Lipidomics Reveals Myocardial Lipid Composition in a Murine Model of Insulin Resistance Induced by a High-Fat Diet.

Ectopic fat accumulation in non-adipose tissues is closely related to diabetes-related myocardial dysfunction. Nevertheless, the complete picture of the lipid metabolites involved in the metabolic-related myocardial alterations is not fully characterized. The aim of this study was to characterize the specific lipid profile in hearts in an animal model of obesity/insulin resistance induced by a high-fat diet (HFD). The cardiac lipidome profiles were assessed via liquid chromatography-mass spectrometry (LC-MS)/MS-MS and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging in hearts from C57BL/6J mice fed with an HFD or standard-diet (STD) for 12 weeks. Targeted lipidome analysis identified a total of 63 lipids (i.e., 48 triacylglycerols (TG), 5 diacylglycerols (DG), 1 sphingomyelin (SM), 3 phosphatidylcholines (PC), 1 DihydroPC, and 5 carnitines) modified in hearts from HFD-fed mice compared to animals fed with STD. Whereas most of the TG were up-regulated in hearts from animals fed with an HFD, most of the carnitines were down-regulated, thereby suggesting a reduction in the mitochondrial ß-oxidation. Roughly 30% of the identified metabolites were oxidated, pointing to an increase in lipid peroxidation. Cardiac lipidome was associated with a specific biochemical profile and a specific liver TG pattern. Overall, our study reveals a specific cardiac lipid fingerprint associated with metabolic alterations induced by HFD.

Lipidomics of triglyceride-rich lipoproteins derived from hyperlipidemic patients on inflammation.

BACKGROUND AND AIM: Triglyceride-rich lipoproteins (TRLs) can have an important role in atherosclerosis development due to their size and ability to penetrate the endothelium. While high plasma triglyceride (TG) levels and chronic inflammation are relevant in metabolic diseases, it remains unclear whether TGs are atherogenic or which TRL-TG-derived metabolites are responsible for inflammation. Here, we aimed to study the lipidome modifications of TRL particles enriched in TG in patients with hyperlipidemia and their associations with a proinflammatory status both in vivo and in vitro. METHODS: Using proton nuclear magnetic resonance (1 H-NMR), we analysed the plasma levels of glycoprotein acetyls and the TRL lipidomic profile of 307 patients with dyslipidemia. THP-1-derived macrophages were used as an in vitro model to explore the molecular inflammatory effects mediated by TRL. RESULTS: In vivo, higher TRL-TG levels were associated with higher circulating levels of NMR-measured glycoproteins (Glyc-A, Glyc-B and Glyc-F; p < .001). Lipidomic analysis showed that TRL-TG enrichment led to decreased cholesterol and phospholipid content (p < .01), an increase in omega-9, and a decrease in saturated fatty acids (p < .001). THP-1 macrophages exposed to increasing TRL particle concentrations augmented the secretion of IL-1ß and TNF-α, which varied based on particle composition. Particles with higher cholesterol and phospholipid contents exerted higher cytokine secretion. The activation of MAPK, Akt/NFκB, and caspase-1 was concurrent with this proinflammatory response. CONCLUSIONS: High TRL-TG levels are associated with a higher systemic inflammatory status and increased particle concentrations. In vitro, higher particle numbers increase proinflammatory cytokine secretion, with cholesterol and phospholipid-rich TRL being more proinflammatory.

Serum branch-chained amino acids are increased in type 2 diabetes and associated with atherosclerotic cardiovascular disease.

BACKGROUND AND AIM: Circulating biomarkers of metabolic and cardiovascular diseases can help in the early detection and prevention of those diseases. Using proton nuclear magnetic resonance (1H-NMR), we aimed to study the plasma levels of low-molecular-weight metabolites (LMWMs) in a cohort of 307 patients with metabolic diseases to assess their relationships with type-2 diabetes (T2D) and incident atherosclerotic cardiovascular disease (ASCVD). METHODS: We conducted a cross-sectional and prospective study. We included 307 patients attending the Lipid Unit of our University Hospital for the treatment of the following metabolic disturbances and associated disorders: T2D (73.9%), obesity (58.7%), and hypertension (55.1%). 1H-NMR was used to study the plasma levels of 13 LMWMs. LMWM serum concentrations were evaluated in patients with and without T2D. and the correlations with several parameters and their associations with T2D were analyzed. The association between LMWM levels at baseline and the development of ASCVD in patients with T2D after 10 years of follow-up was also evaluated. RESULTS: Among the LMWMs measured, the branched-chain amino acids (BCAAs) valine, leucine and isoleucine showed a positive association with several clinical and lipid-related biochemical parameters and inflammatory markers (p < 0.05). Likewise, these three BCAAS were associated with diabetes even after adjusting for covariates (p < 0.05). During the follow-up period of 10 years, 29 of the 185 patients with diabetes at baseline (15.68%) developed ASCVD. After adjusting for clinical covariates, baseline levels of valine and alanine were associated with the development of ASCVD (p < 0.05). CONCLUSION: Overall, our results indicated that plasma levels of LMWMs measured by 1H-NMR could be potential biomarkers associated with T2D. Moreover, alanine and valine can help in the early detection of the cardiovascular risk associated with this metabolic disease.

Fatty Acid Binding Proteins 3 and 4 Predict Both All-Cause and Cardiovascular Mortality in Subjects with Chronic Heart Failure and Type 2 Diabetes Mellitus.

Subjects with type 2 diabetes mellitus (T2D) are at increased risk for heart failure (HF). The cardiac-specific (FABP3) and adipose-tissue-specific (FABP4) types of the fatty acid binding proteins have been associated with both all-cause and cardiovascular (CV) mortality. The aim of this study was to explore the prognosis value of FABP3 and FABP4 in ambulatory subjects with chronic HF (CHF), with and without T2D. A prospective study involving 240 ambulatory CHF subjects was performed. Patients were followed-up for a mean of 5.78 ± 3.30 years and cause of death (if any) was recorded. Primary endpoints were defined as all-cause and CV death, and a composite endpoint that included CV death or hospitalization for HF was included as a secondary endpoint. Baseline serum samples were obtained and the serum FABP3 and FABP4 concentrations were assessed by sandwich enzyme-linked immunosorbent assay. Survival analysis was performed with multivariable Cox regressions, using Fine and Gray competing risks models when needed, to explore the prognostic value of FABP3 and FABP4 concentrations, adjusting for potential confounders. Type 2 diabetes mellitus was highly prevalent, accounting for 47.5% for total subjects with CHF. Subjects with T2D showed higher mortality rates (T2D: 69.30%; non-T2D: 50.79%, p = 0.004) and higher serum FABP3 (1829.3 (1104.9-3440.5) pg/mL vs. 1396.05 (820.3-2362.16) pg/mL, p = 0.007) and FABP4 (45.5 (27.6-79.8) ng/mL vs. 34.1 (24.09-55.3) ng/mL, p = 0.006) concentrations compared with non-T2D CHF subjects. In the whole study cohort, FABP3 was independently associated with all-cause death, and both FABP3 and FABP4 concentrations were associated with CV mortality. The predictive values of these two molecules for all-cause (FABP3: HR 1.25, 95% CI 1.09-1.44; p = 0.002. FABP4: HR 2.21, 95% CI 1.12-4.36; p = 0.023) and CV mortality (FABP3: HR 1.28, 95% CI 1.09-1.50; p = 0.002. FABP4: HR 4.19, 95% CI 2.21-7.95; p < 0.001) were only statistically significant in the subgroup of subjects with T2D. Notably, FABP4 (HR 2.07, 95% CI 1.11-3.87; p = 0.022), but not FABP3, also predicted the occurrence of the composite endpoint (death or hospitalization for HF) only in subjects with T2D. All these associations were not found in CHF subjects without T2D. Our findings support the usefulness of serum FABP3 and FABP4 concentrations as independent predictors for the occurrence of all-cause and CV mortality in ambulatory subjects with CHF with T2D.

DDR1 and Its Ligand, Collagen IV, Are Involved in In Vitro Oligodendrocyte Maturation.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase receptor expressed in epithelial cells from different tissues in which collagen binding activates pleiotropic functions. In the brain, DDR1 is mainly expressed in oligodendrocytes (OLs), the function of which is unclear. Whether collagen can activate DDR1 in OLs has not been studied. Here, we assessed the expression of DDR1 during in vitro OL differentiation, including collagen IV incubation, and the capability of collagen IV to induce DDR1 phosphorylation. Experiments were performed using two in vitro models of OL differentiation: OLs derived from adult rat neural stem cells (NSCs) and the HOG16 human oligodendroglial cell line. Immunocytofluorescence, western blotting, and ELISA were performed to analyze these questions. The differentiation of OLs from NSCs was addressed using oligodendrocyte transcription factor 2 (Olig2) and myelin basic protein (MBP). In HOG16 OLs, collagen IV induced DDR1 phosphorylation through slow and sustained kinetics. In NSC-derived OLs, DDR1 was found in a high proportion of differentiating cells (MBP+/Olig2+), but its protein expression was decreased in later stages. The addition of collagen IV did not change the number of DDR1+/MBP+ cells but did accelerate OL branching. Here, we provide the first demonstration that collagen IV mediates the phosphorylation of DDR1 in HOG16 cells and that the in vitro co-expression of DDR1 and MBP is associated with accelerated branching during the differentiation of primary OLs.

Unveiling the Role of the Fatty Acid Binding Protein 4 in the Metabolic-Associated Fatty Liver Disease.

Metabolic-associated fatty liver disease (MAFLD), the main cause of chronic liver disease worldwide, is a progressive disease ranging from fatty liver to steatohepatitis (metabolic-associated steatohepatitis; MASH). Nevertheless, it remains underdiagnosed due to the lack of effective non-invasive methods for its diagnosis and staging. Although MAFLD has been found in lean individuals, it is closely associated with obesity-related conditions. Adipose tissue is the main source of liver triglycerides and adipocytes act as endocrine organs releasing a large number of adipokines and pro-inflammatory mediators involved in MAFLD progression into bloodstream. Among the adipocyte-derived molecules, fatty acid binding protein 4 (FABP4) has been recently associated with fatty liver and additional features of advanced stages of MAFLD. Additionally, emerging data from preclinical studies propose FABP4 as a causal actor involved in the disease progression, rather than a mere biomarker for the disease. Therefore, the FABP4 regulation could be considered as a potential therapeutic strategy to MAFLD. Here, we review the current knowledge of FABP4 in MAFLD, as well as its potential role as a therapeutic target for this disease.

Relationship Between Fatty Acid Binding Protein 4 and Liver Fat in Individuals at Increased Cardiometabolic Risk.

Background: Liver steatosis is considered the onset of the non-alcoholic fatty liver disease (NAFLD), a major public health challenge. Nevertheless, NAFLD detection and diagnosis remain a difficult task. Fatty acid binding protein 4 (FABP4) has been proposed as potential biomarker for the ectopic fat accumulation in non-adipose tissues, although its role reflecting liver steatosis in metabolic patients is not fully explored. The aim of this study was to assess the relationship between FABP4 and the fatty liver index (FLI) in metabolic patients and to evaluate its potential role in the fatty liver disease. Methods: A cross-sectional study involving 389 participants at increased cardiometabolic risk was performed. FLI was calculated in order to assess liver fatty disease and a FLI ≥ 60 was considered to define liver steatosis. The serum FABP4 levels were assessed by using a sandwich enzyme-linked immunosorbent assay. Multivariable regression models were used to examine the associations of FABP4 with fatty liver after adjusting for demographic and clinical characteristics. Results: Both, FLI and serum FABP4 levels were upregulated in diabetic, obese, and metabolic syndrome patients. Serum FABP4 levels were higher in individuals with liver steatosis. Serum FABP4 were robustly associated with FLI in metabolic patients in both linear and logistic regression analyses. Conclusion: Our findings show that the serum FABP4 is associated to liver steatosis in metabolic patients.

Altered Serum Metabolic Profile Assessed by Advanced 1H-NMR in Breast Cancer Patients.

BACKGROUND: Altered lipid metabolism has been described in some types of cancer. To analyse in depth the metabolic modifications in breast cancer patients, advanced 1H-nuclear magnetic resonance was performed in these patients. The main objective of this paper was to define a specific lipidomic signature for these cancer patients. MATERIALS AND METHODS: Serum from 240 women (171 breast cancer patients and 69 control women) were studied and analysed by nuclear magnetic resonance. RESULTS: Triglyceride-enriched particles, specifically very low-density lipoprotein triglycerides, intermediate-density lipoprotein triglycerides, low-density lipoprotein triglycerides, and high-density lipoprotein triglycerides, were positively associated with breast cancer. Moreover, alanine, tyrosine, and branched amino acids were also associated with increased risk of breast cancer. CONCLUSIONS: Breast cancer patients showed a modified metabolome, giving a very interesting tool to draw different radar charts between control women and breast cancer patients. To our knowledge, this is the first time that advanced nuclear magnetic resonance profiling has been used to identify relevant and specifically altered lipid or amino acid metabolites in BC serum samples. The altered metabolic signature could be analysed for early and reliable BC patient diagnosis and prognosis.

Phosphoproteomic Analysis and Protein-Protein Interaction of Rat Aorta GJA1 and Rat Heart FKBP1A after Secoiridoid Consumption from Virgin Olive Oil: A Functional Proteomic Approach.

Protein functional interactions could explain the biological response of secoiridoids (SECs), main phenolic compounds in virgin olive oil (VOO). The aim was to assess protein-protein interactions (PPIs) of the aorta gap junction alpha-1 (GJA1) and the heart peptidyl-prolyl cis-trans isomerase (FKBP1A), plus the phosphorylated heart proteome, to describe new molecular pathways in the cardiovascular system in rats using nanoliquid chromatography coupled with mass spectrometry. PPIs modified by SECs and associated with GJA1 in aorta rat tissue were calpain, TUBA1A, and HSPB1. Those associated with FKBP1A in rat heart tissue included SUCLG1, HSPE1, and TNNI3. In the heart, SECs modulated the phosphoproteome through the main canonical pathways PI3K/mTOR signaling (AKT1S1 and GAB2) and gap junction signaling (GAB2 and GJA1). PPIs associated with GJA1 and with FKBP1A, the phosphorylation of GAB2, and the dephosphorylation of GJA1 and AKT1S1 in rat tissues are promising protein targets promoting cardiovascular protection to explain the health benefits of VOO.

Low-density lipoprotein from active SLE patients is more atherogenic to endothelial cells than low-density lipoprotein from the same patients during remission.

OBJECTIVES: SLE patients have an enhanced risk of atherosclerosis and cardiovascular disease. However, the increased prevalence of cardiovascular disease is not fully explained by traditional Framingham cardiovascular risk factors. Specific features of low-density lipoprotein (LDL) particles, other than plasma concentration, may induce accelerated atherosclerosis at early stages in these patients. Thus, we aimed to explore the impact of LDL from both active and inactive SLE patients on human aortic endothelial cells. METHODS: Human aortic endothelial cells were stimulated with the same concentration of LDL particles isolated from pooled serum that was collected from 13 SLE patients during both active and inactive states. Gene expression and cell migration assays were performed. RESULTS: Circulating LDL particles obtained from healthy volunteers and SLE patients in both remission and flare states were comparable in terms of number, cholesterol and triglyceride content, and net electric charge. Stimulation of cells with LDL from active SLE patients induced the expression of vascular cell adhesion molecule 1 (∼2.0-fold, P < 0.05), monocyte chemoattractant protein 1 (∼2.0-fold, P < 0.05) and matrix metallopeptidase 2 (∼1.6-fold, P < 0.01) compared with cells stimulated with LDL from inactive SLE patients. Additionally, LDL extracted from active patients increased cell migration in a wound-healing assay (1.4-fold, P < 0.05). CONCLUSION: Our data show that, at the same LDL concentration, LDL from active SLE patients had increased proatherogenic effects on endothelial cells compared with LDL from the same patients when in an inactive or remission state.

Triglyceride-Rich Lipoproteins and Glycoprotein A and B Assessed by 1H-NMR in Metabolic-Associated Fatty Liver Disease.

High plasma triglyceride (TG) levels and chronic inflammation are important factors related to metabolic-associated fatty liver disease in patients at cardiovascular risk. Using nuclear magnetic resonance (1H-NMR), we aimed to study the triglyceride-rich lipoprotein (TRL) and acute-phase glycoprotein profiles of a cohort of patients with metabolic disease and their relationship with fatty liver. Plasma samples of 280 patients (type 2 diabetes, 81.1%; obesity, 63.3%; and metabolic syndrome, 91.8%) from the University Hospital Lipid Unit were collected for the measurement of small, medium and large TRL particle numbers and sizes and glycoprotein profiles (Glyc-A and Glyc-B) by 1H-NMR. Liver function parameters, including the fatty liver index (FLI) and fibrosis-4 (FIB-4) score, were assessed. Hepatic echography assessment was performed in 100 patients, and they were followed up for 10 years. TRL particle concentrations showed a strong positive association with Glyc-A and Glyc-B (ρ=0.895 and ρ=0.654, p<0.001, respectively) and with the liver function-related proteins ALT ρ=0.293, p<0.001), AST (ρ=0.318, p<0.001) and GGT (ρ=0.284, p<0.001). Likewise, TRL concentrations showed a positive association with FLI (ρ=0.425, p<0.001) but not with FIB-4. During the follow-up period of 10 years, 18 new cases of steatosis were observed among 64 patients who were disease-free at baseline. Baseline TRL particle numbers and glycoprotein levels were associated with the new development of metabolic-associated fatty liver disease (MAFLD) (AUC=0.692, p=0.018 and AUC=0.669, p=0.037, respectively). Overall, our results indicated that TRL number and acute-phase glycoproteins measured by 1H-NMR could be potential biomarkers of the development of hepatic steatosis in patients at metabolic risk.

Hepatic Lipidomics and Molecular Imaging in a Murine Non-Alcoholic Fatty Liver Disease Model: Insights into Molecular Mechanisms.

An imbalance between hepatic fatty acid uptake and removal results in ectopic fat accumulation, which leads to non-alcoholic fatty liver disease (NAFLD). The amount and type of accumulated triglycerides seem to play roles in NAFLD progression; however, a complete understanding of how triglycerides contribute to NAFLD evolution is lacking. Our aim was to evaluate triglyceride accumulation in NAFLD in a murine model and its associations with molecular mechanisms involved in liver damage and adipose tissue-liver cross talk by employing lipidomic and molecular imaging techniques. C57BL/6J mice fed a high-fat diet (HFD) for 12 weeks were used as a NAFLD model. Standard-diet (STD)-fed animals were used as controls. Standard liver pathology was assessed using conventional techniques. The liver lipidome was analyzed by liquid chromatography-mass spectrometry (LC-MS) and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging. Liver triglycerides were identified by MS/MS. The transcriptome of genes involved in intracellular lipid metabolism and inflammation was assessed by RT-PCR. Plasma leptin, resistin, adiponectin, and FABP4 levels were determined using commercial kits. HFD-fed mice displayed increased liver lipid content. LC-MS analyses identified 14 triglyceride types that were upregulated in livers from HFD-fed animals. Among these 14 types, 10 were identified in liver cross sections by LDI-MS tissue imaging. The accumulation of these triglycerides was associated with the upregulation of lipogenesis and inflammatory genes and the downregulation of ß-oxidation genes. Interestingly, the levels of plasma FABP4, but not of other adipokines, were positively associated with 8 of these triglycerides in HFD-fed mice but not in STD-fed mice. Our findings suggest a putative role of FABP4 in the liver-adipose tissue cross talk in NAFLD.

Plasma glucose, triglycerides, VLDL, leptin and resistin levels as potential biomarkers for myocardial fat in mice. / Niveles plasmáticos de glucosa, triglicéridos, VLDL, leptina y resistina como potenciales biomarcadores de la grasa miocárdica en ratones.

INTRODUCTION: The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction due to diabetic aetiology, independently of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. METHODS: Correlations between cardiac triglyceride content and plasma levels of major altered molecules during diabetes and cardiac mRNA levels of genes involved in cardiac metabolism (Cd36 and Pdk4) have been explored in a murine model of insulin resistance induced by a high-fat diet. RESULTS: In insulin-resistant mice, the fatty diet increased myocardial triglyceride levels, compared to control animals fed with a standard diet. The content of cardiac triglycerides was directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin. In addition, an inverse correlation was observed between the content of cardiac triglycerides and the cardiac mRNA levels of Cd36 and Pdk4. CONCLUSIONS: Our data reveal that the cardiac triglyceride content is associated with altered plasma biochemical profile and reprogramming of gene expression aimed to mitigate the impact of ectopic lipid accumulation in the myocardium.

The Circulating GRP78/BiP Is a Marker of Metabolic Diseases and Atherosclerosis: Bringing Endoplasmic Reticulum Stress into the Clinical Scenario.

BACKGROUND: Glucose-regulated protein 78/Binding immunoglobulin protein (GRP78/BiP) is a protein associated with endoplasmic reticulum stress and is upregulated by metabolic alterations at the tissue-level, such as hypoxia or glucose deprivation, and it is hyper-expressed in fat tissue of obese individuals. OBJECTIVE: To investigate the role of the GRP78/BiP level as a metabolic and vascular disease biomarker in patients with type 2 diabetes (DM), obesity and metabolic syndrome (MS). METHODS: Four hundred and five patients were recruited, of whom 52.5% were obese, 72.8% had DM, and 78.6% had MS. The intimae media thickness (cIMT) was assessed by ultrasonography. The plasma GRP78/BiP concentration was determined, and its association with metabolic and vascular parameters was assessed. Circulating GRP78/BiP was also prospectively measured in 30 DM patients before and after fenofibrate/niacin treatment and 30 healthy controls. RESULTS: In the cross-sectional study, the GRP78/BiP level was significantly higher in the patients with obesity, DM, and MS. Age-, gender- and BMI-adjusted GRP78/BiP was directly associated with LDL-cholesterol, non-HDL-cholesterol, triglycerides, apoB, and cIMT. GRP78/BiP was positively associated to carotid plaque presence in the adjusted model, irrespective of obesity, DM and MS. In the prospective study, nicotinic acid treatment produced a significant reduction in the GRP78/BiP levels that was not observed with fenofibrate. CONCLUSIONS: GRP78/BiP plasma concentrations are increased in patients with both metabolic derangements and subclinical atherosclerosis. GRP78/BiP could be a useful marker of metabolic and cardiovascular risk.

Fatty acid binding protein 4 (FABP4) as a potential biomarker reflecting myocardial lipid storage in type 2 diabetes.

OBJECTIVE: Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone involved in the crosstalk between adipose and peripheral tissues, and it contributes to widespread insulin resistance in cells, including cardiac cells. However, the role of this adipokine in regulating cardiac metabolism and myocardial neutral lipid content in patients with type 2 diabetes has not been elucidated. METHODS: The impact of circulating FABP4 on the cardiac neutral lipid content was measured by proton magnetic resonance spectroscopy (1H-MRS) in patients with type 2 diabetes. Additionally, circulating FABP4 and the cardiac triglyceride content were analysed in high-fat diet (HFD)-fed mice, and the impact of the exogenous FABP4 was explored in HL-1 cardiac cells. RESULTS: Serum FABP4 levels were higher in type 2 diabetic patients compared to healthy individuals. Circulating FABP4 levels were associated with myocardial neutral lipid content in type 2 diabetic patients. In HFD-fed mice, both serum FABP4 and myocardial triglyceride content were increased. In FABP4-challenged HL-1 cells, extracellular FABP4 increased intracellular lipid accumulation, which led to impairment of the insulin-signalling pathway and reduced insulin-stimulated glucose uptake. However, these effects were partially reversed by FABP4 inhibition with BMS309403, which attenuated the intracellular lipid content and improved insulin signalling and insulin-stimulated glucose uptake. CONCLUSIONS: Taken together, our results identify FABP4 as a molecule involved in diabetic/lipid-induced cardiomyopathy and indicate that this molecule may be an emerging biomarker for diabetic cardiomyopathy-related disturbances, such as myocardial neutral lipid accumulation. Additionally, FABP4 inhibition may be a potential therapeutic target for metabolic-related cardiac dysfunctions.

Review of the scientific evolution of gene therapy for the treatment of homozygous familial hypercholesterolaemia: past, present and future perspectives.

Familial hypercholesterolaemia (FH) is a devastating genetic disease that leads to extremely high cholesterol levels and severe cardiovascular disease, mainly caused by mutations in any of the main genes involved in low-density lipoprotein cholesterol (LDL-C) uptake. Among these genes, mutations in the LDL receptor (LDLR) are responsible for 80%-90% of the FH cases. The severe homozygous variety (HoFH) is not successfully treated with standard cholesterol-lowering therapies, and more aggressive strategies must be considered to mitigate the effects of this disease, such as weekly/biweekly LDL apheresis. However, development of new therapeutic approaches is needed to cure HoFH. Because HoFH is mainly due to mutations in the LDLR, this disease has been proposed as an ideal candidate for gene therapy. Several preclinical studies have proposed that the transference of functional copies of the LDLR gene reduces circulating LDL-C levels in several models of HoFH, which has led to the first clinical trials in humans. Additionally, the recent development of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 technology for genome editing has opened the door to therapies aimed at directly correcting the specific mutation in the endogenous LDLR gene. In this article, we review the genetic basis of the FH disease, paying special attention to the severe HoFH as well as the challenges in its diagnosis and clinical management. Additionally, we discuss the current therapies for this disease and the new emerging advances in gene therapy to target a definitive cure for this disease.

Fluorescent labelling of membrane fatty acid transporter CD36 (SR-B2) in the extracellular loop.

CONTEXT: Upon palmitate oversupply, membrane fatty acid-transporter CD36 (SR-B2) permanently translocates from endosomal storage to the sarcolemma, inducing lipotoxicity. CD36 translocation results from endosomal alkalinisation elicited by palmitate-induced disattachment of the cytoplasmic V1-subcomplex from the membrane-integrated V0-subcomplex of vacuolar-type H+-ATPase. OBJECTIVE: Develop a CD36 fluorescent labeling technique as initial step towards live cell imaging. METHODS: Three human CD36 (hCD36) mutants were constructed via insertion of a tetracysteine motif at different positions within the extracellular domain. Constructs were lentivirally transduced for subsequent CD36 labeling with fluorescein-arsenical hairpin-binder (FlAsH). Cell imaging was combined with V0/V1 immunostaining and Western blotting. RESULTS: Transduction of hCD36-wildtype and mutants yielded corresponding proteins in HL-1 cardiomyocytes. Tetracysteine mutant-2 (hCD36-TC2) showed similar fatty acid uptake to wildtype. FlAsH staining revealed a speckled pattern reminiscent of endosomes. We found decreased V1 co-localization with CD36 upon high-palmitate culturing. Conversely, V0 consistently co-localized with CD36. CONCLUSION: hCD36-TC2 is a possible candidate for application of biarsenical dyes in live imaging studies pending further investigation. Our data is compatible with V0/V1 disassembly in high-palmitate-treated cells.

FABP4 inhibitor BMS309403 decreases saturated-fatty-acid-induced endoplasmic reticulum stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

AIMS: Fatty acid binding protein 4 (FABP4) inhibitors have been proposed as potential therapeutic approaches against insulin resistance-related inflammation and type 2 diabetes mellitus. However, the underlying molecular mechanisms by which these molecules drive these effects in skeletal muscle remain unknown. Here, we assessed whether the FABP4 inhibitor BMS309403 prevented lipid-induced endoplasmic reticulum (ER) stress-associated inflammation in skeletal muscle. MATERIALS AND METHODS: The BMS309403 treatment was assessed both in the skeletal muscle of high-fat diet (HFD)-fed mice and in palmitate-stimulated C2C12 myotubes. RESULTS: HFD feeding promoted insulin resistance, which is characterized by increased plasma levels of glucose, insulin, non-esterified fatty acids, triglycerides, resistin, and leptin and reduced plasma levels of adiponectin compared with control mice fed a standard diet. Additionally, insulin-resistant animals showed increased FABP4 plasma levels. In line with this evidence, recombinant FABP4 attenuated the insulin-induced AKT phosphorylation in C2C12 myotubes. Treatment with BMS309403 reduced lipid-induced ER stress and inflammation in both mouse skeletal muscle and C2C12 myotubes. The effects of the FABP4 inhibitor reducing lipid-induced ER stress-associated inflammation were related to the reduction of fatty acid-induced intramyocellular lipid deposits, ROS and nuclear factor-kappaB (NF-κB) nuclear translocation. Accordingly, BMS309403 reduced lipid-induced p38 MAPK phosphorylation, which is upstream of NF-κB activation. CONCLUSION: Overall, these findings indicate that BMS309403 reduces fatty acid-induced ER stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

Clinical and pathophysiological evidence supporting the safety of extremely low LDL levels-The zero-LDL hypothesis.

While the impact of very low concentrations of low-density lipoprotein cholesterol (LDL-C) on cardiovascular prevention is very reassuring, it is intriguing to know what effect these extremely low LDL-C concentrations have on lipid homoeostasis. The evidence supporting the safety of extremely low LDL levels comes from genetic studies and clinical drug trials. Individuals with lifelong low LDL levels due to mutations in genes associated with increased LDL-LDL receptor (LDLR) activity reveal no safety issues. Patients achieving extremely low LDL levels in the IMPROVE-IT and FOURIER, and the PROFICIO and ODYSSEY programs seem not to have an increased prevalence of adverse effects. The main concern regarding extremely low LDL-C plasma concentrations is the adequacy of the supply of cholesterol, and other molecules, to peripheral tissues. However, LDL proteomic and kinetic studies reaffirm that LDL is the final product of endogenous lipoprotein metabolism. Four of 5 LDL particles are cleared through the LDL-LDLR pathway in the liver. Given that mammalian cells have no enzymatic systems to degrade cholesterol, the LDL-LDLR pathway is the main mechanism for removal of cholesterol from the body. Our focus, therefore, is to review, from a physiological perspective, why such extremely low LDL-C concentrations do not appear to be detrimental. We suggest that extremely low LDL-C levels due to increased LDLR activity may be a surrogate of adequate LDL-LDLR pathway function.

Role of the fatty acid-binding protein 4 in heart failure and cardiovascular disease.

Obesity and ectopic fat accumulation in non-adipose tissues are major contributors to heart failure (HF) and cardiovascular disease (CVD). Adipocytes act as endocrine organs by releasing a large number of bioactive molecules into the bloodstream, which participate in a communication network between white adipose tissue and other organs, including the heart. Among these molecules, fatty acid-binding protein 4 (FABP4) has recently been shown to increase cardiometabolic risk. Both clinical and experimental evidence have identified FABP4 as a relevant player in atherosclerosis and coronary artery disease, and it has been directly related to cardiac alterations such as left ventricular hypertrophy (LVH) and both systolic and diastolic cardiac dysfunction. The available interventional studies preclude the establishment of a direct causal role of this molecule in CVD and HF and propose FABP4 as a biomarker rather than as an aetiological factor. However, several experimental reports have suggested that FABP4 may act as a direct contributor to cardiac metabolism and physiopathology, and the pharmacological targeting of FABP4 may restore some of the metabolic alterations that are conducive to CVD and HF. Here, we review the current knowledge regarding FABP4 in the context of HF and CVD as well as the molecular basis by which this protein participates in the regulation of cardiac function.