Plasmalogens Rescue Neuronal Cell Death through an Activation of AKT and ERK Survival Signaling

Neuronal cells are susceptible to many stresses, which will cause the apoptosis and neurodegenerative diseases. The precise molecular mechanism behind the neuronal protection against these apoptotic stimuli is necessary for drug discovery. In the present study, we have found that plasmalogens (Pls), which are glycerophospholipids containing vinyl ether linkage at sn-1 position, can protect the neuronal cell death upon serum deprivation. Interestingly, caspse-9, but not caspase-8 and caspase-12, was cleaved upon the serum starvation in Neuro-2A cells. Pls treatments effectively reduced the activation of caspase-9. Furthermore, cellular signaling experiments showed that Pls enhanced phosphorylation of the phosphoinositide 3-kinase (PI3K)-dependent serine/threonine-specific protein kinase AKT and extracellular-signal-regulated kinases ERK1/2. PI3K/AKT inhibitor LY294002 and MAPK/ERK kinase (MEK) inhibitor U0126 treatments study clearly indicated that Pls-mediated cell survival was dependent on the activation of these kinases. In addition, Pls also inhibited primary mouse hippocampal neuronal cell death induced by nutrient deprivation, which was associated with the inhibition of caspase-9 and caspase-3 cleavages. It was reported that Pls content decreased in the brain of the Alzheimer’s patients, which indicated that the reduction of Pls content could endanger neurons. The present findings, taken together, suggest that Pls have an anti-apoptotic action in the brain. Further studies on precise mechanisms of Pls-mediated protection against cell death may lead us to establish a novel therapeutic approach to cure neurodegenerative disorders.     

Supplemental feeding of phospholipid-enriched alkyl phospholipid from krill relieves spontaneous atopic dermatitis and strengthens skin intercellular lipid barriers in NC/Nga mice

Plasmalogen (Pls) is a glycerophospholipid derived from alkyl phospholipid (Alk) with antioxidant functions in vivo. The present study investigated the effects of ether phospholipids, such as Pls and Alk, on intercellular lipid barriers in the skin of NC/Nga mice, a model of atopic dermatitis (AD). NC/Nga mice fed Alk showed increased plasma levels of Alk and Pls. The AD-related changes in ceramide composition in the skin were abrogated by oral administration of Alk. Moreover, Alk suppressed skin inflammation in AD mice. These results indicate that Alk partially fortifies the stratum corneum lipid barrier and may be an effective treatment for AD.

Abbreviations: Pls: plasmalogen; PlsCho: choline plasmalogen; PlsEtn: ethanolamine plasmalogen; Alk: alkyl phospholipid; TJ: tight junction; FA: fatty acid; AD: atopic dermatitis; SO: soybean oil; FO: fish oil; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TG: triglyceride; PL: phospholipid; RF: retention factor; AlkCho: choline-type alkyl phospholipid; AlkEtn: ethanolamine-type alkyl phospholipid; LC-MS/MS: liquid chromatography-tandem mass spectrometry; FAR1: fatty acyl-coenzyme (Co)A reductase 1     

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC-chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC-ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC-ClOH.     

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.     

Serum selenium,glutathione peroxidase,lipids, and human liver microsomal enzyme activity

This study evaluated selenium status in relation to lipid peroxidation, liver microsomal function, and serum lipids in humans. Serum selenium concentration, glutathione peroxidase (GSH-Px) activity, liver microsomal enzyme activity, assessed by plasma antipyrine clearance (AP-CL) rate, and serum lipids were determined in 23 healthy subjects in a double-blind placebo-controlled trial of selenium supplementation. The low selenium concentration (74.0±14.2 μg/L, mean±SD) is attributable to the low selenium content of the diet. Subjects with the lowest selenium levels (n=11) had reduced serum GSH-Px activity, AP-CL rate, high-density lipoprotein cholesterol (HDL-C), and total cholesterol (T-C) as compared with subjects with higher selenium concentrations (n=12). Low AP-CL rates were associated with low HDL-C: T-C ratios. Selenium supplementation, 96 μg/d for 2 wk, increased serum selenium, GSH-Px activity, and the HDL-C: T-C ratio. The results suggest that a low serum selenium level is associated with a decrease in liver microsomal enzyme activity and serum HDL-C and T-C concentrations. Selenium supplementation in subjects with low serum selenium may favorably influence relations between serum lipoproteins connected with the development of atherosclerotic vascular disease.     

Serum lipid profiles of young Japanese women with iron deficiency without anemia

PurposeWe aimed to evaluate the association between iron deficiency without anemia (IDNA) and serum lipid profiles in young women of around 20 years of age.MethodsThis study included non-anemic (hemoglobin ≥ 12 g/dL) female volunteers aged 18 to 22 years who were not taking mineral/vitamin supplements and living in the metropolitan area of Tokyo, Japan. These volunteers were classified into two groups based on their sFer (serum ferritin) levels: normal group (sFer ≥ 20 ng/mL, n = 36) and IDNA group (sFer < 20 ng/mL, n = 29). Venous blood samples were obtained from the antecubital veins of these volunteers after 10–12-h fasting to measure the hematological and biochemical parameters, including lipid levels and iron status. The results of each group were compared using Student’s t-test or the Mann–Whitney U test (for inhomogeneous variance).ResultsThe serum cholesterol levels varied depending on the iron status in the women. Serum high-density lipoprotein cholesterol (HDL-C) levels in the IDNA group were significantly higher (P = 0.006) than that in the normal group. However, the levels of total cholesterol (T-CHO), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) were not significantly different between the groups. Serum LDL-C levels were positively and significantly correlated with sFer levels in the IDNA group (Kendall’s rank correlation 0.264, P = 0.044), but not in the normal group. The sFer level was not correlated with serum HDL-C in both groups. The reason for the high serum HDL-C levels in young women with IDNA is not yet clear. Compared to the normal group, the frequency of eating bread containing bran was significantly higher (P = 0.031) and that for yogurt was significantly lower (P = 0.040) in the IDNA group. The proportion of the women who were susceptible to infection, which was measured using the Cornell Medical Index, was significantly higher in the IDNA group than in the normal group. Among those susceptible to infection, the serum HDL-C level of the volunteers in the IDNA group was significantly higher than that of the volunteers in the normal group (P = 0.024).ConclusionsOur findings suggest that lipid parameters may be associated with IDNA and susceptibility to infection. Further research is needed to elucidate the mechanisms underlying the changes in the serum cholesterol levels in individuals with IDNA and the clinical significance of these findings.     

Phase behavior of ethanolamine plasmalogen

In the present study the phase behavior of multilamellar dispersions of 1-O-(1′-alkenyl)-2-oleoyl-glycerophosphoethanolamine (ethanolamine plasmalogen), 1-O-alkyl-2-oleoyl-glycerophosphoethanolamine and 1-acyl-2-oleoyl-glycerophosphoethanolamine was compared using differential scanning calorimetry (DSC) and ³¹P-NMR. The three compounds differed only in the type of bonding (vinyl ether, alkyl ether or acyl ester) linking the aliphatic moiety to position 1 of sn-glycerol.The gel to liquid-crystalline phase transition temperature as determined by DSC was lowest for ethanolamine plasmalogen (26°C) and was similar for the alkylacyl and diacyl analogs (29.5° and 30°C, respectively). Enthalpies of the G → L phase transition were not significantly different for the three phospholipids tested.Ethanolamine plasmalogen undergoes the lamellar to hexagonal phase transition at 30°C, the analogous alkylacyl-glycerophosphoethanolamine(alkylacyl-GPE) and diacyl-GPE at 53°C and 69°C, respectively. Thus, an alkenyl ether bond in position 1 of sn-glycerol, the structural characteristic of plasmalogens, effectively stabilizes the hexagonal H_II arrangement of ethanolamine glycerophospholipids, while it has relatively little effect on destabilization of the lamellar gel state.     

Persistence of an atherogenic lipid profile after treatment of acute infection with brucella

Serum lipid changes during infection may be associated with atherogenesis. No data are available on the effect of Brucellosis on lipids. Lipid parameters were determined in 28 patients with Brucellosis on admission and 4 months following treatment and were compared with 24 matched controls. Fasting levels of total cholesterol (TC), HDL-cholesterol (HDL-C), triglycerides, apolipoproteins (Apo) A, B, E CII, and CIII, and oxidized LDL (oxLDL) were measured. Activities of serum cholesterol ester transfer protein (CETP), paraoxonase 1 (PON1), and lipoprotein-associated phospholipase A₂ (Lp-PLA₂) and levels of cytokines [interleukins (IL)-1β, IL-6, and tumor necrosis factor (TNFa)] were also determined. On admission, patients compared with controls had 1) lower levels of TC, HDL-C, LDL-cholesterol (LDL-C), ApoB, ApoAI, and ApoCIII and higher LDL-C/HDL-C and ApoB/ApoAI ratios; 2) higher levels of IL-1b, IL-6, and TNFa; 3) similar ApoCII and oxLDL levels and Lp-PLA₂ activity, lower PON1, and higher CETP activity; and 4) higher small dense LDL-C concentration. Four months later, increases in TC, HDL-C, LDL-C, ApoB, ApoAI, and ApoCIII levels, ApoB/ApoAI ratio, and PON1 activity were noticed compared with baseline, whereas CETP activity decreased. LDL-C/HDL-C ratio, ApoCII, and oxLDL levels, Lp-PLA₂ activity, and small dense LDL-C concentration were not altered. Brucella infection is associated with an atherogenic lipid profile that is not fully restored 4 months following treatment.     

Alterations in the gut microbiota and metabolite profiles of patients with Kashin-Beck disease,an endemic osteoarthritis in China

Kashin-Beck disease (KBD) is a severe osteochondral disorder that may be driven by the interaction between genetic and environmental factors. We aimed to improve our understanding of the gut microbiota structure in KBD patients of different grades and the relationship between the gut microbiota and serum metabolites. Fecal and serum samples collected from KBD patients and normal controls (NCs) were used to characterize the gut microbiota using 16S rDNA gene and metabolomic sequencing via liquid chromatography-mass spectrometry (LC/MS). To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria in the KBD patients, metagenomic sequencing of fecal samples from grade I KBD, grade II KBD and NC subjects was performed. The KBD group was characterized by elevated levels of Fusobacteria and Bacteroidetes. A total of 56 genera were identified to be significantly differentially abundant between the two groups. The genera Alloprevotella, Robinsoniella, Megamonas, and Escherichia_Shigella were more abundant in the KBD group. Consistent with the 16S rDNA analysis at the genus level, most of the differentially abundant species in KBD subjects belonged to the genus Prevotella according to metagenomic sequencing. Serum metabolomic analysis identified some differentially abundant metabolites among the grade I and II KBD and NC groups that were involved in lipid metabolism metabolic networks, such as that for unsaturated fatty acids and glycerophospholipids. Furthermore, we found that these differences in metabolite levels were associated with altered abundances of specific species. Our study provides a comprehensive landscape of the gut microbiota and metabolites in KBD patients and provides substantial evidence of a novel interplay between the gut microbiome and metabolome in KBD pathogenesis.Subject terms: Metagenomics, Metabolomics     

Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer''s disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA₂ activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA₂ activity accounts for altered GPs in these fractions as neurodegeneration progresses.     

Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.     

Novel stereoconfiguration in lyso-bis-phosphatidic acid of cultured BHK-cells

Lyso-bis-phosphatidic acid purified from cultured hamster kidney fibroblast cells (BHK-cells) was subjected to strong alkaline hydrolysis. The hydrolysate contained phosphorus, free glycerol, total glycerol, α-glycerophosphate, β-glycerophosphate and sn-glycerol-3-phosphate in mole ratios of 1.0:1.0:1.9:0.4:0.6:0.02. The absence of sn-glycerol-3-phosphate indicates that the backbone of this lipid has the uncommon structure of 1-sn-glycerophosphoryl-1′-sn-glycerol. Consequently, the biosynthesis and the degradation of this lipid must differ from the other known mammalian glycerophospholipids.     

Thematic Review Series: Lysophospholipids and their Receptors: Diversity and function of membrane glycerophospholipids generated by the remodeling pathway in mammalian cells

Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained.     

Polymorphism of rs873308 near the transmembrane protein 57 gene is associated with serum lipid levels

SNP (single-nucleotide polymorphism) of rs10903129 near the TMEM (transmembrane protein) 57 locus has been associated with TC (total cholesterol) in a previous GWAS (genome-wide association study), but the association of TMEM57 rs873308 SNP and serum lipid levels has not been previously reported. The current study was undertaken to detect the association of the TMEM57 rs873308 SNP and several environmental factors with serum lipid profiles in the Han Chinese and Mulao populations. The genotypes of the TMEM57 rs873308 SNP in 865 individuals of Han Chinese and 902 participants of Mulao nationality were determined by PCR and RFLP (restriction-fragment-length polymorphism) combined with gel electrophoresis and then confirmed by direct sequencing. The T allele frequency of TMEM57 rs873308 SNP was not different between Han and Mulao (23.18% versus 25.72%, P>0.05), but different between males and females in the two ethnic groups (P<0.05). The T allele carriers had lower serum TC, Apo (apolipoprotein) B, HDL-C (high-density lipoprotein cholesterol) levels, ApoA1/ApoB ratio in Han; and lower TAG (triacylglycerol), LDL-C (low-density lipoprotein cholesterol), ApoA1 levels and the ApoA1/ApoB ratio and higher HDL-C levels in Mulao than the T allele non-carriers. There was also different association of the TMEM57 rs873308 SNP and serum lipid profiles between males and females in the both ethnic groups. Serum lipid parameters in the two ethnic groups were also associated with several environmental factors. The association of the TMEM57 rs873308 SNP and serum lipid levels was different in the Han Chinese and Mulao populations and between males and females in the both ethnic groups. There may be a sex-specific association of the TMEM57 rs873308 SNP and serum lipid levels in our study populations.     

Molecular basis of phospholipase A2 activity toward phospholipids with sn-1 substitutions

We studied secretory phospholipase A₂ type IIA (sPLA₂) activity toward phospholipids that are derivatized in the sn-1 position of the glycerol backbone. We explored what type of side group (small versus bulky groups, hydrophobic versus polar groups) can be introduced at the sn-1 position of the glycerol backbone of glycerophospholipids and at the same time be hydrolyzed by sPLA₂. The biophysical characterization revealed that the modified phospholipids can form multilamellar vesicles, and several of the synthesized sn-1 functionalized phospholipids were hydrolyzed by sPLA₂. Molecular dynamics simulations provided detailed insight on an atomic level that can explain the observed sPLA₂ activity toward the different phospholipid analogs. The simulations revealed that, depending on the nature of the side chain located at the sn-1 position, the group may interfere with an incoming water molecule that acts as the nucleophile in the enzymatic reaction. The simulation results are in agreement with the experimentally observed sPLA₂ activity toward the different phospholipid analogs.     

Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study

HDL is strongly inversely related to cardiovascular risk. Hepatic HDL uptake is controlled by ecto-F₁-ATPase activity, and potentially inhibited by mitochondrial inhibitor factor 1 (IF1). We recently found that IF1 is present in serum and correlates with HDL-cholesterol (HDL-C). Here, we have evaluated the relationship between circulating IF1 and plasma lipoproteins, and we determined whether IF1 concentration is associated with the risk of coronary heart disease (CHD). Serum IF1 was measured in 648 coronary patients ages 45–74 and in 669 matched male controls, in the context of a cross-sectional study on CHD. Cardiovascular risk factors were documented for each participant, including life-style habits and biological and clinical markers. In controls, multivariate analysis demonstrated that IF1 was independently positively associated with HDL-C and apoA-I (r = 0.27 and 0.28, respectively, P < 0.001) and negatively with triglycerides (r = −0.23, P < 0.001). Mean IF1 concentration was lower in CHD patients than in controls (0.43 mg/l and 0.53 mg/l, respectively, P < 0.001). In multivariate analyses, following adjustments on cardiovascular risk factors or markers, IF1 was negatively related to CHD (P < 0.001). This relationship was maintained after adjustment for HDL-C or apoA-I. This study identifies IF1 as a new determinant of HDL-C that is inversely associated with CHD.     

In Vivo Incorporation of a Polyunsaturated Fatty Acid into the sn-C-1 and sn-C-2 Positions of Tetrahymena Glycerophospholipids1

The glycerophospholipids of the protozoon Tetrahymena pyriformis W are unique in that the polyunsaturated fatty acid γ-linolenate (18:3Δ^6,9,12) is a major component of both the sn-C-1 and sn-C-2 positions. Tetrahymena were incubated with [1-¹⁴C]γ-linolenate. The positional distribution of the radiolabeled fatty acid in the three major glycerophospholipids was determined. [1-¹⁴C]γ-linolenate was found at both carbons of the three lipids, in general agreement with the mass distribution of γ-linolenate, except for markedly greater labeling at the sn-C-2 position of phosphatidylcholine. We hypothesize that an acyltransferase exists in Tetrahymena that can esterify γ-linolenate at both carbons during glycerophospholipid biosynthesis.     

Association between Serum C-Peptide as a Risk Factor for Cardiovascular Disease and High-Density Lipoprotein Cholesterol Levels in Nondiabetic Individuals

Objective

Objective: Although serum C-peptide has increasingly received attention as a new and important risk factor for cardiovascular disease (CVD), the potential mechanisms remain unclear. This study aimed to investigate the association between serum C-peptide as a risk factor for CVD and high-density lipoprotein cholesterol (HDL-C) levels.

Methods

The present study included 13,185 participants aged ≥20 years. Serum C-peptide and HDL-C levels were measured according to a standard protocol. Stratified analysis of covariance was used to compare serum HDL-C levels between different quartiles of serum C-peptide levels. Logistic regression analysis was used to determine the association between serum C-peptide and HDL-C levels. Cox proportional hazard regression analysis was conducted to determine the hazard ratio of serum HDL-C for CVD-related mortality.

Results

The results of the ANCOVA analysis showed a significant linear trend between the mean serum HDL-C level and the different quartiles of serum C-peptide. Compared to the first quartile (25th percentile), the second, third, and fourth quartiles had gradual reduction in serum HDL-C levels. Logistic regression analyses showed a strong negative association between serum C-peptide levels and HDL-C levels; the p value for the linear trend was <0.001. In men, compared with the lowest quartile of the serum C-peptide level, the relative risk was 1.75, 2.79, and 3.07 for the upper three quartiles of the serum C-peptide level. The relative risk was 1.60, 2.61, and 3.67 for women. The results of the survival analysis showed that serum HDL-C levels were negatively associated with CVD-related death in both men and women.

Conclusion

Serum C-peptide as a risk factor for CVD was significantly and negatively associated with serum HDL-C levels in individuals without diabetes. These findings suggest that serum C-peptide levels association with CVD death can be caused, at least in part, by the low serum HDL-C level.