Subclinical atherosclerosis and its progression are modulated by PLIN2 through a feed-forward loop between LXR and autophagy.

BACKGROUND: Hyperlipidaemia is a major risk factor for cardiovascular disease, and atherosclerosis is the underlying cause of both myocardial infarction and stroke. We have previously shown that the Pro251 variant of perilipin-2 reduces plasma triglycerides and may therefore be beneficial to reduce atherosclerosis development. OBJECTIVE: We sought to delineate putative beneficial effects of the Pro251 variant of perlipin-2 on subclinical atherosclerosis and the mechanism by which it acts. METHODS: A pan-European cohort of high-risk individuals where carotid intima-media thickness has been assessed was adopted. Human primary monocyte-derived macrophages were prepared from whole blood from individuals recruited by perilipin-2 genotype or from buffy coats from the Karolinska University hospital blood central. RESULTS: The Pro251 variant of perilipin-2 is associated with decreased intima-media thickness at baseline and over 30 months of follow-up. Using human primary monocyte-derived macrophages from carriers of the beneficial Pro251 variant, we show that this variant increases autophagy activity, cholesterol efflux and a controlled inflammatory response. Through extensive mechanistic studies, we demonstrate that increase in autophagy activity is accompanied with an increase in liver-X-receptor (LXR) activity and that LXR and autophagy reciprocally activate each other in a feed-forward loop, regulated by CYP27A1 and 27OH-cholesterol. CONCLUSIONS: For the first time, we show that perilipin-2 affects susceptibility to human atherosclerosis through activation of autophagy and stimulation of cholesterol efflux. We demonstrate that perilipin-2 modulates levels of the LXR ligand 27OH-cholesterol and initiates a feed-forward loop where LXR and autophagy reciprocally activate each other; the mechanism by which perilipin-2 exerts its beneficial effects on subclinical atherosclerosis.

Integrative studies implicate matrix metalloproteinase-12 as a culprit gene for large-artery atherosclerotic stroke.

BACKGROUND: Ischaemic stroke and coronary heart disease are important contributors to the global disease burden and share atherosclerosis as the main underlying cause. Recent evidence from a genome-wide association study (GWAS) suggested that single nucleotide polymorphisms (SNP) near the MMP12 gene at chromosome 11q22.3 were associated with large-vessel ischaemic stroke. Here, we evaluated and extended these results by examining the relationship between MMP12 and atherosclerosis in clinical and experimental studies. METHODS AND RESULTS: Plasma concentrations of MMP12 were measured at baseline in 3394 subjects with high-risk for cardiovascular disease (CVD) using the Olink ProSeek CVD I array. The plasma MMP12 concentration showed association with incident cardiovascular and cerebrovascular events (130 and 67 events, respectively, over 36 months) and carotid intima-media thickness progression (P = 3.6 × 10-5 ). A GWAS of plasma MMP12 concentrations revealed that SNPs rs499459, rs613084 and rs1892971 at chr11q22.3 were independently associated with plasma MMP12 (P < 5 × 10-8 ). The lead SNPs showed associations with mRNA levels of MMP12 and adjacent MMPs in atherosclerotic plaques. MMP12 transcriptomic and proteomic levels were strongly significantly increased in carotid plaques compared with control arterial tissue and in plaques from symptomatic versus asymptomatic patients. By combining immunohistochemistry and proximity ligation assay, we demonstrated that MMP12 localizes to CD68 + macrophages and interacts with elastin in plaques. MMP12 silencing in human THP-1-derived macrophages resulted in reduced macrophage migration. CONCLUSIONS: Our study supports the notion that MMP12 is implicated in large-artery atherosclerotic stroke, functionally by enhancing elastin degradation and macrophage invasion in plaques.

Fatty acid desaturases in human adipose tissue: relationships between gene expression, desaturation indexes and insulin resistance.

AIMS/HYPOTHESIS: Fatty acid desaturases introduce double bonds into growing fatty acid chains. The key desaturases in humans are Delta5-desaturase (D5D), Delta6-desaturase (D6D) and stearoyl-CoA desaturase (SCD). Animal and human data implicate hepatic desaturase activities in insulin resistance, obesity and dyslipidaemia. However, the role of desaturase activity in adipose tissue is uncertain. We therefore evaluated relationships between adipose mRNA expression, estimated desaturase activities (fatty acid ratios) in adipose tissue and insulin resistance. METHODS: Subcutaneous adipose tissue mRNA expression of D5D (also known as FADS1), D6D (also known as FADS2) and SCD was determined in 75 individuals representative of the study population of 294 healthy 63-year-old men. Desaturation indexes (product/substrate fatty acid ratios) were generated from adipose tissue fatty acid composition in all individuals. Insulin resistance was defined as the upper quartile of the updated homeostasis model assessment (HOMA-2) index. RESULTS: The relevant desaturation indexes (16:1/16:0, 18:1/18:0, 20:4/20:3 and 18:3/18:2) reflected expression of SCD, but not of D5D or D6D in adipose tissue. Insulin-resistant individuals had a higher adipose tissue 18:1/18:0, but not 16:1/16:0 ratio than insulin-sensitive individuals. Individuals with a high adipose tissue 18:1/18:0 ratio were 4.4-fold (95% CI 1.8-11.8) more likely to be insulin resistant [threefold (95% CI 1.1-8.6) after adjustment for waist circumference and plasma triacylglycerol]. In a multiple regression model predicting HOMA-2, the independent effect of the 18:1/18:0 ratio was borderline (p=0.086). CONCLUSIONS/INTERPRETATION: Adipose tissue desaturation indexes of SCD reflect the expression of the gene encoding the enzyme in this tissue. Elevated SCD activity within adipose tissue is closely coupled to the development of insulin resistance.

Fatty acid handling protein expression in adipose tissue, fatty acid composition of adipose tissue and serum, and markers of insulin resistance.

OBJECTIVE: Proteins involved in cellular fatty acid (FA) uptake and metabolism may be of relevance in the context of disturbed FA metabolism associated with insulin resistance. Therefore this study investigated relationships between FA handling protein mRNA expression in adipose tissue, FA composition of adipose tissue and serum, and markers of insulin resistance. SUBJECTS: 75 subjects with a range of insulin sensitivities recruited from a cohort of 294 healthy 63-year-old Swedish men. MEASUREMENTS: Anthropometric and biochemical variables (e.g. waist-hip-ratio (WHR) and homeostasis model assessment (HOMA) index of insulin sensitivity), FA composition of the subcutaneous (s.c.) gluteal adipose tissue, serum nonesterified FA (NEFA) and serum phospholipid compartments (by gas-liquid chromatography; n = 294), and mRNA levels of FA handling proteins (adipocyte and keratinocyte lipid binding proteins, fatty acid transport protein (FATP) -1 and -4, CD36/fatty acid translocase, plasma membrane fatty acid binding protein, and acyl-CoA synthase-1 (ACS1)) in s.c. gluteal adipose tissue (by quantitative real-time polymerase chain reaction; n = 75). RESULTS: ACS1 expression was negatively correlated with measures of insulin resistance and central obesity (ACS1 versus HOMA: r = -0.28, P<0.05; ACS1 versus WHR: r = -0.23, P<0.05), with an opposite trend for FATP4. Further analysis of ACS1 expression levels revealed correlations with adipose tissue 16:0 (r = -0.27, P<0.05) and NEFA 16:1 (r = 0.29, P<0.05), FA composition variables which in turn correlated with HOMA index (r = 0.39, P<0.001 and r = -0.23, P<0.05, respectively, n = 75). Moreover, NEFA 16:1 predicted ACS1 expression independently of HOMA, WHR and adipose tissue 16:0 in multiple regression analysis (standardized coefficient = 0.27, P<0.05). CONCLUSION: Significant associations were found between measures of insulin sensitivity, adipose tissue FA handling protein expression, and specific FA composition variables. Although causal relationships could not be identified these findings suggest a role of FA handling proteins in relation to insulin sensitivity, via their involvement in FA trafficking and metabolism. In particular they indicate links between ACS1 activity, the distribution of 16:0 and 16:1, and insulin sensitivity, which may be of physiological relevance.

In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.

Previous studies in several laboratories have demonstrated inadvertent chromosomal abnormalities in long-term cultured human embryonic stem cells (HESC). Here, using a two-step selection process we report a functional adaptation of a HESC line, HS181, towards a decreased dependence of extra cellular matrix (ECM) for in vitro survival, that is for growth directly onto a plastic surface. Successful adaptation was paralleled with a karyotype change in 100% of the cells to 47,XX,del(7)(q11.2),+i(12)(p10). The resulting adapted population showed increased survival and growth on plastic and also maintained expression of HESC markers, but showed a decreased pluripotency, as demonstrated by results from embryoid body (EB) formation in vitro. The finding of reduced pluripotency may not be totally unexpected since the variant cells were selected for self-renewal and proliferation, not differentiation during the adaptation to growth on plastic. In the light of recent models of a germ cell origin of HESC it is of particular interest that similar to many of the reported spontaneous HESC mutants, one of the identified specific chromosome abnormalities, i(12p), has also been strongly implicated for human germ cell cancer. However, the mutated HESC variant carrying this mutation failed to grow as a xeno-graft in a mouse model in vivo. This is surprising and needs a further mechanistic analysis for its explanation. Increased knowledge of genetic integrity of HESC may have significance on the understanding of mechanisms for tumor progression and thus strategy for treatments, particularly for tumors occurring in early life.

Expression of fatty-acid-handling proteins in human adipose tissue in relation to obesity and insulin resistance.

AIMS/HYPOTHESIS: Protein-mediated trans-membrane and intracellular fatty acid trafficking are becoming increasingly recognised as biochemically and physiologically important concepts. Obesity and insulin resistance are polygenic disorders, heavily influenced by environmental and life-style factors, and are virtually always associated with disturbed fatty acid metabolism in adipose and other tissues. The aim of this study was to investigate mRNA expression levels of fatty-acid-handling proteins in adipose tissue in relation to markers of genetic and acquired obesity and insulin resistance. METHODS: We quantified mRNA expression of subcutaneous adipose tissue fatty-acid-handling proteins (ALBP, KLBP, FATP1, FATP4, CD36, ACS1) in 17 monozygotic twin-pairs with a range of intra-pair differences (Delta) in BMI and detailed measures of obesity and insulin resistance, allowing influences of genetic and non-genetic factors to be distinguished. RESULTS: In acquired obesity FATP4 expression was up-regulated independently of genetic background (DeltaFATP4 versus DeltaBMI; r=0.50, p=0.04; DeltaFATP4 versus Deltabody fat; r=0.59, p=0.01). Similarly, CD36 and FATP1 expression correlated with acquired differences in HDL cholesterol and non-esterified fatty acid concentrations respectively. Moreover, FATP4 and CD36 expression levels correlated with measures of obesity and insulin resistance that are influenced by both genetic and non-genetic factors (FATP4 versus BMI: r=0.53, p=0.0001; FATP4 versus body fat: r=0.51, p=0.002; FATP4 versus homeostasis model assessment [HOMA]: r=0.49, p=0.001; CD36 versus BMI: r=0.50, p=0.02; CD36 versus body fat: r=0.63, p=0.001; CD36 versus HOMA: r=0.34, p=0.06). CONCLUSIONS/INTERPRETATION: These findings indicate that expression of specific adipose tissue fatty-acid-handling proteins is related to obesity and insulin resistance, and that, in particular, FATP4 plays a role in acquired obesity. Our results suggest that facilitated fatty acid trafficking is a physiologically and pathologically relevant phenomenon in man.

Genetic and structural evaluation of fatty acid transport protein-4 in relation to markers of the insulin resistance syndrome.

Disturbances in fatty acid metabolism are involved in the etiology of insulin resistance and the related dyslipidemia, hypertension, and procoagulant state. The fatty acid transport proteins (FATPs) are implicated in facilitated cellular uptake of nonesterified fatty acids (NEFAs), thus potentially regulating NEFA concentrations and metabolism. The aim of this study was to investigate polymorphic loci in the FATP4 gene with respect to associations with fasting and postprandial lipid and lipoprotein variables and markers of insulin resistance in 608 healthy, middle-aged Swedish men and to evaluate possible mechanisms behind any associations observed. Heterozygotes for a Gly209Ser polymorphism (Ser allele frequency 0.05) had significantly lower body mass index and, correcting for body mass index, significantly lower triglyceride concentrations, systolic blood pressure, insulin concentrations, and homeostasis model assessment index compared with common homozygotes. A three-dimensional model of the FATP4 protein based on structural and functional similarity with adenylate-forming enzymes revealed that the variable residue 209 is exposed in a region potentially involved in protein-protein interactions. Furthermore, the model indicated functional regions with respect to NEFA transport and acyl-coenzyme A synthase activity and membrane association. These findings propose FATP4 as a candidate gene for the insulin resistance syndrome and provide a structural basis for understanding FATP function in NEFA transport and metabolism.

A common polymorphism in the fatty acid transport protein-1 gene associated with elevated post-prandial lipaemia and alterations in LDL particle size distribution.

The fatty acid transport proteins (FATPs) have been implicated in facilitated cellular uptake of non-esterified fatty acids (NEFAs), thus having the potential to regulate local and systemic NEFA concentrations and metabolism. Hypothesising that genetic variation within the FATP genes may affect lipid metabolism, we investigated a G/A substitution at position 48 in intron 8 of the fatty acid transport-1 (FATP1) gene with respect to associations with fasting and post-prandial plasma lipid and lipoprotein variables in 628 healthy 50-year-old Swedish men and 426 Swedish women, aged 37-65 years. A subset of 105 men with the apoE3/E3 genotype underwent an oral fat tolerance test. Although fasting plasma TG concentrations were not different, male A/A individuals had significantly higher post-prandial TG concentrations and VLDL(1) (S(f) 60-400 apoB100)-to-VLDL(2) (S(f) 20-60 apoB100) ratio compared to male G/A and G/G individuals. A/A individuals apparently failed to suppress plasma NEFA concentrations during the oral fat tolerance test. Furthermore, fasting plasma concentrations of the largest, most buoyant LDL subfraction (LDL-I) were significantly lower in carriers of the A allele in the male cohort. Electromobility shift assays and reporter gene studies indicated that binding of nuclear factors and effect on transcriptional activity differ between the intron 8 alleles. These findings suggest that through regulation of NEFA trafficking, FATP1 might play a role in post-prandial lipid metabolism and development of cardiovascular disease.

Determination of interaction kinetic constants for HIV-1 protease inhibitors using optical biosensor technology.

The interaction between HIV-1 protease and inhibitors has been studied with optical biosensor technology. Optimized experimental procedures and mathematical analysis permitted determination of association and dissociation rate constants. A sensor surface with native enzyme was unstable and exhibited a drift that was influenced by the binding of inhibitor. This was hypothesized to be due to a specific mechanism involving autoproteolysis and/or dimer dissociation. The use of a mutant predicted to be less susceptible to autoproteolysis (Q7K) than wild-type enzyme resulted in a minor effect on surface stability, while a completely stable surface was obtained by treatment of the immobilized enzyme with N-ethyl-N'-(dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide; the most stable surface was achieved by chemically modifying the Q7K enzyme. The stabilized surface was enzymatically active and the interaction with inhibitors was similar to that for native enzyme. Several of the inhibitors had very high association rates, and estimation of kinetic constants was therefore performed with a binding equation accounting for limited mass transport. Of the clinical inhibitors studied, saquinavir had the highest affinity for the enzyme, a result of the lowest dissociation rate. Although the dissociation rate for ritonavir was sixfold faster, the affinity was only twofold lower than that for saquinavir since the association rate was threefold faster. Nelfinavir and indinavir exhibited lower affinities relative to the other inhibitors, a consequence of a slower association for nelfinavir and a relatively fast dissociation for indinavir. These results show that biosensor-based interaction studies can resolve affinity into association and dissociation rates, and that these are characteristic parameters for the interaction between enzymes and inhibitors.

Enhanced detection and distinction of RNA by enzymatic probe ligation.

It is important that RNA molecules representing members of gene families are distinguished in expression analyses, and even greater resolving power may be required to identify allelic variants of transcripts in order to investigate imprinting or to study the distribution of mutant genes in tissues. Ligase-mediated gene detection allows precise distinction of DNA sequence variants, but it is not known if ligases can also be used to distinguish variants of RNA sequences. Here we present conditions for efficient ligation of pairs of DNA oligonucleotides hybridizing next to one another on RNA strands, permitting discrimination of any single nucleotide probe-target mismatch by a factor of between 20- and 200-fold. The mechanism allows padlock probes to be used to distinguish single-nucleotide variants in RNA. Ligase-mediated gene detection could therefore provide highly sensitive and accurate ligase-mediated detection and distinction of RNA sequence variants in solution, on DNA microarrays, and in situ.