Circadian organization of lipid landscape is perturbed in type 2 diabetic patients.

Lipid homeostasis in humans follows a diurnal pattern in muscle and pancreatic islets, altered upon metabolic dysregulation. We employ tandem and liquid-chromatography mass spectrometry to investigate daily regulation of lipid metabolism in subcutaneous white adipose tissue (SAT) and serum of type 2 diabetic (T2D) and non-diabetic (ND) human volunteers (n = 12). Around 8% of ≈440 lipid metabolites exhibit diurnal rhythmicity in serum and SAT from ND and T2D subjects. The spectrum of rhythmic lipids differs between ND and T2D individuals, with the most substantial changes observed early morning, as confirmed by lipidomics in an independent cohort of ND and T2D subjects (n = 32) conducted at a single morning time point. Strikingly, metabolites identified as daily rhythmic in both serum and SAT from T2D subjects exhibit phase differences. Our study reveals massive temporal and tissue-specific alterations of human lipid homeostasis in T2D, providing essential clues for the development of lipid biomarkers in a temporal manner.

Sphingosine-1-phosphate as a key player of insulin secretion induced by high-density lipoprotein treatment.

Beta cell failure is one of the most important features of type 2 diabetes mellitus (T2DM). High-density lipoprotein (HDL) has been proposed to improve ß-cell function. However, the mechanisms involved in this process are still poorly understood. The aim of this study was to investigate the contribution of sphingosine-1-phosphate (S1P) in the impact of HDL treatment on insulin secretion by pancreatic ß-cells and to determine its mechanisms. Primary cultures of ß-cells isolated from rat were treated with or without HDL in the presence or absence of S1P pathway inhibitors and insulin secretion response was analyzed. The S1P content of HDL (HDL-S1P) isolated from T2DM patients was analyzed and correlated to the HDL-induced insulin secretion. The expression of genes involved in the biosynthesis of the insulin was also evaluated. HDL as well as S1P treatment enhanced glucose-stimulated insulin secretion (GSIS). In HDL isolated from T2DM patients, while HDL-S1P was strongly correlated to its pro-secretory capacity (r = 0.633, p = 0.005), HDL-cholesterol and apolipoprotein AI levels were not. HDL-induced GSIS was blocked by the S1P1/3 antagonist but not by the S1P2 antagonist, and was also accompanied by increased intracellular S1P in ß-cells. We also observed that HDL improved GSIS without significant changes in expression levels of insulin biosynthesis genes. Our present study highlights the importance HDL-S1P in GSIS in T2DM patients and demonstrates that HDL induces insulin secretion by a process involving both intra- and extra-cellular sources of S1P independently of an effect on insulin biosynthesis genes.

Ether lipids, sphingolipids and toxic 1-deoxyceramides as hallmarks for lean and obese type 2 diabetic patients.

AIM: The worldwide increase in obesity and type 2 diabetes (T2D) represents a major health challenge. Chronically altered lipids induced by obesity further promote the development of T2D, and the accumulation of toxic lipid metabolites in serum and peripheral organs may contribute to the diabetic phenotype. METHODS: To better understand the complex metabolic pattern of lean and obese T2D and non-T2D individuals, we analysed the lipid profile of human serum, skeletal muscle and visceral adipose tissue of two cohorts by systematic mass spectrometry-based lipid analysis. RESULTS: Lipid homeostasis was strongly altered in a disease- and tissue-specific manner, allowing us to define T2D signatures associated with obesity from those that were obesity independent. Lipid changes encompassed lyso-, diacyl- and ether-phospholipids. Moreover, strong changes in sphingolipids included cytotoxic 1-deoxyceramide accumulation in a disease-specific manner in serum and visceral adipose tissue. The high amounts of non-canonical 1-deoxyceramide present in human adipose tissue most likely come from cell-autonomous synthesis because 1-deoxyceramide production increased upon differentiation to adipocytes in mouse cell culture experiments. CONCLUSION: Taken together, the observed lipidome changes in obesity and T2D will facilitate the identification of T2D patient subgroups and represent an important step towards personalized medicine in diabetes.

Validation of molecular biomarkers for preoperative diagnostics of human papillary thyroid carcinoma in fine needle aspirates.

BACKGROUND: Despite substantial efforts, reliable preoperative diagnostic for human thyroid malignancies in case of cytologically indeterminate nodules is still missing, resulting in high number of unnecessary thyroidectomies. In an attempt to increase precision of existing preoperative diagnostics, we aimed at validating the panel of molecular biomarkers predictive for papillary thyroid carcinoma (PTC) in preoperative fine needle aspirate (FNA) samples. METHODS: In this prospective study conducted in preoperative thyroid FNA from 44 thyroid nodules, expression levels of 11 molecular biomarkers previously validated on the postoperative samples of PTCs were measured by Cell-to-CT and QuantiGene Plex methods and correlated with final diagnosis. RESULTS: The QuantiGene Plex resulted in reliable gene expression measurements for FNA and core-needle biopsy (CNB) samples, however this method was less sensitive than pre-amplification based Cell-to-CT. Measurements conducted on the same samples by the two methods significantly correlated for most of the genes. Expression levels of TIMP1, c-MET and ARNTL were upregulated in PTC nodules as compared to benign counterparts, supporting previous post-operative studies. Strong correlation was observed between these biomarker alterations in the same samples. Within the sub-group of 15 indeterminate nodules (Bethesda II-V), TIMP1 had 100% specificity and 83% sensitivity for PTC cases. CONCLUSIONS: Assessment of TIMP1, c-MET and core-clock gene ARNTL expression levels by QuantiGene Plex assay in FNA samples holds promise as an ancillary method to the cytological preoperative diagnostics.

HDL protects against myocardial ischemia reperfusion injury via miR-34b and miR-337 expression which requires STAT3.

PURPOSE: High density lipoprotein (HDL) protects against myocardial infarction via mechanisms that remain unclear. STAT3 (signal transducer and activator of transcription 3) plays a key role in HDL-induced cardioprotection. In the heart, microRNAs (miRNAs) are involved in ischemia reperfusion injury. We therefore investigated whether the cardioprotective effect of HDL modulates miRNAs as a downstream target of STAT3 activation. METHODS: STAT3 cardiomyocyte deficient mice (STAT3-KO) and wildtype littermates (STAT3-WT) were submitted to left coronary ligature and reperfused (IR) with or without injection of HDL. Infarct size (IS) was determined and cardiac miRNA expression was evaluated after reperfusion in sham, IR and IR+HDL hearts by microarray analysis. In vitro, neonatal rat ventricular cardiomyocytes were submitted to hypoxia with or without HDL incubation. Cell viability and miRNA expression were analysed. RESULTS: In vivo, HDL reduced IS from 40.5±4.3% to 24.4±2.1% (p<0.05) in STAT3-WT mice. HDL failed to protect in STAT3-KO mice. In STAT3-WT mice, both miR-34b and miR-337 were increased in IR compared to sham and IR+HDL groups (p<0.05). These miRNAs were not modulated in STAT3-KO mice. In vitro, incubation with HDL improved cell viability against hypoxia (p<0.05). The expression of miR-34b and miR-337 was increased by hypoxia and reduced by HDL treatment (p<0.05). In cardiomyocytes transfected with miRNA mimics, HDL failed to improve cell viability against hypoxia. CONCLUSIONS: Our study, performed both in vivo and in vitro, delineates a novel cardioprotective signalling pathway activated by HDL, involving STAT3-mediated decrease of miR-34b and miR-337 expression.

Identification of Differential Transcriptional Patterns in Primary and Secondary Hyperparathyroidism.

Context: Hyperparathyroidism is associated with hypercalcemia and the excess of parathyroid hormone secretion; however, the alterations in molecular pattern of functional genes during parathyroid tumorigenesis have not been unraveled. We aimed at establishing transcriptional patterns of normal and pathological parathyroid glands (PGs) in sporadic primary (HPT1) and secondary hyperparathyroidism (HPT2). Objective: To evaluate dynamic alterations in molecular patterns as a function of the type of PG pathology, a comparative transcript analysis was conducted in subgroups of healthy samples, sporadic HPT1 adenoma and hyperplasia, and HPT2. Design: Normal, adenomatous, HPT1, and HPT2 hyperplastic PG formalin-fixed paraffin-embedded samples were subjected to NanoString analysis. In silico microRNA (miRNA) analyses and messenger RNA-miRNA network in PG pathologies were conducted. Individual messenger RNA and miRNA levels were assessed in snap-frozen PG samples. Results: The expression levels of c-MET, MYC, TIMP1, and clock genes NFIL3 and PER1 were significantly altered in HPT1 adenoma compared with normal PG tissue when assessed by NanoString and quantitative reverse transcription polymerase chain reaction. RET was affected in HPT1 hyperplasia, whereas CaSR and VDR transcripts were downregulated in HPT2 hyperplastic PG tissue. CDH1, c-MET, MYC, and CaSR were altered in adenoma compared with hyperplasia. Correlation analyses suggest that c-MET, MYC, and NFIL3 exhibit collective expression level changes associated with HPT1 adenoma development. miRNAs, predicted in silico to target these genes, did not exhibit a clear tendency upon experimental validation. Conclusions: The presented gene expression analysis provides a differential molecular characterization of PG adenoma and hyperplasia pathologies, advancing our understanding of their etiology.

High-density lipoprotein from end-stage renal disease patients exhibits superior cardioprotection and increase in sphingosine-1-phosphate.

BACKGROUND: Chronic kidney disease (CKD) exacerbates the risk of death due to cardiovascular disease (CVD). Modifications to blood lipid metabolism which manifest as increases in circulating triglycerides and reductions in high-density lipoprotein (HDL) cholesterol are thought to contribute to increased risk. In CKD patients, higher HDL cholesterol levels were not associated with reduced mortality risk. Recent research has revealed numerous mechanisms by which HDL could favourably influence CVD risk. In this study, we compared plasma levels of sphingosine-1-phosphate (S1P), HDL-associated S1P (HDL-S1P) and HDL-mediated protection against oxidative stress between CKD and control patients. METHODS: High-density lipoprotein was individually isolated from 20 CKD patients and 20 controls. Plasma S1P, apolipoprotein M (apoM) concentrations, HDL-S1P content and the capacity of HDL to protect cardiomyocytes against doxorubicin-induced oxidative stress in vitro were measured. RESULTS: Chronic kidney disease patients showed a typical profile with significant reductions in plasma HDL cholesterol and albumin and an increase in triglycerides and pro-inflammatory cytokines (TNF-alpha and IL-6). Unexpectedly, HDL-S1P content (P = .001) and HDL cardioprotective capacity (P = .034) were increased significantly in CKD patients. Linear regression analysis of which factors could influence HDL-S1P content showed an independent, negative and positive association with plasma albumin and apoM levels, respectively. DISCUSSION: The novel and unexpected observation in this study is that uremic HDL is more effective than control HDL for protecting cardiomyocytes against oxidative stress. It is explained by its higher S1P content which we previously demonstrated to be the determinant of HDL-mediated cardioprotective capacity. Interestingly, lower concentrations of albumin in CKD are associated with higher HDL-S1P.

High-density lipoprotein-associated sphingosine-1-phosphate activity in heterozygous familial hypercholesterolaemia.

BACKGROUND: Patients with heterozygous familial hypercholesterolaemia (FH) suffer from high plasma cholesterol and an environment of increased oxidative stress. We examined its potential effects on high-density lipoprotein (HDL)-associated sphingosine-1-phosphate (S1P) content (HDL-S1P) and HDL-mediated protection against oxidative stress, both with and without statin treatment. MATERIALS AND METHODS: In a case-control study, HDL was isolated from 12 FH patients with and without statin treatment and from 12 healthy controls. The HDL-S1P content and the capacity of HDL to protect cardiomyocytes against oxidative stress in vitro were measured. RESULTS: HDL-associated S1P was significantly correlated with cell protection, but not with HDL-cholesterol or apolipoprotein AI. The latter did not correlate with HDL-mediated cell protection. Neither the HDL-S1P content nor HDL protective capacity differed between nontreated FH patients and controls. The relative amounts of apolipoprotein AI and apolipoprotein M were similar between controls and FH patients. Statin treatment had no effect on any of these measures. CONCLUSIONS: The FH environment is not detrimental to HDL-S1P content or HDL-S1P-mediated cell protection. Statin treatment does not modulate HDL function in this regard.

Diabetes Mellitus Is Associated With Reduced High-Density Lipoprotein Sphingosine-1-Phosphate Content and Impaired High-Density Lipoprotein Cardiac Cell Protection.

OBJECTIVE: The dyslipidemia of type 2 diabetes mellitus has multiple etiologies and impairs lipoprotein functionality, thereby increasing risk for cardiovascular disease. High-density lipoproteins (HDLs) have several beneficial effects, notably protecting the heart from myocardial ischemia. We hypothesized that glycation of HDL could compromise this cardioprotective effect. APPROACH AND RESULTS: We used in vitro (cardiomyocytes) and ex vivo (whole heart) models subjected to oxidative stress together with HDL isolated from diabetic patients and nondiabetic HDL glycated in vitro (methylglyoxal). Diabetic and in vitro glycated HDL were less effective (P<0.05) than control HDL in protecting from oxidative stress. Protection was significantly, inversely correlated with the degree of in vitro glycation (P<0.001) and the levels of hemoglobin A1c in diabetic patients (P<0.007). The ability to activate protective, intracellular survival pathways involving Akt, Stat3, and Erk1/2 was significantly reduced (P<0.05) using glycated HDL. Glycation reduced the sphingosine-1-phosphate (S1P) content of HDL, whereas the S1P concentrations of diabetic HDL were inversely correlated with hemoglobin A1c (P<0.005). The S1P contents of in vitro glycated and diabetic HDL were significantly, positively correlated (both <0.01) with cardiomyocyte survival during oxidative stress. Adding S1P to diabetic HDL increased its S1P content and restored its cardioprotective function. CONCLUSIONS: Our data demonstrate that glycation can reduce the S1P content of HDL, leading to increased cardiomyocyte cell death because of less effective activation of intracellular survival pathways. It has important implications for the functionality of HDL in diabetes mellitus because HDL-S1P has several beneficial effects on the vasculature.

Improving reconstituted HDL composition for efficient post-ischemic reduction of ischemia reperfusion injury.

BACKGROUND: New evidence shows that high density lipoproteins (HDL) have protective effects beyond their role in reverse cholesterol transport. Reconstituted HDL (rHDL) offer an attractive means of clinically exploiting these novel effects including cardioprotection against ischemia reperfusion injury (IRI). However, basic rHDL composition is limited to apolipoprotein AI (apoAI) and phospholipids; addition of bioactive compound may enhance its beneficial effects. OBJECTIVE: The aim of this study was to investigate the role of rHDL in post-ischemic model, and to analyze the potential impact of sphingosine-1-phosphate (S1P) in rHDL formulations. METHODS AND RESULTS: The impact of HDL on IRI was investigated using complementary in vivo, ex vivo and in vitro IRI models. Acute post-ischemic treatment with native HDL significantly reduced infarct size and cell death in the ex vivo, isolated heart (Langendorff) model and the in vivo model (-48%, p<0.01). Treatment with rHDL of basic formulation (apoAI + phospholipids) had a non-significant impact on cell death in vitro and on the infarct size ex vivo and in vivo. In contrast, rHDL containing S1P had a highly significant, protective influence ex vivo, and in vivo (-50%, p<0.01). This impact was comparable with the effects observed with native HDL. Pro-survival signaling proteins, Akt, STAT3 and ERK1/2 were similarly activated by HDL and rHDL containing S1P both in vitro (isolated cardiomyocytes) and in vivo. CONCLUSION: HDL afford protection against IRI in a clinically relevant model (post-ischemia). rHDL is significantly protective if supplemented with S1P. The protective impact of HDL appears to target directly the cardiomyocyte.

The scavenger receptor class B, type I is a primary determinant of paraoxonase-1 association with high-density lipoproteins.

OBJECTIVE: To examine the contribution of the scavenger receptor (SR) BI to the mechanism by which high-density lipoprotein (HDL) acquires paraoxonase-1 (PON1). METHODS AND RESULTS: Serum PON1 activity contributes to the antioxidant capacity of HDLs and is suggested to be an independent risk factor for atherosclerosis. The association of PON1 with HDL is a major determinant of its serum activity levels. PON1 secretion was studied in stably transfected Chinese hamster ovary and HepG2 models. Complementary analyses were performed in transgenic models. Modulation of SR-BI expression, by SR-BI small and interfering RNA knockdown and pharmacologically, correlated with significant changes (P<0.01) in PON1 secretion to HDLs and very-low-density lipoproteins. Block lipid transport-1 (BLT1), which increases the affinity of HDL for SR-BI without modulating its expression, was associated with significant increases in secretion. Downregulating postsynaptic density 95/disc-large/zona occludens kinase in HepG2 reduced cell SR-BI protein and lowered enzyme secretion. Serum PON1 activity was significantly reduced in postsynaptic density 95/disc-large/zona occludens kinase knockout mice. CONCLUSIONS: The present study identifies SR-BI as a major determinant of the capacity of HDL to acquire PON1. It reinforces the concept of the receptor as a docking molecule, allowing communication between HDL and the cell, and extends the importance of SR-BI to HDL metabolism and function.

Expression of the hepatic Niemann-Pick C1 like 1 protein gene is sensitive to rosuvastatin treatment of primary human hepatocytes.

Statins act by reducing hepatic cholesterol synthesis, thus stimulating uptake of serum cholesterol. Statin therapy modulates a number of genes involved in hepatic cholesterol homeostasis. These have rarely been analyzed simultaneously in the same experimental setting, with virtually no studies of primary human hepatocytes. This study analyzed the efficacy of rosuvastatin in the coordinated regulation of a number of genes implicated in cholesterol metabolism in primary human hepatocytes. Expression of five cholesterol-related genes were significantly upregulated, notably the Niemann-Pick C1 like 1 protein, for whom functional studies have been essentially limited to the intestine. Two genes were significantly downregulated, including sterol recognition element binding protein-1 gene that is implicated in control of hepatic lipogenesis. The results show the coordinated regulation of several genes implicated in hepatic cholesterol homeostasis and suggest therapeutic targets that could complement that clinical action of statins.

Paraoxonase-1 promoter polymorphism C--107T and serum apolipoprotein AI interact to modulate serum paraoxonase-1 status.

OBJECTIVES: The objective was to examine the hypothesis that modifications to paraoxonase-1 specific activity (SP, activity per unit mass peptide) could contribute to serum paraoxonase-1 status, a determinant of the clinical efficacy of the enzyme. METHODS: Enzyme activities and concentrations were determined in a large population (n=912) of patients and controls. SP were subsequently examined as a function of paraoxonase-1 gene polymorphisms, plasma lipids and lipoproteins, and physiological and pathophysiological parameters. RESULTS: Pathophysiological parameters (diabetes, metabolic syndrome, smoking, aging) did not promote variations in paraoxonase-1 SP, whilst coronary disease lowered SP (P<0.003). No serum lipid, apolipoprotein or lipoprotein component had an impact on specific activity, with the exception of apolipoprotein AI (P<0.005, both substrates). The paraoxonase-1 promoter C--107T and Q192R polymorphisms influenced SP and, together with apolipoprotein AI, were highly significant, independent determinants in regression models. There was an interaction between apolipoprotein AI and the C--107T polymorphism, which significantly modulated SP and serum paraoxonase-1 status. CONCLUSIONS: Enzyme inactivation giving rise to modulated activity per unit mass of peptide is not a major contributor to pathological effects of disease on serum paraoxonase-1 status. The C--107T polymorphism and serum apolipoprotein AI have major impacts individually on SP and also provide an example of gene-environment interaction to modulate such activities. These effects accentuate the differences between--107C and--107T allele carriers in terms of serum paraoxonase-1 status. The data underline the complexity of the factors that determine serum paraoxonase-1 status and suggest that the latter would benefit from therapeutic modulation of serum high density lipoproteins.

Paraoxonase-1 promoter haplotypes and serum paraoxonase: a predominant role for polymorphic position - 107, implicating the Sp1 transcription factor.

Accumulating data suggest that paraoxonase-1 (PON1) is a primary determinant of the antioxidant and anti-inflammatory capacities of high-density lipoproteins (HDLs). Variations in HDLs and PON1 have been shown to influence the functions of both. There is a wide spectrum of serum PON1 mass in humans, to which promoter polymorphisms make an important contribution. The present studies attempted to define: (i) the relevance in vivo of promoter polymorphisms by analysing haplotype structure; and (ii) molecular mechanisms implicated in promoter activity. Highly significant differences (P <0.0001) in serum mass and activity were observed as a function of haplotype sequence. Of three promoter polymorphisms (-107, -824 and -907), the -107 site was shown to be of predominant importance to serum PON1. Significant increases in serum PON1 mass and activities between haplotype subgroups could be explained by unit increases in the number of high-expresser variants of the -107 site (-107C) alone. No significant contribution was observed for the -824 and -907 sites. The coding-region Leu(55)-->Met (L55M) polymorphism made an independent contribution to serum PON1 mass, which may account for variations in serum PON1 mass and activity within haplotype subgroups defined by the -107 site. A molecular basis for the effect of the -107 polymorphism on serum PON1 was indicated by the greater affinity of the high-expresser variant (-107C) for hepatocyte nuclear extracts, indicating higher affinity for transcription factors. Competition studies with oligonucleotides representing the consensus (and mutated) sequence for Sp1, and the use of Sp1 antibodies, confirmed formation of complexes between the transcription factor and the PON1 promoter during incubation with nuclear extracts. The data underline the importance of the region containing the C(-107)T polymorphism for gene expression in vivo. Differences in the affinity of the -107C and -107T polymorphic fragments for nuclear extracts have been demonstrated, and coincide with their impact on gene expression. A potential role for the transcription factor Sp1 has been demonstrated, which is consistent with the disruption of an Sp1 recognition sequence by the -107 polymorphism.

Paraoxonase-1 L55M polymorphism is associated with an abnormal oral glucose tolerance test and differentiates high risk coronary disease families.

Polymorphisms of the gene for the antioxidant enzyme, paraoxonase-1 (PON1), have been identified as risk factors for coronary disease (CHD), notably in diabetic patients. The polymorphisms have also been linked with other diabetic complications. The present study analyzed glucose metabolism as a function of PON1 polymorphisms in young healthy nondiabetic men from families with premature CHD and matched controls. The L55M PON1 polymorphism was independently associated with the glucose response to an oral glucose tolerance test. LL homozygotes had significantly impaired glucose disposal (P = 0.0007) compared with (LM+MM) genotypes. It was particularly marked for subjects from high CHD risk families and differentiated them from matched controls (P = 0.049). The area under the glucose curve (P = 0.0036) and the time to peak glucose value (P = 0.026) were significantly higher in the LL carriers, whereas the insulin response was slower (P = 0.013). Insulin resistance did not differ between L55M genotypes. There was a trend for reduced pancreatic beta-cell function as measured by glucose-induced insulin secretion (LL vs. LM vs. MM, 20.26 vs. 23.74 vs. 25.60; P = 0.077). The frequency of the L55 allele decreased significantly (P = 0.028) across regions defining a north-south European axis. No significant differences for the glucose response or case-control populations were observed as a function of the PON1 Q192R polymorphism. The study demonstrates an association between PON1 gene polymorphisms and glucose metabolism. The L55M-glucose interaction differentiated offspring of high CHD risk families, suggesting that it may be of particular relevance for vascular disease and possibly other diabetic complications.