic analysis further identified markedly reduced glycerolipids, glycerophospholipids, NEFA and acylcarnitines in Bscl2cKO hearts, which were partially normalised by TMZ or HFD. CONCLUSIONS: We identified a new form of cardiac dysfunction with excessive lipid utilisation which ultimately causes cardiac substrate depletion and bioenergetics failure. Our findings also uncover a crucial role of BSCL2 in controlling cardiac lipid catabolism and contractile function and provide novel insights into metabolically treating energy-starved HF using FAO inhibitor or HFD.

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1ß, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 µM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Regulation of lipid metabolism in pancreatic beta cells by interferon gamma: A link to anti-viral function.

Interferons (IFN) have been shown to alter lipid metabolism in immune and some non-hematopoietic cells and this affects host cell response to pathogens. In type 1 diabetes, IFNγ acts as a proinflammatory cytokine that, along with other cytokines, is released during pancreatic beta cell autoinflammation and contributes to immune response and beta cell dysfunction. The hypothesis tested herein is that IFN modifies beta cell lipid metabolism and this is associated with enhanced anti-viral response and beta cell stress. Treatment of INS-1 cells with IFNγ for 6 to 24 h led to a dynamic change in TAG and lipid droplet (LD) levels, with a decrease at 6 h and an increase at 24 h. The later accumulation of TAG was associated with increased de novo lipogenesis (DNL), and impaired mitochondrial fatty acid oxidation (FAO). Gene expression results suggested that IFNγ regulates lipolytic, lipogenic, LD and FAO genes in a temporal manner. The changes in lipid gene expression are dependent on the classical Janus kinase (JAK) pathway. Pretreatment with IFNγ robustly enhanced anti-viral gene expression induced by the viral mimetic polyinosinic: polycytidylic acid (PIC), and this potentiating effect of IFNγ was markedly attenuated by inhibitors of DNL. The IFNγ-induced accumulation of lipid, however, was insufficient to cause endoplasmic reticulum (ER) stress. These studies demonstrated a non-canonical effect of IFNγ in regulation of pancreatic beta cell lipid metabolism that is intimately linked with host cell defense and might alter cellular function early in the progression to type 1 diabetes.

ELOVL4-Mediated Production of Very Long-Chain Ceramides Stabilizes Tight Junctions and Prevents Diabetes-Induced Retinal Vascular Permeability.

Tight junctions (TJs) involve close apposition of transmembrane proteins between cells. Although TJ proteins have been studied in detail, the role of lipids is largely unknown. We addressed the role of very long-chain (VLC ≥26) ceramides in TJs using diabetes-induced loss of the blood-retinal barrier as a model. VLC fatty acids that incorporate into VLC ceramides are produced by elongase elongation of very long-chain fatty acids protein 4 (ELOVL4). ELOVL4 is significantly reduced in the diabetic retina. Overexpression of ELOVL4 significantly decreased basal permeability, inhibited vascular endothelial growth factor (VEGF)- and interleukin-1ß-induced permeability, and prevented VEGF-induced decrease in occludin expression and border staining of TJ proteins ZO-1 and claudin-5. Intravitreal delivery of AAV2-hELOVL4 reduced diabetes-induced increase in vascular permeability. Ultrastructure and lipidomic analysis revealed that ω-linked acyl-VLC ceramides colocalize with TJ complexes. Overall, normalization of retinal ELOVL4 expression could prevent blood-retinal barrier dysregulation in diabetic retinopathy through an increase in VLC ceramides and stabilization of TJs.

The Mechanism of Diabetic Retinopathy Pathogenesis Unifying Key Lipid Regulators, Sirtuin 1 and Liver X Receptor.

Diabetic retinopathy (DR) is a complication secondary to diabetes and is the number one cause of blindness among working age individuals worldwide. Despite recent therapeutic breakthroughs using pharmacotherapy, a cure for DR has yet to be realized. Several clinical trials have highlighted the vital role dyslipidemia plays in the progression of DR. Additionally, it has recently been shown that activation of Liver X receptor (LXRα/LXRß) prevents DR in diabetic animal models. LXRs are nuclear receptors that play key roles in regulating cholesterol metabolism, fatty acid metabolism and inflammation. In this manuscript, we show insight into DR pathogenesis by demonstrating an innovative signaling axis that unifies key metabolic regulators, Sirtuin 1 and LXR, in modulating retinal cholesterol metabolism and inflammation in the diabetic retina. Expression of both regulators, Sirtuin 1 and LXR, are significantly decreased in diabetic human retinal samples and in a type 2 diabetic animal model. Additionally, activation of LXR restores reverse cholesterol transport, prevents inflammation, reduces pro-inflammatory macrophages activity and prevents the formation of diabetes-induced acellular capillaries. Taken together, the work presented in this manuscript highlights the important role lipid dysregulation plays in DR progression and offers a novel potential therapeutic target for the treatment of DR.

Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy.

Activation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies.

Liver-specific loss of Perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.

Hepatic inflammation and fibrosis are key elements in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive liver disease initiated by excess hepatic lipid accumulation. Lipid droplet protein Perilipin 2 (Plin2) alleviates dietary-induced hepatic steatosis when globally ablated; however, its role in the progression of NASH remains unknown. To investigate this further, we challenged Plin2 liver-specific knockout mice (designated L-KO) and their respective wild-type (WT) controls with a methionine-choline-deficient (MCD) diet for 15 days to induce a NASH phenotype of increased hepatic triglyceride levels through impaired phosphatidylcholine (PC) synthesis and very-low-density lipoprotein (VLDL) secretion. Results on liver weights, body weights, fat tissue mass, and histology in WT and L-KO mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation; however, these effects were blunted in L-KO mice. In addition, levels of PC and VLDL were unchanged, and hepatic steatosis was reduced in L-KO mice fed the MCD diet, due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). These mice also exhibited decreased hepatic expression of proinflammatory markers cyclooxygenase 2, IL-6, TNF-α, IL-1ß, and reduced expression of endoplasmic reticulum (ER) stress proteins C/EBP homologous protein and cleaved caspase-1. Taken together, these results suggest that Plin2 liver-specific ablation alleviates diet-induced hepatic steatosis and inflammation via a PEMT-mediated mechanism that involves compensatory changes in proteins involved in phospholipid remodeling, inflammation, and ER stress that work to alleviate diet-induced NASH. Overall, these findings support a role for Plin2 as a target for NASH therapy.

Rapid and comprehensive 'shotgun' lipidome profiling of colorectal cancer cell derived exosomes.

There is an increasing recognition of the role that cancer cell derived exosomes play in intercellular signaling upon fusion or uptake with a target cell, including immune system evasion, tumor growth and metastasis. To date, however, although exosomal membrane and cargo lipids are expected to play a pivotal role in exosome biogenesis and secretion, as well as in fusion or uptake and target cell functional response, the detailed characterization of cancer cell derived exosome lipids across a range of different cancers has not yet been broadly explored. Here, a simple and straightforward lipidome analysis strategy consisting of optimized sample extraction and novel sample derivatization techniques, coupled with high-resolution 'shotgun' mass spectrometry and 'targeted' tandem mass spectrometry methods, is demonstrated for the rapid identification of >520 individual lipids in 36 lipid classes and sub classes from exosomes secreted by the colorectal cancer cell line, LIM1215. Relative quantification and comparison of exosome versus cellular lipid profiles reveals significant enrichment of certain lipid classes, as well as substantial lipid subclass remodeling and changes in abundance of individual lipids, including sphingolipids, sterol lipids, glycerolipids and glycerophospholipids, and particularly plasmalogen- and alkyl ether-containing glycerophospholipids. This analysis strategy therefore provides a platform for comprehensive lipidome profiling across a wide range of cancer cell or tissue derived exosomes, that will facilitate subsequent functional studies aimed at elucidating the role of specific cellular or exosome lipids in the onset and progression of colorectal cancer, or to identify specific lipid(s) that could serve as effective diagnostic or prognostic disease biomarkers.

N-3 polyunsaturated Fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function.

OBJECTIVE: The vasodegenerative phase of diabetic retinopathy is characterized by not only retinal vascular degeneration but also inadequate vascular repair due to compromised bone marrow derived endothelial progenitor cells (EPCs). We propose that n-3 polyunsaturated fatty acid (PUFA) deficiency in diabetes results in activation of the central enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM) and that ASM represents a molecular metabolic link connecting the initial damage in the retina and the dysfunction of EPCs. RESEARCH DESIGN AND METHODS: Type 2 diabetic rats on control or docosahexaenoic acid (DHA)-rich diet were studied. The number of acellular capillaries in the retinas was assessed by trypsin digest. mRNA levels of interleukin (IL)-1ß, IL-6, intracellular adhesion molecule (ICAM)-1 in the retinas from diabetic animals were compared to controls and ASM protein was assessed by western analysis. EPCs were isolated from blood and bone marrow and their numbers and ability to form colonies in vitro, ASM activity and lipid profiles were determined. RESULTS: DHA-rich diet prevented diabetes-induced increase in the number of retinal acellular capillaries and significantly enhanced the life span of type 2 diabetic animals. DHA-rich diet blocked upregulation of ASM and other inflammatory markers in diabetic retina and prevented the increase in ASM activity in EPCs, normalized the numbers of circulating EPCs and improved EPC colony formation. CONCLUSIONS: In a type 2 diabetes animal model, DHA-rich diet fully prevented retinal vascular pathology through inhibition of ASM in both retina and EPCs, leading to a concomitant suppression of retinal inflammation and correction of EPC number and function.

The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models.

OBJECTIVE: Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. RESEARCH DESIGN AND METHODS: Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. RESULTS: We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α-induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM(-/-) mice or inhibition of ASM activity by DHA prevents acellular capillary formation. CONCLUSIONS: This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.

Remodeling of retinal Fatty acids in an animal model of diabetes: a decrease in long-chain polyunsaturated fatty acids is associated with a decrease in fatty acid elongases Elovl2 and Elovl4.

OBJECTIVE: The results of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid metabolism in diabetes. This study determined retinal-specific fatty acid metabolism in control and diabetic animals. RESEARCH DESIGN AND METHODS: Tissue gene and protein expression profiles were determined by quantitative RT-PCR and Western blot in control and streptozotocin-induced diabetic rats at 3-6 weeks of diabetes. Fatty acid profiles were assessed by reverse-phase high-performance liquid chromatography, and phospholipid analysis was performed by nano-electrospray ionization tandem mass spectrometry. RESULTS: We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturase gene expression in the retina compared with liver. Elovl4, an elongase expressed in the retina but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1, and Elovl6. Importantly, early-stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2, and Elovl6. Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very-long-chain polyunsaturated fatty acids (PUFAs), particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of proinflammatory markers interleukin-6, vascular endothelial growth factor, and intercellular adhesion molecule-1. CONCLUSIONS: This is the first comprehensive study demonstrating diabetes-induced changes in retinal fatty acid metabolism. Normalization of retinal fatty acid levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.