Lipid Subspecies Research Articles

Circulating total and H-specific GDF15 levels are elevated in subjects with MASLD but not in hyperlipidemic but otherwise metabolically healthy subjects with obesity

BackgroundGrowth differentiation factor 15 (GDF15) is a mitokine, the role of which, total or H-specific, in modulating energy metabolism and homeostasis in obesity-related diseases, such as metabolic dysfunction associated steatotic liver disease (MASLD), has not been fully elucidated in adult humans. We aimed to investigate the fasting and stimulated levels of GDF15, total and H-specific, glucose-dependent insulinotropic polypeptide (GIP) and C-peptide, in two physiology interventional studies: one focusing on obesity, and the other on MASLD.MethodsStudy 1 investigated individuals with normal weight or with obesity, undergoing a 3-h mixed meal test (MMT); and study 2, examined adults with MASLD and controls undergoing a 120-min oral glucose tolerance test (OGTT). Exploratory correlations of total and H-specific GDF15 with clinical, hormonal and metabolomic/lipidomic parameters were also performed.ResultsIn study 1, 15 individuals were included per weight group. Fasting and postprandial total and H-specific GDF15 were similar between groups, whereas GIP was markedly higher in leaner individuals and was upregulated following a MMT. Baseline and postprandial C-peptide were markedly elevated in people with obesity compared with lean subjects. GIP was higher in leaner individuals and was upregulated after a MMT, while C-peptide and its overall AUC after a MMT was markedly elevated in people with obesity compared with lean subjects. In study 2, 27 individuals were evaluated. Fasting total GDF15 was similar, but postprandial total GDF15 levels were significantly higher in MASLD patients compared to controls. GIP and C-peptide remained unaffected. The postprandial course of GDF15 was clustered among those of triglycerides and molecules of the alanine cycle, was robustly elevated under MASLD, and constituted the most notable differentiating molecule between healthy and MASLD status. We also present robust positive correlations of the incremental area under the curve of total and H-specific GDF15 with a plethora of lipid subspecies, which remained significant after adjusting for confounders.ConclusionSerum GDF15 levels do not differ in relation to weight status in hyperlipidemic but otherwise metabolically healthy individuals. In contrast, GDF15 levels are significantly increased in MASLD patients at baseline and they remain significantly higher compared to healthy participants during OGTT, pointing to a role for GDF15 as a mitokine with important roles in the pathophysiology and possibly therapeutics of MASLD.Trial registration ClinicalTrials.gov NCT03986684, NCT04430946.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus

BackgroundIn this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state.MethodsAn untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D.ResultsA total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D.ConclusionsOur findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.

Plasma Lipidomic Profiling Using Mass Spectrometry for Multiple Sclerosis Diagnosis and Disease Activity Stratification (LipidMS).

This investigation explores the potential of plasma lipidomic signatures for aiding in the diagnosis of Multiple Sclerosis (MS) and evaluating the clinical course and disease activity of diseased patients. Plasma samples from 60 patients with MS (PwMS) were clinically stratified to either a relapsing-remitting (RRMS) or a chronic progressive MS course and 60 age-matched controls were analyzed using state-of-the-art direct infusion quantitative shotgun lipidomics. To account for potential confounders, data were filtered for age and BMI correlations. The statistical analysis employed supervised and unsupervised multivariate data analysis techniques, including a principal component analysis (PCA), a partial least squares discriminant analysis (oPLS-DA) and a random forest (RF). To determine whether the significant absolute differences in the lipid subspecies have a relevant effect on the overall composition of the respective lipid classes, we introduce a class composition visualization (CCV). We identified 670 lipids across 16 classes. PwMS showed a significant increase in diacylglycerols (DAG), with DAG 16:0;0_18:1;0 being proven to be the lipid with the highest predictive ability for MS as determined by RF. The alterations in the phosphatidylethanolamines (PE) were mainly linked to RRMS while the alterations in the ether-bound PEs (PE O-) were found in chronic progressive MS. The amount of CE species was reduced in the CPMS cohort whereas TAG species were reduced in the RRMS patients, both lipid classes being relevant in lipid storage. Combining the above mentioned data analyses, distinct lipidomic signatures were isolated and shown to be correlated with clinical phenotypes. Our study suggests that specific plasma lipid profiles are not merely associated with the diagnosis of MS but instead point toward distinct clinical features in the individual patient paving the way for personalized therapy and an enhanced understanding of MS pathology.

Diacylglycerols and Lysophosphatidic Acid, Enriched on Lipoprotein(a), Contribute to Monocyte Inflammation.

Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1β after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.

Changes in lipids and inflammation in adults with super-refractory status epilepticus on a ketogenic diet.

Introduction: This study aims to test the hypothesis that increased ketone body production resulting from a ketogenic diet (KD) will correlate with reductions in pro-inflammatory cytokines and lipid subspecies and improved clinical outcomes in adults treated with an adjunctive ketogenic diet for super-refractory status epilepticus (SRSE). Methods: Adults (18years or older) were treated with a 4:1 (fat: carbohydrate and protein) ratio of enteral KD as adjunctive therapy to pharmacologic seizure suppression in SRSE. Blood and urine samples and clinical measurements were collected at baseline (n = 10), after 1week (n = 8), and after 2weeks of KD (n = 5). In addition, urine acetoacetate, serum β-hydroxybutyrate, lipidomics, pro-inflammatory cytokines (IL-1β and IL-6), chemokines (CCL3, CCL4, and CXCL13), and clinical measurements were obtained at these three time points. Univariate and multivariate data analyses were performed to determine the correlation between ketone body production and circulating lipids, inflammatory biomarkers, and clinical outcomes. Results: Changes in lipids included an increase in ceramides, mono-hexosylceramide, sphingomyelin, phosphocholine, and phosphoserines, and there was a significant reduction in pro-inflammatory mediators, IL-6 and CXCL13, seen at 1 and 2weeks of KD. Higher blood β-hydroxybutyrate levels at baseline correlated with better clinical outcomes; however, ketone body production did not correlate with other variables during treatment. Higher chemokine CCL3 levels following treatment correlated with a longer stay in the intensive care unit and a higher modified Rankin Scale score (worse neurologic disability) at discharge and 6-month follow up. Discussion: Adults receiving an adjunctive enteral ketogenic diet for super-refractory status epilepticus exhibit alterations in select pro-inflammatory cytokines and lipid species that may predict their response to treatment.

Untargeted and Targeted Blood Lipidomic Signature Profile of Gestational Alcohol Exposure.

Alcohol consumption has a close relationship with blood lipid levels in a nonpregnant state, with a myriad of effects on the liver; however, little is known about the interaction of alcohol and lipids in the context of fetal alcohol spectrum disorders (FASD). We herein aimed to determine the effect of alcohol on the lipid profile in a pregnant rat model, with a focus on FASD. Dry blood spots (50 µL) were obtained from rat maternal blood collected on gestational day (GD) 20, two hours after the last binge alcohol exposure (4.5 g/kg, GD 5-10; 6 g/kg, GD 11-20). The samples were then analyzed using high-throughput untargeted and targeted lipid profiling via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In untargeted lipidomics, 73 of 315 identified lipids were altered in the alcohol group compared to the pair-fed controls; 67 were downregulated and 6 were upregulated. In targeted analysis, 57 of the 260 studied lipid subspecies were altered, including Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylglycerol (PG), Phosphatidic Acid (PA), Phosphatidylinositol (PI), and Phosphatidylserine (PS); 36 of these were downregulated and 21 lipid subspecies were upregulated. These findings suggest alcohol-induced dysregulation of lipids in the maternal blood of rats and provide novel insights into possible FASD mechanisms.

Ether lipid transfer across the blood-brain and placental barriers does not improve by inactivation of the most abundant ABC transporters

Phospholipid transport from the periphery to the brain is an understudied topic. When certain lipid species are deficient due to impaired synthesis, though, transfer across the blood-brain barrier is essential for replenishing lipids in the brain. For example, the deficiency in plasmalogens, the most abundant ether lipids in mammals, has detrimental effects on the brain, which is a major issue in inherited peroxisomal disorders but also contributes to more common disorders like Alzheimer’s disease. Oral administration of alkylglycerols like batyl alcohol, which carry a pre-formed ether bond, enables replenishment of ether lipids in various peripheral tissues. However, plasmalogen deficiency in the brain cannot be overcome by this approach. Here, we tried to increase cerebral plasmalogen uptake by modulating the efflux transport across the blood-brain barrier. We hypothesized, based on previous literature, that at least some ether lipid species readily enter endothelial cells of the barrier through the transporter MFSD2A but are re-exported by ATP-binding cassette (ABC) transporters. By crossbreeding Mdr1a-/-/Mdr1b-/-/Bcrp-/- and ether lipid-deficient Gnpat-/- mice as well as pharmacological inhibition with MK-571 to inactivate the major ABC transporters at the blood-brain barrier, we evaluated the potential of combined ABC transporter inhibition and oral batyl alcohol administration for the treatment of plasmalogen deficiency. We found that even in the absence of the most abundant ABC transporters, batyl alcohol supplementation did not restore plasmalogen levels in the brain, despite the presence of a wide spectrum of ether lipid subspecies in the plasma as demonstrated by lipidomic analysis. Surprisingly, batyl alcohol treatment of pregnant Gnpat+/- dams had beneficial effects on the plasmalogen levels of Gnpat-/- offspring with defective ether lipid biosynthesis, independently of ABC transporter status at the placental barrier. Our results underline the autonomy of brain lipid homeostasis and indicate that peripheral supplementation of ether lipids is not sufficient to supply the brain with larger amounts of plasmalogens. Yet, the findings suggest that alkylglycerol treatment during pregnancy may pose a viable option to ameliorate some of the severe developmental defects of inborn ether lipid deficiency.

Distinct cord blood C-peptide, adipokine, and lipidomic signatures by in utero HIV exposure.

Background:Early-life metabolic derangements in HIV-exposed uninfected (HEU) infants have been reported.Methods:Pregnant women with HIV and HIV-uninfected pregnant women were enrolled with their newborns in a US cohort from 2011–15. We measured cord insulin, C-peptide, and metabolic cytokines of HEU and HIV-unexposed uninfected (HUU) newborns using ELISA and metabolites, lipid subspecies, and eicosanoids via liquid chromatography/mass spectrometry. Linear regression was employed to assess the association of intrauterine HIV/ART with insulin and C-peptide. Graphical lasso regression was used to identify differences between metabolite/lipid subspecies networks associated with C-peptide.Results:Of 118 infants, 56 were HEU, ART-exposed. In adjusted analyses, mean cord insulin (β=0.295, p=0.03) and C-peptide (β=0.522, p<0.01) were significantly higher in HEU vs. HUU newborns. HEU neonates exhibited primarily positive associations between complex lipids and C-peptide, indicative of fuel storage, and augmented associations between cord eicosanoids and cytokines. HUU neonates exhibited negative associations with lipids and C-peptide indicative of increased fuel utilization.Conclusion:Higher cord insulin and C-peptide in HEU vs. HUU newborns as well as differences in cord metabolites, metabolic-related cytokines, and eicosanoids may reflect a propensity for fuel storage and an inflammatory milieu suggestive of fetal metabolic changes associated with in utero HIV/ART exposure.

Mineralocorticoid receptor modulation by dietary sodium influences NAFLD development in mice

Introduction and ObjectivesNonalcoholic-fatty-liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome (MetS). Mineralocorticoid receptor (MR) activation is associated with increased risk of MetS but few studies have assessed the role of liver MR on NAFLD. We aimed to evaluate the effect of MR modulation by sodium intake in liver injury in experimental models of NAFLD. Materials and MethodsC57BL/6J mice were fed either a high-fat-diet (HFD) or a choline/methionine deficient (MCD) diet with different sodium concentrations. Hepatic concentration of lipid species, serum aldosterone levels, expression of MR, proinflammatory and profibrotic markers and liver histology were assessed. ResultsMice fed with High-Na+/HFD showed a lower MR expression in liver (p = 0.01) and less steatosis on histology (p = 0.04). Consistently, animals from this group exhibited lower levels of serum aldosterone (p = 0.028) and lower hepatic triglyceride content (p = 0.008). This associated to a reduced expression of lipogenic genes, significant changes in lipid subspecies, lower HOMA-IR (p < 0.05), and lower expression of pro-inflammatory and profibrotic markers compared to those mice fed a Low-Na+/HFD. Additionally, mice fed a High-Na+/HFD showed higher expression of salt-inducible kinase (SIK)-1 and lower expression of serum-and-glucocorticoid-inducible kinase (SGK)-1. Similar results were observed with the MCD diet model. ConclusionWe identified in two experimental models of NAFLD that High-Na+ diet content is associated to lower serum aldosterone levels and hepatic MR downregulation, associated to decreased steatosis and reduced de novo hepatic lipogenesis, proinflammatory and profibrotic markers. Decreased activation of hepatic MR seems to generate beneficial downstream inhibition of lipogenesis in experimental NAFLD.

Human epidermal stem cell differentiation is modulated by specific lipid subspecies

While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these-ceramides and glucosylceramides-induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.

Non-invasive assessment of hepatic lipid subspecies matched with non-alcoholic fatty liver disease phenotype

Background and aimsNonalcoholic fatty liver disease (NAFLD) is characterized by excessive hepatic fat accumulation. Increased hepatic saturated fats and decreased hepatic polyunsaturated fats may be particularly lipotoxic, contributing to metabolic dysfunction. We compared hepatic lipid subspecies in adults with and without NAFLD, and examined links with hallmark metabolic and clinical characteristics of NAFLD. Methods and resultsNineteen adults with NAFLD (total hepatic fat:18.8 ± 0.1%) were compared to sixteen adults without NAFLD (total hepatic fat: 2.1 ± 0.01%). 1H-MRS was used to assess hepatic lipid subspecies. Methyl, allylic, methylene, and diallylic proton peaks were measured. Saturation, unsaturation, and polyunsaturation indices were calculated. Whole-body phenotyping in a subset of participants included insulin sensitivity (40 mU/m2 hyperinsulinemic-euglycemic clamps), CT-measured abdominal adipose tissue depots, exercise capacity, and serum lipid profiles. Participants with NAFLD exhibited more saturated and less unsaturated hepatic fat, accompanied by increased insulin resistance, total and visceral adiposity, triglycerides, and reduced exercise capacity compared to controls (all P < 0.05). All proton lipid peaks were related to insulin resistance and hypertriglyceridemia (P < 0.05). ConclusionParticipants with NAFLD preferentially stored excess hepatic lipids as saturated fat, at the expense of unsaturated fat, compared to controls. This hepatic lipid profile was accompanied by an unhealthy metabolic phenotype.

Effect of a Mediterranean Diet with Varying Quantities of Lean Beef on non-HDL and HDL Lipid Particles: A Randomized Controlled Feeding Cross-Over Trial (OR36-05-19)

ObjectivesTo evaluate the effects of a Mediterranean (Med) style diet with varying quantities of lean beef on non-HDL and HDL lipid subspecies. We hypothesized that a Med diet with lean beef would confer cardiovascular benefits beyond a standard lipid panel and be superior to an average American diet (AAD). MethodsWe conducted a multicenter, 4-period controlled feeding, randomized crossover study at Penn State University and USDA-Beltsville to evaluate the effects of a Med diet (CHO 42%, PRO 17%, FAT 41%, SFA 8%, MUFA 26%, PUFA 8%) with different quantities of lean beef (0.5, 2.5 and 5.5 oz/day) compared to an average American diet (AAD; CHO 52%, PRO 15%, FAT 33%, SFA 12%, MUFA 13%, PUFA 8%) on CVD risk factors. Participants (n = 66) included generally healthy normal to overweight/obese males and females (BMI = 20–38 kg/m2) 30 to 67 years. Participants were randomized to each of the 4 diets for 4 weeks with an approximate 2-week break between treatments. Fasting blood samples were collected on two consecutive days at baseline (start of study), and at the end of each diet period. ResultsAll three Med diets decreased LDL-C versus AAD (–10.5 ± 2.0, –9.0 ± 2.0, –6.8 ± 2.0 mg/dL, P < 0.0001 for the 0.5, 2.5 and 5.5 oz., respectively). All Med diets elicited similar reductions in total LDL particle number and large particle number (P < 0.01 for both) compared to baseline, however only the Med diets with 0.5 oz./day (–91.2 ± 23 nmol/L) and 2.5 oz./day (–85.3 ± 23 nmol/L) were significantly decreased versus AAD (P < 0.01). There were no treatment differences for IDL or small LDL particles. All diets reduced HDL-C and HDL particle number from baseline (P < 0.01). ConclusionsA healthy Med style diet containing 2.5 oz./day of lean beef elicits similar improvements in lipid subspecies compared to a traditional Med style diet containing 0.5 oz./day. The Med style diet containing 5.5 oz./day of lean beef had similar effects on lipid subspecies to the AAD, therefore our findings suggest that £2.5 oz./day of lean beef can be included in a Med diet and not compromise the cardiovascular benefits of a Med diet. Funding SourcesThis study was funded by the Beef Checkoff. This study also was supported by the USDA, ARS and the Penn State Clinical and Translational Research Institute.

Ceramide as a risk factor of nonalcoholic fatty liver disease

At present, one of the factors of the pathogenesis of nonalcoholic fatty liver disease, which deserve particular attention, is the accumulation of specific lipid subspecies in the liver – ceramides. This article shows what the ceramides are, how they are formed, and how they influence on apoptosis and development of insulin.

Effect of high fat diet on phenotype, brain transcriptome and lipidome in Alzheimer\u2019s model mice

We examined the effect of chronic high fat diet (HFD) on amyloid deposition and cognition of 12-months old APP23 mice, and correlated the phenotype to brain transcriptome and lipidome. HFD significantly increased amyloid plaques and worsened cognitive performance compared to mice on normal diet (ND). RNA-seq results revealed that in HFD mice there was an increased expression of genes related to immune response, such as Trem2 and Tyrobp. We found a significant increase of TREM2 immunoreactivity in the cortex in response to HFD, most pronounced in female mice that correlated to the amyloid pathology. Down-regulated by HFD were genes related to neuron projections and synaptic transmission in agreement to the significantly deteriorated neurite morphology and cognition in these mice. To examine the effect of the diet on the brain lipidome, we performed Shotgun Lipidomics. While there was no difference in the total amounts of phospholipids of each class, we revealed that the levels of 24 lipid sub-species in the brain were significantly modulated by HFD. Network visualization of correlated lipids demonstrated overall imbalance with most prominent effect on cardiolipin molecular sub-species. This integrative approach demonstrates that HFD elicits a complex response at molecular, cellular and system levels in the CNS.

Circulating MicroRNA-122 Is Associated With the Risk of New-Onset Metabolic Syndrome and Type 2 Diabetes.

MicroRNA-122 (miR-122) is abundant in the liver and involved in lipid homeostasis, but its relevance to the long-term risk of developing metabolic disorders is unknown. We therefore measured circulating miR-122 in the prospective population-based Bruneck Study (n = 810; survey year 1995). Circulating miR-122 was associated with prevalent insulin resistance, obesity, metabolic syndrome, type 2 diabetes, and an adverse lipid profile. Among 92 plasma proteins and 135 lipid subspecies quantified with mass spectrometry, it correlated inversely with zinc-α-2-glycoprotein and positively with afamin, complement factor H, VLDL-associated apolipoproteins, and lipid subspecies containing monounsaturated and saturated fatty acids. Proteomics analysis of livers from antagomiR-122–treated mice revealed novel regulators of hepatic lipid metabolism that are responsive to miR-122 inhibition. In the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT, n = 155), 12-month atorvastatin reduced circulating miR-122. A similar response to atorvastatin was observed in mice and cultured murine hepatocytes. Over up to 15 years of follow-up in the Bruneck Study, multivariable adjusted risk ratios per one-SD higher log miR-122 were 1.60 (95% CI 1.30–1.96; P < 0.001) for metabolic syndrome and 1.37 (1.03–1.82; P = 0.021) for type 2 diabetes. In conclusion, circulating miR-122 is strongly associated with the risk of developing metabolic syndrome and type 2 diabetes in the general population.

Mendelian randomization to assess causal effects of blood lipids on coronary heart disease: lessons from the past and applications to the future.

Mendelian randomization is a technique for judging the causal impact of a risk factor on an outcome from observational data using genetic variants. Although evidence from Mendelian randomization for the effects of major lipids and lipoproteins on coronary heart disease (CHD) risk has been around for a relatively long time, new data resources and new methodological approaches have given fresh insight into these relationships. The lessons from these analyses are likely to be highly relevant when it comes to lipidomics and the analyses of lipid subspecies whose biology is less well understood. Although analyses of low-density lipoprotein cholesterol and lipoprotein(a) are unambiguous as there are genetic variants that associate exclusively with these risk factors and have well understood biology, analyses for triglycerides, and high-density lipoprotein cholesterol (HDL-c) are less clear. For example, a subset of genetic variants having specific associations with HDL-c are not associated with CHD risk, but an allele score including all variants associated with HDL-c does associate with CHD risk. Recently developed methods, such as multivariable Mendelian randomization, Mendelian randomization-Egger, and a weighted median method, suggest that the relationship between HDL-c and CHD risk is null, thus confirming experimental evidence. Robust methods for Mendelian randomization have important utility for understanding the causal relationships between major lipids and CHD risk, and are likely to play an important role in judging the causal relevance of lipid subspecies and other metabolites measured on high-dimensional phenotyping platforms.

Proteomic and lipidomic analyses of paraoxonase defined high density lipoprotein particles: Association of paraoxonase with the anti-coagulant, protein S.

Characterizing high density lipoprotein (HDL) particles and their relevance to HDL function is a major research objective. One aim is to identify functionally distinct particles. To try to limit both functional and compositional heterogeneity the present study focused on paraoxonase-1 (PON1) as a target for isolation of a minor HDL subfraction. Immunoaffinity techniques were applied to isolate PON1-containing HDL (P-HDL) and total HDL (T-HDL), which were subsequently characterized and compared. Analyses of the lipidomes showed significant differences between the fractions in the relative concentrations of individual lipid subspecies, notably reduced levels of unsaturated lysophosphatidylcholine (p < 0.05) in P-HDL (reflected in a significantly reduced total lysophosphatidylcholine polyunsaturated fatty acid content, p < 0.004). Significant differences were also observed for the proteomes. P-HDL was highly enriched in the anti-coagulant, vitamin K activated protein S (prot S) (p < 0.0001), and alpha2 macroglobulin (p < 0.01), compared to T-HDL. Conversely, procoagulant proteins kininogen 1 and histidine-rich glycoprotein were largely excluded from P-HDL. Immunoabsorption of PON1 from plasma significantly reduced prot S anti-coagulant activity. The P-HDL lipidome and proteome showed significant differences from T-HDL. Enrichment in anti-coagulation proteins indicates complementary functionalities within P-HDL particles and underlines their anti-atherosclerotic potential.

Abstract 17961: Circulating MicroRNA-122 is Associated With Incident Metabolic Syndrome and Type-2-diabetes

Background: MicroRNA-122 (miR-122) is highly abundant in the liver and has been implicated in lipid and glucose homeostasis. Methods: Circulating miR-122 levels were measured in the prospective population-based Bruneck Study (n=810) and the ASCOT trial (n=155 pre/post atorvastatin initiation). Using mass spectrometry, we measured 135 lipid subspecies and 93 plasma proteins in Bruneck participants. We also conducted mechanistic studies in pre-clinical models and cultured hepatocytes. We calculated risk ratios over up to 15 years follow-up for cardiometabolic diseases using pooled logistic and Cox regression, incorporating repeated miR-122 measurements. Results: In the Bruneck Study, miR-122 was associated with an adverse metabolic profile, including insulin resistance, obesity, and prevalent metabolic syndrome and diabetes. MiR-122 positively correlated with lipid subspecies containing fatty acids that can be derived from hepatic de novo lipogenesis. In mice, miR-122 inhibition with antagomiR treatment down-regulated hepatic enzymes implicated in de novo lipogenesis and cholesterol synthesis. In the ASCOT trial, 10mg atorvastatin treatment led to a reduction in circulating miR-122 levels. A similar statin response was observed in mice and in primary hepatocytes. In the Bruneck Study, miR-122 was closely related to circulating levels of afamin, zinc-alpha-2-glycoprotein, and a wide range of apolipoproteins. The adjusted relative risk per SD higher log miR-122 was 1.60 (1.30-1.96; P&lt;0.001) for metabolic syndrome, 1.37 (1.03-1.82; P=0.021) for type-2 diabetes, and 1.10 (0.90-1.33; P=0.330) for cardiovascular disease. Risk ratios were similar upon further adjustment, for women and men, and by clinical categories of adiposity. Conclusion: This study provides strong evidence for an involvement of miR-122 in glucose and lipid metabolism. High miR-122 levels correlate with an adverse lipid profile and multiple metabolic abnormalities and are associated with a higher risk of new-onset metabolic syndrome and diabetes in the general community.

Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manduca sexta: synthesis of sn-1,2-diacylglycerols.

The pathway for the adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerols in the adult Manduca sexta fat body was studied. Adult fat body lipids were labeled by feeding 5th instar larvae either with [9,10(n)-3H]oleic acid or [1(3)-3H] glycerol and after 32 days insects at the adult stage were used. This long-term prelabeling led to labeled fat body acylglycerols in which triacylglycerols comprised the main radioactive lipid component (95.5%), regardless of the radiolabeled compound used. Because the distribution of radioactivity among the lipid classes was very close to the mass distribution of the fat body lipid subspecies, it was concluded that homogeneous labeling of fat body lipids was obtained. After adipokinetic hormone treatment, an accumulation of radioactivity in the sn-1,2-diacylglycerol fraction was the only significant change found in the distribution of radioactivity among fat body lipids. The size of diacylglycerol pool increased 280% 60 min after adipokinetic hormone stimulation, whereas the fatty acid, monoacylglycerol and phosphatidic acid pool sizes remained constant. These results support the hypothesis that adipokinetic hormone-stimulated synthesis of sn-1,2-diacylglycerol in the fat body involves stereospecific hydrolysis of the triacylglycerol stores.