Plasma apoM Levels and Progression to Kidney Dysfunction in Patients With Type 1 Diabetes.

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.

Deoxysphingolipids Upregulate MMP-1, Downregulate TIMP-1, and Induce Cytotoxicity in Human Schwann Cells.

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.

Transcriptomics Reveal Altered Metabolic and Signaling Pathways in Podocytes Exposed to C16 Ceramide-Enriched Lipoproteins.

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.

Sphingosine 1-phosphate distribution in human plasma: associations with lipid profiles.

The physiological significance of sphingosine 1-phosphate (S1P) transport in blood has been debated. We have recently reported a comprehensive sphingolipid profile in human plasma and lipoprotein particles (VLDL, LDL, and HDL) using HPLC-MS/MS (Hammad et al., 2010). We now determined the relative concentrations of sphingolipids including S1P in the plasma subfraction containing lipoproteins compared to those in the remaining plasma proteins. Sphingomyelin and ceramide were predominantly recovered in the lipoprotein-containing fraction. Total plasma S1P concentration was positively correlated with S1P concentration in the protein-containing fraction, but not with S1P concentration in the lipoprotein-containing fraction. The percentage of S1P transported in plasma lipoproteins was positively correlated with HDL cholesterol (HDL-C) concentration; however, S1P transport in lipoproteins was not limited by the concentration of HDL-C in the individual subject. Thus, different plasma pools of S1P may have different contributions to S1P signaling in health and disease.

HDL3, but not HDL2, stimulates plasminogen activator inhibitor-1 release from adipocytes: the role of sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that regulates numerous key cardiovascular functions. High-density lipoproteins (HDLs) are the major plasma lipoprotein carriers of S1P. Fibrinolysis is a physiological process that allows fibrin clot dissolution, and decreased fibrinolytic capacity may result from increased circulating levels of plasminogen activator inhibitor-1 (PAI-1). We examined the effect of S1P associated with HDL subfractions on PAI-1 secretion from 3T3 adipocytes. S1P concentration in HDL3 averaged twice that in HDL2. Incubation of adipocytes with increasing concentrations of S1P in HDL3, but not HDL2, or with S1P complexed to albumin stimulated PAI-I secretion in a concentration-dependent manner. Quantitative RT-PCR revealed that S1P(1-3) are expressed in 3T3 adipocytes, with S1P(2) expressed in the greatest amount. Treatment of adipocytes with the S1P(1) and S1P(3) antagonist VPC23019 did not block PAI-1 secretion. Inhibiting S1P(2) with JTE-013 or reducing the expression of the gene coding for S1P(2) using silencing RNA (siRNA) technology blocked PAI-1 secretion, suggesting that the S1P(2) receptor mediates PAI-1 secretion from adipocytes exposed to HDL3 or S1P. Treatment with the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor RO-318425, or the Rho-associated protein kinase (ROCK) inhibitor Y27632 all significantly inhibited HDL3- and S1P-mediated PAI-1 release, suggesting that HDL3- and/or S1P-stimulated PAI-1 secretion from 3T3 cells is mediated by activation of multiple, downstream signaling pathways of S1P(2).

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells.

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Apolipoprotein C-III protein concentrations and gene polymorphisms in Type 1 diabetes: associations with microvascular disease complications in the DCCT/EDIC cohort.

AIM: We investigated the associations of apolipoprotein C-III (apoCIII) protein and apoCIII gene variation with microvascular disease complications in Type 1 diabetes. METHODS: The serum apoCIII concentration, and both a T(-455)-->C and a SacI gene polymorphisms were determined in 409 patients in the DCCT/EDIC cohort of patients with Type 1 diabetes. Correlations with albumin excretion rate (AER) and the severity of retinopathy were investigated. RESULTS: Higher apoCIII concentrations were associated (P<.0001) with increased triglycerides (r=.78), total (r=.61) and LDL (r=.40) cholesterol, apoAI (r=.26), and apoB (r=.50), AER (r=.08), and the severity of retinopathy (ETDRS score, r=.11), and these relationships persisted after controlling for age, gender, body mass index (BMI), and HbA1c level. The apoCIII concentration was significantly higher in the group of patients with macroalbuminuria (AERs 300 mg/24 h) compared to the groups with microalbuminuria (AER 40-299 mg/24 h; P<.0001) or normoalbuminuria (AER <40 mg/24 h) (P<.0001). The apoCIII concentration also was significantly higher in the group of patients with severe retinopathy (ETDRS 10-23) compared to those with moderate (ETDRS 4-9; P<.02) or mild retinopathy (ETDRS 1-3; P<.0001). Neither the T(-455)-->C polymorphism nor a SacI polymorphism in the 3' UTR were associated with circulating apoCIII concentrations, nor the severity of nephropathy or retinopathy. CONCLUSIONS: Elevated apoCIII levels have been associated with increased macrovascular disease risk. In the DCCT/EDIC cohort of patients, there was an independent positive association of apoCIII level with microvascular complications of Type 1 diabetes.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes: associations with lipoprotein subclasses.

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P < .0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

Fibrinogen is a marker for nephropathy and peripheral vascular disease in type 1 diabetes: studies of plasma fibrinogen and fibrinogen gene polymorphism in the DCCT/EDIC cohort.

We examined whether plasma fibrinogen levels and the beta-fibrinogen gene G(-455)-->A polymorphism were related to microvascular or macrovascular disease in patients (n = 909) with type 1 diabetes enrolled in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/ EDIC). Univariate regression showed that fibrinogen levels were correlated with BMI (r = 0.15; P < 0.0001), HbA(1c) (r = 0.11; P = 0.0014), total cholesterol (r = 0.17; P < 0.0001), and LDL cholesterol (r = 0.16; P < 0.0001) in all patients. In men, but not women, waist-to-hip ratio (r = 0.20; P < 0.0001) and triglycerides (r = 0.13; P = 0.0047) also became powerful predictors of fibrinogen level; in women, but not men, fibrinogen was correlated with both diastolic (r = 0.16; P = 0.0011) and systolic (r = 0.11; P = 0.0241) blood pressure. Fibrinogen was correlated with urinary albumin excretion rates in men (r = 0.13; P = 0.0033), but not in women. In both sexes, however, the development of proteinuria (albumin excretion >300 mg/24 h) was accompanied by 1.5-fold increment in plasma fibrinogen compared with patients with normal excretion or microalbuminuria. In addition, high fibrinogen levels were associated with a lower average ankle-brachial index in women (r = -0.13; P = 0.0075), but not men. Multiple regression analyses demonstrated that plasma fibrinogen was independently correlated with high albumin excretion rate in men, and with low average ankle-brachial index in women. Fibrinogen was not correlated with the severity of retinopathy. Carotid artery intima-medial thickness was not correlated with fibrinogen, and the G(-455)-->A polymorphism in the 5' promoter region of the beta-fibrinogen gene did not influence circulating fibrinogen levels. However, the presence of the more common G(-455) allele was associated with greater intima-medial thickness in the internal carotid artery (ANCOVA P = 0.045). Last, hyperfibrinogenemia in type 1 diabetes is associated with components of the insulin resistance syndrome trait cluster, and the association is influenced by sex.