Introduction Diabetic kidney disease (DKD) is characterized by impairment of renal glomerular and tubular cells. Low plasma levels of ceramides and lactosylceramides containing very long-chain (VLC) fatty acid were found to be predictive of DKD development. Elongase 1 (Elovl1) is a ubiquitous elongase that elongates C20-C22 fatty acids to generate very long-chain C24 fatty acids. Using a novel transgenic mouse overexpressing Elovl1, we investigated whether modification to sphingolipid fatty acid composition averts DKD development. Methods A transgenic (TG) mouse overexpressing Elovl1 was created at the Medical University of South Carolina/Transgenic Core. Elovl1 TG and wild type (WT) mice were rendered diabetic using serial streptozotocin injections. Plasma, kidney, liver, and urine sphingolipidomics of diabetic and non-diabetic TG and WT mice were analyzed using mass spectroscopy and Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry. Sphingolipid metabolizing enzymes were analyzed using immunohistochemical & multispectral imaging coupled with digital analysis. Results Plasma sphingomyelins were higher, but lactosylceramides were lower in diabetic TG than in diabetic WT mice. Kidney lactosylceramides were also lower in diabetic TG mice. In urine, diabetic TG mice excreted more VLC lactosylceramides than diabetic WT mice, but less VLC sphingomyelins, VLC ceramides, sphingosine and sphingosine 1-phosphate. There was extensive damage to proximal tubules in kidneys of diabetic WT mice compared to diabetic TG mice. Glomeruli in diabetic WT kidneys appeared also abnormal, whereas no obvious abnormality of glomeruli in diabetic TG was observed. In diabetic TG, Elovl1 overexpression resulted in decreased kidneys levels of both ceramide synthase and acid sphingomyelinase, but increased acid ceramidase levels compared to non-diabetic TG mice. Conclusion The diabetic Elovl1 TG mouse revealed interaction between Elovl1 overexpression and DKD development, and showed that distinct VLC sphingolipids could be involved in maintaining cell membrane integrity of renal cells.

Abnormalities of sphingolipid metabolism play an important role in diabetes. We compared sphingolipid levels in plasma and in isolated lipoproteins between healthy control subjects and two groups of patients, one with chronic kidney disease without diabetes (ND-CKD), and the other with type 2 diabetes and macroalbuminuria (D-MA). Ceramides, sphingomyelins, and sphingoid bases and their phosphates in LDL were higher in ND-CKD and in D-MA patients compared to controls. However, ceramides and sphingoid bases in HDL2 and HDL3 were lower in ND-CKD and in D-MA patients than in controls. Sphingomyelins in HDL2 and HDL3 were lower in D-MA patients than in controls but were normal in ND-CKD patients. Compared to controls, lactosylceramides in LDL and VLDL were higher in ND-CKD patients but not in D-MA patients. However, lactosylceramides in HDL2 and HDL3 were lower in both ND-CKD and D-MA patients than in controls. Plasma hexosylceramides in ND-CKD patients were increased and sphingoid bases decreased in both ND-CKD and D-MA patients. However, hexosylceramides in LDL, HDL2, and HDL3 were higher in ND-CKD patients than in controls. In D-MA patients, only C16:0 hexosylceramide in LDL was higher than in controls. The data suggest that sphingolipid measurement in lipoproteins, rather than in whole plasma, is crucial to decipher the role of sphingolipids in kidney disease.

Aims:We aimed to determine whether plasma advanced glycation end products or oxidation products (AGE/oxidation-P) predict altered renal function and/or preeclampsia (PE) in pregnant women with type 1 diabetes.Methods:Prospectively, using a nested case-control design, we studied 47 pregnant women with type 1 diabetes, of whom 23 developed PE and 24 did not. Nineteen nondiabetic, normotensive pregnant women provided reference values. In plasma obtained at ~12, 22, and 32 weeks' gestation (visits 1, 2, and 3; V1-V3), we measured five AGE products (carboxymethyllysine (CML), carboxyethyl-lysine (CEL), methylglyoxal-hydroimidazolone (MGH1), 3-deoxyglucosone hydroimidazolone (3DGH), and glyoxal-hydroimidazolone (GH1)) and four oxidation products (methionine sulfoxide (MetSO), 2-aminoadipic acid (2-AAA), 3-nitrotyrosine (3NT), and dityrosine (DT)), by liquid chromatography/mass spectroscopy. Clinical outcomes were "estimated glomerular filtration rate" (eGFR) at each visit and onset of PE.Results:In diabetic women, associations between AGE/oxidation-P and eGFR were found only in those who developed PE. In this group, CEL, MGH1, and GH1 at V2 and CML, CEL, MGH1, and GH1 at V3 were inversely associated with contemporaneous eGFR, while CEL, MGH1, 3DGH, and GH1 at V2 were inversely associated with eGFR at V3 (all p < 0.05). There were no associations of plasma AGE or oxidation-P with pregnancy-related development of proteinuria or PE.Conclusions:Inverse associations of second and early third trimester plasma AGE with eGFR among type 1 diabetic women who developed PE suggest that these patients constitute a subset susceptible to AGE-mediated injury and thus to cardiorenal complications later in life. However, AGE/oxidation-P did not predict PE in type 1 diabetic women.

Sphingolipids play an important role in the development of diabetes, both type 1 and type 2 diabetes, as well as in the development of both micro- and macro-vascular complications. Several reviews have been published concerning the role of sphingolipids in diabetes but most of the emphasis has been on the possible mechanisms by which sphingolipids, mainly ceramides, contribute to the development of diabetes. Research on circulating levels of the different classes of sphingolipids in serum and in lipoproteins and their importance as biomarkers to predict not only the development of diabetes but also of its complications has only recently emerged and it is still in its infancy. This review summarizes the previously published literature concerning sphingolipid-mediated mechanisms involved in the development of diabetes and its complications, focusing on how circulating plasma sphingolipid levels and the relative content carried by the different lipoproteins may impact their role as possible biomarkers both in the development of diabetes and mainly in the development of diabetic complications. Further studies in this field may open new therapeutic avenues to prevent or arrest/reduce both the development of diabetes and progression of its complications.

BACKGROUND:We have previously shown that very long ceramides/lactosylceramides predicted the development of macroalbuminuria (MA) in type 1 diabetes and expanded our studies into type 2 diabetes. OBJECTIVE:This study proposes comparing the levels of plasma sphingolipids and their distribution in circulating lipoproteins (VLDL/IDL, LDL, HDL2 and HDL3) between a healthy control group and two groups of subjects with type 2 diabetes, one with and other without MA. METHODS:Plasma and lipoprotein sphingolipids/glycosphingolipids were measured using HPLC-MS/MS in 114 subjects (40 controls; 74 type 2 diabetes, 40 without MA; and 34 with MA) and the levels were compared between controls and the two groups of diabetes. Group effect sizes were calculated using Cohen's d. RESULTS:Sphingomyelin species carried by LDL are significantly higher in diabetic patients with MA than in those with normal albumin excretion rate (AER). Compared to controls, significant decreases in the levels of sphingolipids carried by HDL in patients with diabetes with normal AER or MA were observed for all sphingolipid classes except for hexosylceramide, which was normal in diabetic patients without MA. Although lower than in controls, the levels of lactosylceramides carried by HDL2/HDL3 were significantly higher in diabetes with MA. CONCLUSIONS:Considering the critical role sphingolipids play in major cell biological responses and cell signaling pathways, the consequences for disease development of changes in the distribution of sphingolipids/glycosphingolipids carried by lipoproteins could be considerable. Our work is just a first step to address a considerable gap in our present knowledge in this important field.

Apolipoprotein M (apoM), primarily carried by HDL, has been associated with several conditions, including cardiovascular disease and diabetic nephropathy. This study proposes to examine whether plasma apoM levels are associated with the development of diabetic kidney disease, assessed as progression to macroalbuminuria (MA) and chronic kidney disease (CKD). Plasma apoM was measured using an enzyme immunoassay in 386 subjects from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) cohort at DCCT entry and closeout and the concentrations used to determine the association with risk of progression to kidney dysfunction from the time of measurement through 18 years of EDIC follow-up. apoM levels, at DCCT baseline, were higher in patients who developed CKD than in those who retained normal renal function. At DCCT closeout, participants who progressed to MA, CKD, or both MA and CKD also had significantly higher apoM levels than those who remained normal, and increased levels of apoM were associated with increased risk of progression to both MA (risk ratio [RR] 1.30 [95% CI 1.01, 1.66]) and CKD (RR 1.69 [95% CI 1.18, 2.44]). Our results strongly suggest that alterations in apoM and therefore in the composition and function of HDL in type 1 diabetes are present early in the disease process and are associated with the development of nephropathy.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects females more than males, with African Americans developing more severe manifestation of the disease. SLE patients are at increased risk for cardiovascular disease (CVD), and SLE women 35-44 years old have 50 fold the incidence rate of CVD. Because SLE patients do not follow the typical age and gender pattern for CVD, but instead an accelerated disease course, the traditional biomarkers of elevated LDL and total cholesterol levels do not accurately assess their CVD risk. Recently, we have reported that African American SLE patients had higher ceramide, hexosylceramide, sphingosine and dihydrosphingosine 1-phosphate levels compared to their healthy controls, and those with atherosclerosis had higher sphingomyelin and sphingoid bases levels than those without (PLoS One. 2019; e0224496). In the current study, we sought to identify sphingolipid species that correlate with and pose the potential to predict atherosclerosis severity in African American SLE patients. Plasma samples from a group of African American predominantly female SLE patients with well-defined carotid atherosclerotic plaque burden were analyzed for sphingolipidomics using targeted mass spectroscopy. The data demonstrated that at baseline, plaque area and C3 values correlated inversely with most lactoceramide species. After one-year follow-up visit, values of the change of plaque area correlated positively with the lactoceramide species. There was no correlation between LDL-C concentrations and lactoceramide species. Taken together, lactocylcermide levels may have a 'predictive' value and sphingolipidomics have an added benefit to currently available tools in early diagnosis and prognosis of African American SLE patients with CVD.

Sphingolipids are a heterogeneous class of lipids and essential components of the plasma membrane and plasma lipoproteins. Studies have shown that plasma deoxysphingolipid (DSL), a newly identified sphingolipid class, is increased in diabetic patients and associated with diabetic neuropathy. However, it remains unknown if there is a causal relationship between plasma DSL increase and diabetic neuropathy. Since matrix metalloproteinases (MMPs) play an important role in diabetic neuropathy by degrading extracellular matrix in the peripheral nervous system, we investigated the effect of DSLs on the expression of MMPs and tissue inhibitor of metalloproteinase (TIMPs), and cytotoxicity in human Schwann cells. We quantified protein secretion, gene expression, and collagenase activity, and performed cytotoxicity assays. Results showed that DSLs upregulated MMP-1, downregulated TIMP-1, and induced cytotoxicity in Schwann cells. Furthermore, we quantified DSLs in VLDL, LDL, HDL2, and HDL3 isolated from type 2 diabetes mellitus (T2DM) patients with or without neuropathy. Interestingly, lipidomic analysis showed that only HDL2 isolated from T2DM patients with neuropathy contains significantly higher level of DSLs than that isolated from T2DM patients without neuropathy. Additionally, results showed that HDL2 isolated from T2DM patients with neuropathy was more potent than that isolated from T2DM patients without neuropathy in upregulating MMP-1, downregulating TIMP-1, and stimulating collagenase activity in Schwann cell. Taken together, this study demonstrated for the first time a potential causal relationship between DSLs and diabetic neuropathy and that DSL-containing HDL2 from T2DM patients with neuropathy was more potent than that from T2DM patients without neuropathy in stimulating collagenase activity.

The risk for preeclampsia (PE) is enhanced ~4-fold by the presence of maternal type 1 diabetes (T1DM). Vitamin D is essential for healthy pregnancy. We assessed the total, bioavailable, and free concentrations of plasma 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D), and vitamin D binding protein (VDBP) at ~12, ~22, and ~32 weeks' gestation ("Visits" (V) 1, 2, and 3, respectively) in 23 T1DM women who developed PE, 24 who remained normotensive, and 19 non-diabetic, normotensive women (reference controls). 25(OH)D deficiency was more frequent in diabetic than non-diabetic women (69% vs. 22%, p < 0.05), but no measure of 25(OH)D predicted PE. By contrast, higher 1,25(OH)2D concentrations at V2 (total, bioavailable, and free: p < 0.01) and V3 (bioavailable: p < 0.05; free: p < 0.01), lower concentrations of VDBP at V3 (p < 0.05), and elevated ratios of 1,25(OH)2D/VDBP (V2, V3: p < 0.01) and 1,25(OH)2D/25(OH)D (V3, p < 0.05) were all associated with PE, and significance persisted in multivariate analyses. In summary, in women with T1DM, concentrations of 1,25(OH)2D were higher, and VDBP lower, in the second and third trimesters in women who later developed PE than in those who did not. 1,25(OH)2D may serve as a new marker for PE risk and could be implicated in pathogenesis.

Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that involves multiple organs and disproportionality affects females, especially African Americans from 15 to 44 years of age. SLE can lead to end organ damage including kidneys, lungs, cardiovascular and neuropsychiatric systems, with cardiovascular complications being the primary cause of death. Usually, SLE is diagnosed and its activity is assessed using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), Systemic Lupus International Collaborating Clinics Damage Index (SLICC/ACR), and British Isles Lupus Assessment Group (BILAG) Scales, which unfortunately often occurs after a certain degree of systemic involvements, disease activity or organ damage already exists. There is certainly a need for the identification of early biomarkers to diagnose and assess disease activity as well as to evaluate disease prognosis and response to treatment earlier in the course of the disease. Here we review advancements made in the area of sphingolipidomics as a diagnostic/prognostic tool for SLE and its co-morbidities. We also discuss recent reports on differential sphingolipid metabolism and blood sphingolipid profiles in SLE-prone animal models as well as in diverse cohorts of SLE patients. In addition, we address targeting sphingolipids and their metabolism as a method of treating SLE and some of its complications. Although such treatments have already shown promise in preventing organ-specific pathology caused by SLE, further investigational studies and clinical trials are warranted.

Preeclampsia (PE) occurs more frequently in pregnant diabetic than nondiabetic women (∼20% vs. ∼5%). Early identification of high risk women is needed. Dyslipidemia is implicated: we previously showed that early in pregnancy, increased cholesterol-rich lipoproteins are associated with subsequent PE in type 1 diabetes (T1D). Haptoglobin (Hp) is a plasma protein that binds free hemoglobin, and has two allelic forms, Hp-1, Hp-2, hence three phenotypes. Among people with diabetes, Hp 2-2 phenotype (present in ≈50%) has been associated with oxidative stress, cardiovascular risk and renal decline. We investigated whether maternal Hp phenotype is associated with PE in T1D, and/or modulates lipoprotein-related risks for PE. A prospective study of pregnancy included 23 T1DM women (cases) who developed PE, and 24 T1DM (controls) who remained normotensive. All were free of microalbuminuria and hypertension at enrolment. Hp phenotype was determined by ELISA (Savyon Diagnostics Ltd.). Lipid profiles were measured at three study visits (V1-V3), all preceding PE onset: (mean ± SD) 12.4 ± 1.8, 21.7 ± 1.4, and 31.3 ± 1.4 weeks gestation. Results: Hp phenotype did not differ between women with and without PE, and lipid profiles did not differ by Hp phenotype. In PE cases vs. controls, by univariate analysis, HDL-C was lower at V1 (1.9±0.4 vs. 2.2±0.5 mmol/l (mean ± SD)), while LDL-C was higher at V2 (3.1± 1.0 vs. 2.6± 0.8 mmol/l), as were triacylglycerols (1.6± 0.4 vs. 1.3± 0.4 mmol/l) (p<0.05). In Hp 2-2 women, these associations were stronger: in 2-2 cases (n=11) vs. 2-2 controls (n=9), HDL-C was lower at all visits (V1: p<0.05; V2, V3: p<0.01) and LDL-C was higher at V1 (p<0.01) and V2 (p<0.05). In contrast, in women with one or two Hp-1 alleles, no associations between lipids and PE were observed. Conclusion: In T1D women, lipoprotein-related risks for PE may be limited to those with Hp 2-2 phenotype. The data provide further evidence of a role for Hp phenotype as a modulator of vascular risk in diabetes. Disclosure C.B. Kelly: None. J. Yu: None. A. Jenkins: Research Support; Self; Medtronic, Mylan, Sanofi-Aventis. A.J. Nankervis: None. K.F. Hanssen: None. S.K. Garg: Research Support; Self; Dexcom, Inc., Eli Lilly and Company, Sanofi US. Advisory Panel; Self; Sanofi US. Research Support; Self; MannKind Corporation, Diasome Pharmaceuticals, Inc., Labstyle Innovations, Lexicon Pharmaceuticals, Inc., Medtronic. Advisory Panel; Self; Novo Nordisk A/S. C.E. Aston: None. T. Lyons: None.

Background Population-wide reductions in cardiovascular disease (CVD) have not been equally shared in the African American community due to a higher burden of CVD risk factors such as metabolic disorders and obesity. Differential concentrations of sphingolipids such as ceramide, sphingosine, and sphingosine 1-phosphate (S1P) has been associated with the development of CVD, metabolic disorders (MetD), and obesity. Whether African Americans have disparate expression levels of sphingolipids that explain higher burdens of CVD remains unknown. Methods A cross sectional analysis of plasma concentrations of ceramides, sphingosine, and S1P were measured from 8 whites and 7 African Americans without metabolic disorders and 7 whites and 8 African Americans with metabolic disorders using high performance liquid chromatography/tandem mass spectrometry methodology (HPLC/MS-MS). Subjects were stratified by both race and metabolic status. Subjects with one or more of the following physician confirmed diagnosis: diabetes, hypertension, hypercholesterolemia, or dyslipidemia were classified as having metabolic disease (MetD). Data was analyzed using a Two-Way ANOVA and Tukey's post hoc test. Results Total ceramide levels were increased in African Americans compared to African Americans with MetD. Ceramide C16 levels were higher in whites with MetD compared to African Americans with MetD (p<0.05). Ceramide C20 levels were higher in whites with MetD compared to whites. Ceramide C20 levels were higher in African Americans compared to African Americans with MetD. Furthermore, whites with MetD had higher levels of C20 compared to African Americans with MetD (p<0.0001). Ceramide C24:0 and C24:1 in African Americans was higher compared to African Americans with MetD (p<0.05). The plasma concentration of Sph-1P ceramide was higher in African Americans vs whites (p = 0.01). Lastly, ceramide C20 negatively correlated with hemoglobin A1c (HbA1c) levels in our study cohort. Conclusions Plasma ceramide concentration patterns are distinct in African Americans with MetD. Further research with larger samples sizes are needed to confirm these findings and to understand whether racial disparities in sphingolipid concentrations have potential therapeutic implications for CVD-related health outcomes.

Statin therapy reduces cardiovascular risk in type 2 diabetes (T2DM), partly by improving dyslipidemia, and partly through ‘pleiotropic’ effects. Sphingolipids, including ceramides, are amphiphilic components of lipoprotein surface monolayers. Sphingolipids regulate lipoprotein surface fluidity, and include key signaling molecules: ceramides are specifically associated with apoptosis and risk for premature coronary heart disease. Effects of statins on the sphingolipid content of lipoproteins are poorly defined. Using liquid chromatography/electrospray ionization-tandem mass spectrometry, we determined fasting plasma concentrations of individual ceramides (Cer), sphingomyelins (SM), and glycosphingolipids (hexosyl- and lactosyl-Cer) in plasma from 19 men and 21 women with T2DM, before and after treatment with rosuvastatin (10 mg/day, 6 weeks). Exclusion criteria were use of ‘statins’ in the preceding 6 months, and any current use of lipid-modifying agents. Using existing lipoprotein compositional data, we calculated effects of rosuvastatin therapy on non-HDL sphingolipid content. Results: Rosuvastatin reduced total cholesterol (C) (by 33%), triglycerides (20%), LDL-C (49%), and non-HDL particle number (34%); HDL-C was unaffected. It decreased total SM (15%), Cer (29%), and hexosyl- (30%) and lactosyl- (34%) Cer, independent of gender. Assuming HDL composition was unaffected, rosuvastatin reduced non-HDL SM by 42% and Cer by 61%: these reductions remained significant after correction for non-HDL-C and/or non-HDL lipoprotein surface area. Conclusions: In T2DM, rosuvastatin lowers not only the number of non-HDL particles, but also the sphingolipid, and especially the Cer, content of each particle. This effect is independent of gender, potentially anti-atherogenic, and may represent a new pleiotropic effect of statins. Further studies are needed to confirm these findings in isolated lipoprotein classes, and to determine mechanism(s). Disclosure C.B. Kelly: None. S.M. Hammad: None. R.L. Klein: None. M.F. Lopes-Virella: None. A. Gosmanova: None. M.J. Leyva: None. C.E. Aston: None. T. Lyons: None.

The risk for preeclampsia (PE) is increased 4-fold in women with type 1 diabetes (T1D), in whom we previously demonstrated that elevated cholesterol-rich lipoproteins confer risk at the 1st trimester. Sphingolipids, including ceramides (Cer) and glycosphingolipids (hexosyl- (HexCer) and lactosyl-ceramides (LacCer)), are lipoprotein constituents that regulate cell signaling. We propose that these bio-active lipids could be modulators of PE risk. In a prospective T1D pregnancy cohort (studied at 12, 22, 32 weeks’ gestation), we used liquid chromatography/electrospray ionization-tandem mass spectrometry to determine plasma levels of 12 species of Cer, HexCer, and LacCer in 23 women with T1DM who did vs. 24 who did not develop PE, and in 19 normotensive nondiabetic pregnant women. All were free of microalbuminuria and hypertension at enrolment; all visits preceded clinical PE onset. Results: Plasma levels of C22-Cer (1st and 3rd study visits); C26:1-Cer (1st and 2nd visits); C26-Cer (1st visit); C18-Cer and C18:1-Cer (2nd visit) were significantly higher in women with T1DM who did vs. did not develop PE. C22-Cer and C26-Cer were significantly lower in T1DM vs. non-T1DM (1st visit). There were no overall PE- or diabetes-related differences in any HexCer- or LacCer species. Longitudinally, most Cer and HexCer species increased throughout pregnancy in all groups (except C14- and C18-Cer; and C14 and C26-HexCer). C16-LacCer increased, while C22:1-, C26-, and C26:1-LacCer decreased as pregnancy advanced, independent of PE status. Independent of LDL-C, total Cer increased throughout pregnancy in all groups. Conclusions: Maternal Cer species may serve as early biomarkers for PE in women with T1DM. T1DM was associated with lower levels of two Cer species. Like cholesterol-rich lipoproteins, Cer, HexCer and some LacCer species increase as pregnancy advances in all groups, but other LacCer species decrease: the significance of these findings requires further study. Disclosure C.B. Kelly: None. S.M. Hammad: None. R.L. Klein: None. M.F. Lopes-Virella: None. M.J. Leyva: None. J. Yu: None. A.J. Jenkins: Advisory Panel; Self; Abbott, Australian Diabetes Society, Medtronic. Research Support; Self; Abbott, GlySens Incorporated, Medtronic, Mylan. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc. A.J. Nankervis: None. K.F. Hanssen: None. S.K. Garg: Advisory Panel; Self; Eli Lilly and Company, Roche Pharma, Sanofi-Aventis, Zealand Pharma A/S. Research Support; Self; Eli Lilly and Company, WebMD. J.A. Scardo: None. C.E. Aston: None. T. Lyons: None. Funding JDRF; Novo Nordisk; National Institutes of Health

Preeclampsia (PE) occurs four times more frequently in pregnant women with type 1 diabetes (T1D) than in nondiabetic women. We hypothesized that elevated plasma advanced glycoxidation products (AGEs) predict PE in T1D women. In a prospective T1D pregnancy cohort (study visits at 12, 22, 32 weeks' gestation), we used liquid chromatography/mass spectrometry, with internal stable heavy isotope substituted standards, to determine plasma levels of nine AGE and oxidation products (carboxymethyl-lysine [CML], carboxyethyl-lysine [CEL], methylglyoxal-hydroimidazolone [MGHI], 3-deoxyglucosone hydroimidazolones [3DGH], methionine sulfoxide [MetSO], glyoxal hydroimidazolone [GHI], 3-nitrotyrosine [3NT], dityrosine [DT], 2-aminoadipic acid [2AAA]) in 23 women with T1DM who developed PE vs. 24 who did not, and in 19 nondiabetic normotensive pregnant women. These products have been associated with CVD and renal failure in non-pregnant cohorts. All women were free of microalbuminuria and hypertension at enrolment, and all visits preceded clinical PE onset. GFR was estimated (Chronic Kidney Disease Epidemiology Collaboration equation). Results: Plasma CML, CEL, MGHI, 3DGH, MetSO, GHI, and 2AAA did not differ between the three groups at any study visit, and thus did not predict preeclampsia in T1D. In T1D women who later developed PE, eGFR was inversely associated at V3 with CML (p=0.008), CEL (p=0.03), MGH1 (p=0.03), 3DGH (p=0.03), GHI (p<0.001), and 2AAA (p<0.05); and at V2 with CEL (p=0.04), MGHI (p=0.02), and GHI (p=0.02). No such associations were observed in normotensive T1D or in nondiabetic pregnant women. Plasma levels of 3NT and DT were undetectable in all participants. Conclusions: Plasma AGEs did not predict PE in well-controlled, previously healthy T1D women, and levels did not differ from healthy nondiabetic controls. Uniquely, in T1D women who developed PE, plasma AGEs were associated with renal function, consistent with the notion that subclinical alterations in renal function precede PE. Disclosure H. Karanchi: None. C.B. Kelly: None. S.M. Hammad: None. R.L. Klein: None. M.F. Lopes-Virella: None. M.J. Leyva: None. J. Yu: None. A.J. Jenkins: Advisory Panel; Self; Abbott, Australian Diabetes Society, Medtronic. Research Support; Self; Abbott, GlySens Incorporated, Medtronic, Mylan. Speaker’s Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc. A.J. Nankervis: None. K.F. Hanssen: None. C.E. Aston: None. S. Howell: Employee; Self; PreventAGE Health Care, LLC. P.J. Beisswenger: Other Relationship; Self; PreventAGE Health Care, LLC. T. Lyons: None.

Preeclampsia (PE), a major cause of maternal and fetal morbidity and mortality worldwide, has significantly higher incidence in women with type 1 diabetes mellitus (T1DM) than in nondiabetic populations (∼20% vs. 5%, respectively). Sphingolipids are key signaling molecules, and have many roles in the structure and regulation of cellular functions. Sphingomyelin (SM) is the most abundant sphingolipid in plasma lipoproteins. There is evidence that altered SM metabolism may contribute to cardiovascular and renal complications in diabetes. We aimed to determine whether plasma concentrations of 12 SM species were associated with PE in T1DM in a prospective study of pregnancy of 23 T1DM women who developed PE and 24 T1DM who remained normotensive. Additionally, 19 normotensive nondiabetic pregnant women were included for reference. All subjects were free of microalbuminuria and hypertension at enrolment, and all visits (12, 22, 32 weeks’ gestation) preceded clinical PE onset. Results: There were no cross-sectional differences in total SM, or in any individual SM species, between diabetic women with and without PE, or between normotensive women with and without diabetes at any stage of gestation. With advancing gestation in all groups, C18-SM, C18:1-SM, C20-SM, C20:1-SM, C22-SM, C22:1-SM, C24-SM, and C24:1-SM increased (all p<0.001). LDL- and VLDL-cholesterol also increased, while HDL-cholesterol did not change. In contrast, C14-SM, C16-SM, C26-SM and C26:1-SM decreased (all p<0.001). There were no correlations between any SM species and conventional lipid profiles. Conclusions: With advancing gestation, plasma concentrations of most SM species increased, as did LDL and VLDL; however, two long-chain and two very long-chain SM species decreased. Although there were no differences in SM according to diabetes or PE status at any time point, further studies should address the significance of differential changes of SM species during pregnancy. Disclosure C.B. Kelly: None. S.M. Hammad: None. R.L. Klein: None. M.F. Lopes-Virella: None. M.J. Leyva: None. J. Yu: None. A.J. Jenkins: Advisory Panel; Self; Abbott, Australian Diabetes Society, Medtronic. Research Support; Self; Abbott, GlySens Incorporated, Medtronic, Mylan. Speaker's Bureau; Self; Eli Lilly and Company, Novo Nordisk Inc. A.J. Nankervis: None. K.F. Hanssen: None. S.K. Garg: Advisory Panel; Self; Eli Lilly and Company, Roche Pharma, Sanofi-Aventis, Zealand Pharma A/S. Research Support; Self; Eli Lilly and Company, WebMD. J.A. Scardo: None. C.E. Aston: None. T. Lyons: None. Funding JDRF; Novo Nordisk; National Institutes of Health

Systemic lupus erythematous (SLE) is a chronic multi-organ autoimmune disease. Genetic and environmental factors contribute to disease onset and severity. Sphingolipids are signaling molecules involved in regulating cell functions and have been associated with multiple genetic disease processes. African-Americans are more likely to suffer from SLE morbidity than Whites. The Medical University of South Carolina has banked plasma samples from a well-characterized lupus cohort that includes African-Americans and Whites. This study examined the influence of race on plasma sphingolipid profiles in SLE patients and association of sphingolipid levels with comorbid atherosclerosis and SLE disease activity. Mass spectrometry revealed that healthy African-Americans had higher sphingomyelin levels and lower lactosylcermide levels compared to healthy Whites. SLE patients, irrespective of race, had higher levels of ceramides, and sphingoid bases (sphingosine and dihydrosphingosine) and their phosphates compared to healthy subjects. Compared to African-American controls, African-American SLE patients had higher levels of ceramides, hexosylceramides, sphingosine and dihydrosphingosine 1-phosphate. Compared to White controls, White SLE patients exhibited higher levels of sphingoid bases and their phosphates, but lower ratios of C16:0 ceramide/sphingosine 1-phosphate and C24:1 ceramide/sphingosine 1-phosphate. White SLE patients with atherosclerosis exhibited lower levels of sphingoid bases compared to White SLE patients without atherosclerosis. In contrast, African-American SLE patients with atherosclerosis had higher levels of sphingoid bases and sphingomyelins compared to African-American SLE patients without atherosclerosis. Compared to White SLE patients with atherosclerosis, African-American SLE patients with atherosclerosis had higher levels of select sphingolipids. Plasma levels of sphingosine, C16:0 ceramide/sphingosine 1-phosphate ratio and C24:1 ceramide/sphingosine 1-phosphate ratio significantly correlated with SLEDAI in the African-American but not White SLE patients. The C16:0 ceramide/sphingosine 1-phosphate ratio in SLE patients, and levels of C18:1 and C26:1 lactosylcermides, C20:0 hexosylceramide, and sphingoid bases in SLE patients with atherosclerosis could be dependent on race. Further ethnic studies in SLE cohorts are necessary to verify use of sphingolipidomics as complementary diagnostic tool.

3-ketodihydrosphingosine reductase (KDSR) is the key enzyme in the de novo sphingolipid synthesis. We identified a novel missense kdsrI105R mutation in zebrafish that led to a loss of function, and resulted in progression of hepatomegaly to steatosis, then hepatic injury phenotype. Lipidomics analysis of the kdsrI105R mutant revealed compensatory activation of the sphingolipid salvage pathway, resulting in significant accumulation of sphingolipids including ceramides, sphingosine and sphingosine 1-phosphate (S1P). Ultrastructural analysis revealed swollen mitochondria with cristae damage in the kdsrI105R mutant hepatocytes, which can be a cause of hepatic injury in the mutant. We found elevated sphingosine kinase 2 (sphk2) expression in the kdsrI105R mutant. Genetic interaction analysis with the kdsrI105R and the sphk2wc1 mutants showed that sphk2 depletion suppressed liver defects observed in the kdsrI105R mutant, suggesting that liver defects were mediated by S1P accumulation. Further, both oxidative stress and ER stress were completely suppressed by deletion of sphk2 in kdsrI105R mutants, linking these two processes mechanistically to hepatic injury in the kdsrI105R mutants. Importantly, we found that the heterozygous mutation in kdsr induced predisposed liver injury in adult zebrafish. These data point to kdsr as a novel genetic risk factor for hepatic injury.

BACKGROUND: Glycosphingolipids are important components of cell membranes, modulators of cell-cell interactions and cell recognition, and have recently emerged as bioactive molecules and important players in nearly all cell biological processes. We previously have shown that decreased plasma levels of long and very long species of ceramides were able to predict the development of macroalbuminuria (MA) in type 1 diabetes. OBJECTIVE: This study proposed to examine whether plasma glycosphingolipids could predict development of diabetic nephropathy, assessed as MA or chronic kidney disease (CKD). METHODS: Measurement of plasma hexosylceramides (H) and lactosylceramides (L) were conducted in the Lipidomics Core Facility of our Institution in a subcohort of 432 patients from the DCCT/Epidemiology of Diabetes Interventions and Complications cohort in plasma collected at entry into the study. Inverse probability weighted Cox proportional hazards regression models were used to assess the effect of glycosphingolipids levels on the risk of developing MA (albumin excretion rate >= 300 mg/24 hours) or CKD (glomerular filtration rate <60 mL/min) over a period of 21 to 28 years. RESULTS: Decreases of several long and very long chain lactosylceramides were significantly associated with increased risk of progression to MA but not CKD. Among the hexosylceramides, the only significant association observed was between one of its minor species C18:1-H and CKD. CONCLUSION: Our findings showed that decreased levels of long and very long lactosylceramides were able to predict the development of MA in type I diabetes. This finding is similar to previous findings showing that low levels of long and very long ceramides were also able to predict development of MA in the same cohort. Further studies are needed to determine the changes in sphingolipid metabolism leading to the development of complications. (C) 2019 Published by Elsevier Inc. on behalf of National Lipid Association.