Inhibition of CERS1 in skeletal muscle exacerbates age-related muscle dysfunction.

Age-related muscle wasting and dysfunction render the elderly population vulnerable and incapacitated, while underlying mechanisms are poorly understood. Here, we implicate the CERS1 enzyme of the de novo sphingolipid synthesis pathway in the pathogenesis of age-related skeletal muscle impairment. In humans, CERS1 abundance declines with aging in skeletal muscle cells and, correlates with biological pathways involved in muscle function and myogenesis. Furthermore, CERS1 is upregulated during myogenic differentiation. Pharmacological or genetic inhibition of CERS1 in aged mice blunts myogenesis and deteriorates aged skeletal muscle mass and function, which is associated with the occurrence of morphological features typical of inflammation and fibrosis. Ablation of the CERS1 orthologue lagr-1 in Caenorhabditis elegans similarly exacerbates the age-associated decline in muscle function and integrity. We discover genetic variants reducing CERS1 expression in human skeletal muscle and Mendelian randomization analysis in the UK biobank cohort shows that these variants reduce muscle grip strength and overall health. In summary, our findings link age-related impairments in muscle function to a reduction in CERS1, thereby underlining the importance of the sphingolipid biosynthesis pathway in age-related muscle homeostasis.

Ryanodine receptor type 1 content decrease-induced endoplasmic reticulum stress is a hallmark of myopathies.

BACKGROUND: Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders. METHODS: We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels. RESULTS: RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P = 0.026), inclusion body myopathy (P = 0.003), polymyositis (P < 0.001) and juvenile dermatomyositis (P < 0.001) and in muscle samples of myotonic dystrophy type 2 (P < 0.001), presymptomatic (P < 0.001) and symptomatic (P < 0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P = 0.004) and limb-girdle muscular dystrophy type 2A (P = 0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P < 0.001) and mitochondrial myopathy (-71%, P < 0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P < 0.001), Ca2+ transfer to mitochondria (-48%, P = 0.002) and the mitophagy gene Parkinson protein 2 transcripts (P = 0.037) and induces mitochondrial accumulation (+99%, P = 0.005) and dysfunction (P < 0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P = 0.046), in mouse RyR1 recessive muscles (+58%, P = 0.001) and in human inflammatory (+96%, P = 0.006) and mitochondrial (+64%, P = 0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P < 0.001), mouse RyR1 recessive muscles (+63%, P = 0.009), human inflammatory (+50%, P = 0.038) and mitochondrial (+51%, P = 0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population. CONCLUSIONS: Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies.

Inhibiting de novo ceramide synthesis restores mitochondrial and protein homeostasis in muscle aging.

Disruption of mitochondrial function and protein homeostasis plays a central role in aging. However, how these processes interact and what governs their failure in aging remain poorly understood. Here, we showed that ceramide biosynthesis controls the decline in mitochondrial and protein homeostasis during muscle aging. Analysis of transcriptome datasets derived from muscle biopsies obtained from both aged individuals and patients with a diverse range of muscle disorders revealed that changes in ceramide biosynthesis, as well as disturbances in mitochondrial and protein homeostasis pathways, are prevalent features in these conditions. By performing targeted lipidomics analyses, we found that ceramides accumulated in skeletal muscle with increasing age across Caenorhabditis elegans, mice, and humans. Inhibition of serine palmitoyltransferase (SPT), the rate-limiting enzyme of the ceramide de novo synthesis, by gene silencing or by treatment with myriocin restored proteostasis and mitochondrial function in human myoblasts, in C. elegans, and in the skeletal muscles of mice during aging. Restoration of these age-related processes improved health and life span in the nematode and muscle health and fitness in mice. Collectively, our data implicate pharmacological and genetic suppression of ceramide biosynthesis as potential therapeutic approaches to delay muscle aging and to manage related proteinopathies via mitochondrial and proteostasis remodeling.

COX7A2L genetic variants determine cardiorespiratory fitness in mice and human.

Mitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3' untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and improved cardiorespiratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality.

Inhibition of sphingolipid de novo synthesis counteracts muscular dystrophy.

Duchenne muscular dystrophy (DMD), the most common muscular dystrophy, is a severe muscle disorder, causing muscle weakness, loss of independence, and premature death. Here, we establish the link between sphingolipids and muscular dystrophy. Transcripts of sphingolipid de novo biosynthesis pathway are up-regulated in skeletal muscle of patients with DMD and other muscular dystrophies, which is accompanied by accumulation of metabolites of the sphingolipid pathway in muscle and plasma. Pharmacological inhibition of sphingolipid synthesis by myriocin in the mdx mouse model of DMD ameliorated the loss in muscle function while reducing inflammation, improving Ca2+ homeostasis, preventing fibrosis of the skeletal muscle, heart, and diaphragm, and restoring the balance between M1 and M2 macrophages. Myriocin alleviated the DMD phenotype more than glucocorticoids. Our study identifies inhibition of sphingolipid synthesis, targeting multiple pathogenetic pathways simultaneously, as a strong candidate for treatment of muscular dystrophies.

Sphingolipids accumulate in aged muscle, and their reduction counteracts sarcopenia.

Age-related muscle dysfunction and sarcopenia are major causes of physical incapacitation in older adults and currently lack viable treatment strategies. Here we find that sphingolipids accumulate in mouse skeletal muscle upon aging and that both genetic and pharmacological inhibition of sphingolipid synthesis prevent age-related decline in muscle mass while enhancing strength and exercise capacity. Inhibition of sphingolipid synthesis confers increased myogenic potential and promotes protein synthesis. Within the sphingolipid pathway, we show that accumulation of dihydroceramides is the culprit disturbing myofibrillar homeostasis. The relevance of sphingolipid pathways in human aging is demonstrated in two cohorts, the UK Biobank and Helsinki Birth Cohort Study in which gene expression-reducing variants of SPTLC1 and DEGS1 are associated with improved and reduced fitness of older individuals, respectively. These findings identify sphingolipid synthesis inhibition as an attractive therapeutic strategy for age-related sarcopenia and co-occurring pathologies.

The exercise-induced long noncoding RNA CYTOR promotes fast-twitch myogenesis in aging.

Skeletal muscle displays remarkable plasticity upon exercise and is also one of the organs most affected by aging. Despite robust evidence that aging is associated with loss of fast-twitch (type II) muscle fibers, the underlying mechanisms remain to be elucidated. Here, we identified an exercise-induced long noncoding RNA, CYTOR, whose exercise responsiveness was conserved in human and rodents. Cytor overexpression in mouse myogenic progenitor cells enhanced myogenic differentiation by promoting fast-twitch cell fate, whereas Cytor knockdown deteriorated expression of mature type II myotubes. Skeletal muscle Cytor expression was reduced upon mouse aging, and Cytor expression in young mice was required to maintain proper muscle morphology and function. In aged mice, rescuing endogenous Cytor expression using adeno-associated virus serotype 9 delivery of CRISPRa reversed the age-related decrease in type II fibers and improved muscle mass and function. In humans, CYTOR expression correlated with type II isoform expression and was decreased in aged myoblasts. Increased CYTOR expression, mediated by a causal cis­expression quantitative trait locus located within a CYTOR skeletal muscle enhancer element, was associated with improved 6-min walk performance in aged individuals from the Helsinki Birth Cohort Study. Direct CYTOR overexpression using CRISPRa in aged human donor myoblasts enhanced expression of type II myosin isoforms. Mechanistically, Cytor reduced chromatin accessibility and occupancy at binding motifs of the transcription factor Tead1 by binding, and hence sequestering, Tead1. In conclusion, the long noncoding RNA Cytor was found to be a regulator of fast-twitch myogenesis in aging.

Hematopoietic upstream stimulating factor 1 deficiency is associated with increased atherosclerosis susceptibility in LDL receptor knockout mice.

Total body upstream stimulatory factor 1 (USF1) deficiency in mice is associated with brown adipose tissue activation and a marked protection against the development of obesity and atherosclerotic lesions. Functional expression of USF1 has also been detected in monocytes and monocyte-derived macrophages. In the current study we therefore tested whether selective hematopoietic USF1 deficiency can also beneficially impact the development of atherosclerosis. For this purpose, LDL receptor knockout mice were transplanted with bone marrow from USF1 knockout mice or their wild-type littermate controls and subsequently fed a Western-type diet for 20 weeks to stimulate atherosclerotic lesion development. Strikingly, absence of USF1 function in bone marrow-derived cells was associated with exacerbated blood leukocyte (+ 100%; P < 0.01) and peritoneal leukocyte (+ 50%; P < 0.05) lipid loading and an increased atherosclerosis susceptibility (+ 31%; P < 0.05). These effects could be attributed to aggravated hyperlipidemia, i.e. higher plasma free cholesterol (+ 33%; P < 0.001) and cholesteryl esters (+ 39%; P < 0.001), and the development of hepatosteatosis. In conclusion, we have shown that hematopoietic USF1 deficiency is associated with an increased atherosclerosis susceptibility in LDL receptor knockout mice. These findings argue against a contribution of macrophage-specific USF1 deficiency to the previously described beneficial effect of total body USF1 deficiency on atherosclerosis susceptibility in mice.

Urolithin A improves muscle function by inducing mitophagy in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, and despite advances in genetic and pharmacological disease-modifying treatments, its management remains a major challenge. Mitochondrial dysfunction contributes to DMD, yet the mechanisms by which this occurs remain elusive. Our data in experimental models and patients with DMD show that reduced expression of genes involved in mitochondrial autophagy, or mitophagy, contributes to mitochondrial dysfunction. Mitophagy markers were reduced in skeletal muscle and in muscle stem cells (MuSCs) of a mouse model of DMD. Administration of the mitophagy activator urolithin A (UA) rescued mitophagy in DMD worms and mice and in primary myoblasts from patients with DMD, increased skeletal muscle respiratory capacity, and improved MuSCs' regenerative ability, resulting in the recovery of muscle function and increased survival in DMD mouse models. These data indicate that restoration of mitophagy alleviates symptoms of DMD and suggest that UA may have potential therapeutic applications for muscular dystrophies.

Transcription Factor USF1 Is Required for Maintenance of Germline Stem Cells in Male Mice.

A prerequisite for lifelong sperm production is that spermatogonial stem cells (SSCs) balance self-renewal and differentiation, yet factors required for this balance remain largely undefined. Using mouse genetics, we now demonstrate that the ubiquitously expressed transcription factor upstream stimulatory factor (USF)1 is critical for the maintenance of SSCs. We show that USF1 is not only detected in Sertoli cells as previously reported, but also in SSCs. Usf1-deficient mice display progressive spermatogenic decline as a result of age-dependent loss of SSCs. According to our data, the germ cell defect in Usf1-/- mice cannot be attributed to impairment of Sertoli cell development, maturation, or function, but instead is likely due to an inability of SSCs to maintain a quiescent state. SSCs of Usf1-/- mice undergo continuous proliferation, which provides an explanation for their age-dependent depletion. The proliferation-coupled exhaustion of SSCs in turn results in progressive degeneration of the seminiferous epithelium, gradual decrease in sperm production, and testicular atrophy. We conclude that the general transcription factor USF1 is indispensable for the proper maintenance of mammalian spermatogenesis.

USF1 deficiency alleviates inflammation, enhances cholesterol efflux and prevents cholesterol accumulation in macrophages.

BACKGROUND: The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. METHODS: We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. RESULTS: We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1ß and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. CONCLUSIONS: Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.

The Contribution of GWAS Loci in Familial Dyslipidemias.

Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined.

USF1 deficiency activates brown adipose tissue and improves cardiometabolic health.

USF1 (upstream stimulatory factor 1) is a transcription factor associated with familial combined hyperlipidemia and coronary artery disease in humans. However, whether USF1 is beneficial or detrimental to cardiometabolic health has not been addressed. By inactivating USF1 in mice, we demonstrate protection against diet-induced dyslipidemia, obesity, insulin resistance, hepatic steatosis, and atherosclerosis. The favorable plasma lipid profile, including increased high-density lipoprotein cholesterol and decreased triglycerides, was coupled with increased energy expenditure due to activation of brown adipose tissue (BAT). Usf1 inactivation directs triglycerides from the circulation to BAT for combustion via a lipoprotein lipase-dependent mechanism, thus enhancing plasma triglyceride clearance. Mice lacking Usf1 displayed increased BAT-facilitated, diet-induced thermogenesis with up-regulation of mitochondrial respiratory chain complexes, as well as increased BAT activity even at thermoneutrality and after BAT sympathectomy. A direct effect of USF1 on BAT activation was demonstrated by an amplified adrenergic response in brown adipocytes after Usf1 silencing, and by augmented norepinephrine-induced thermogenesis in mice lacking Usf1. In humans, individuals carrying SNP (single-nucleotide polymorphism) alleles that reduced USF1 mRNA expression also displayed a beneficial cardiometabolic profile, featuring improved insulin sensitivity, a favorable lipid profile, and reduced atherosclerosis. Our findings identify a new molecular link between lipid metabolism and energy expenditure, and point to the potential of USF1 as a therapeutic target for cardiometabolic disease.

Novel hepatic microRNAs upregulated in human nonalcoholic fatty liver disease.

MicroRNAs (miRNAs) control gene expression by reducing mRNA stability and translation. We aimed to identify alterations in human liver miRNA expression/function in nonalcoholic fatty liver disease (NAFLD). Subjects with the highest (median liver fat 30%, n = 15) and lowest (0%, n = 15) liver fat content were selected from >100 obese patients for miRNA profiling of liver biopsies on microarrays carrying probes for 1438 human miRNAs (a cross-sectional study). Target mRNAs and pathways were predicted for the miRNAs most significantly upregulated in NAFLD, their cell-type-specific expression was investigated by quantitative PCR (qPCR), and the transcriptome of immortalized human hepatocytes (IHH) transfected with the miRNA with the highest number of predicted targets, miR-576-5p, was studied. The screen revealed 42 miRNAs up- and two downregulated in the NAFLD as compared to non-NAFLD liver. The miRNAs differing most significantly between the groups, miR-103a-2*, miR-106b, miR-576-5p, miRPlus-I137*, miR-892a, miR-1282, miR-3663-5p, and miR-3924, were all upregulated in NAFLD liver. Target pathways predicted for these miRNAs included ones involved in cancer, metabolic regulation, insulin signaling, and inflammation. Consistent transcriptome changes were observed in IHH transfected with miR-576-5p, and western analysis revealed a marked reduction of the RAC1 protein belonging to several miR-576-5p target pathways. To conclude, we identified 44 miRNAs differentially expressed in NAFLD versus non-NAFLD liver, 42 of these being novel in the context of NAFLD. The study demonstrates that by applying a novel study set-up and a broad-coverage array platform one can reveal a wealth of previously undiscovered miRNA dysregulation in metabolic disease.

Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice.

Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [(3)H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [(3)H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.

Silencing of ANGPTL 3 (angiopoietin-like protein 3) in human hepatocytes results in decreased expression of gluconeogenic genes and reduced triacylglycerol-rich VLDL secretion upon insulin stimulation.

Homozygosity of loss-of-function mutations in ANGPTL3 (angiopoietin-like protein 3)-gene results in FHBL2 (familial combined hypolipidaemia, OMIM #605019) characterized by the reduction of all major plasma lipoprotein classes, which includes VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein), HDL (high-density lipoprotein) and low circulating NEFAs (non-esterified fatty acids), glucose and insulin levels. Thus complete lack of ANGPTL3 in humans not only affects lipid metabolism, but also affects whole-body insulin and glucose balance. We used wild-type and ANGPTL3-silenced IHHs (human immortalized hepatocytes) to investigate the effect of ANGPTL3 silencing on hepatocyte-specific VLDL secretion and glucose uptake. We demonstrate that both insulin and PPARγ (peroxisome-proliferator-activated receptor γ) agonist rosiglitazone down-regulate the secretion of ANGPTL3 and TAG (triacylglycerol)-enriched VLDL1-type particles in a dose-dependent manner. Silencing of ANGPTL3 improved glucose uptake in hepatocytes by 20-50% and influenced down-regulation of gluconeogenic genes, suggesting that silencing of ANGPTL3 improves insulin sensitivity. We further show that ANGPTL3-silenced cells display a more pronounced shift from the secretion of TAG-enriched VLDL1-type particles to secretion of lipid poor VLDL2-type particles during insulin stimulation. These data suggest liver-specific mechanisms involved in the reported insulin-sensitive phenotype of ANGPTL3-deficient humans, featuring lower plasma insulin and glucose levels.

Osbpl8 deficiency in mouse causes an elevation of high-density lipoproteins and gender-specific alterations of lipid metabolism.

OSBP-related protein 8 (ORP8) encoded by Osbpl8 is an endoplasmic reticulum sterol sensor implicated in cellular lipid metabolism. We generated an Osbpl8(-/-) (KO) C57Bl/6 mouse strain. Wild-type and Osbpl8KO animals at the age of 13-weeks were fed for 5 weeks either chow or high-fat diet, and their plasma lipids/lipoproteins and hepatic lipids were analyzed. The chow-fed Osbpl8KO male mice showed a marked elevation of high-density lipoprotein (HDL) cholesterol (+79%) and phospholipids (+35%), while only minor increase of apolipoprotein A-I (apoA-I) was detected. In chow-fed female KO mice a less prominent increase of HDL cholesterol (+27%) was observed, while on western diet the HDL increment was prominent in both genders. The HDL increase was accompanied by an elevated level of HDL-associated apolipoprotein E in male, but not female KO animals. No differences between genotypes were observed in lecithin:cholesterol acyltransferase (LCAT) or hepatic lipase (HL) activity, or in the fractional catabolic rate of fluorescently labeled mouse HDL injected in chow-diet fed animals. The Osbpl8KO mice of both genders displayed reduced phospholipid transfer protein (PLTP) activity, but only on chow diet. These findings are consistent with a model in which Osbpl8 deficiency results in altered biosynthesis of HDL. Consistent with this hypothesis, ORP8 depleted mouse hepatocytes secreted an increased amount of nascent HDL into the culture medium. In addition to the HDL phenotype, distinct gender-specific alterations in lipid metabolism were detected: Female KO animals on chow diet showed reduced lipoprotein lipase (LPL) activity and increased plasma triglycerides, while the male KO mice displayed elevated plasma cholesterol biosynthetic markers cholestenol, desmosterol, and lathosterol. Moreover, modest gender-specific alterations in the hepatic expression of lipid homeostatic genes were observed. In conclusion, we report the first viable OsbplKO mouse model, demonstrating a HDL elevating effect of Osbpl8 knock-out and additional gender- and/or diet-dependent impacts on lipid metabolism.

Genomic, transcriptomic, and lipidomic profiling highlights the role of inflammation in individuals with low high-density lipoprotein cholesterol.

OBJECTIVE: Low high-density lipoprotein cholesterol (HDL-C) is associated with cardiometabolic pathologies. In this study, we investigate the biological pathways and individual genes behind low HDL-C by integrating results from 3 high-throughput data sources: adipose tissue transcriptomics, HDL lipidomics, and dense marker genotypes from Finnish individuals with low or high HDL-C (n=450). APPROACH AND RESULTS: In the pathway analysis of genetic data, we demonstrate that genetic variants within inflammatory pathways were enriched among low HDL-C associated single-nucleotide polymorphisms, and the expression of these pathways upregulated in the adipose tissue of low HDL-C subjects. The lipidomic analysis highlighted the change in HDL particle quality toward putatively more inflammatory and less vasoprotective state in subjects with low HDL-C, as evidenced by their decreased antioxidative plasmalogen contents. We show that the focal point of these inflammatory pathways seems to be the HLA region with its low HDL-associated alleles also associating with more abundant local transcript levels in adipose tissue, increased plasma vascular cell adhesion molecule 1 (VCAM1) levels, and decreased HDL particle plasmalogen contents, markers of adipose tissue inflammation, vascular inflammation, and HDL antioxidative potential, respectively. In a population-based look-up of the inflammatory pathway single-nucleotide polymorphisms in a large Finnish cohorts (n=11 211), no association of the HLA region was detected for HDL-C as quantitative trait, but with extreme HDL-C phenotypes, implying the presence of low or high HDL genes in addition to the population-genomewide association studies-identified HDL genes. CONCLUSIONS: Our study highlights the role of inflammation with a genetic component in subjects with low HDL-C and identifies novel cis-expression quantitative trait loci (cis-eQTL) variants in HLA region to be associated with low HDL-C.

Novel Loci for metabolic networks and multi-tissue expression studies reveal genes for atherosclerosis.

Association testing of multiple correlated phenotypes offers better power than univariate analysis of single traits. We analyzed 6,600 individuals from two population-based cohorts with both genome-wide SNP data and serum metabolomic profiles. From the observed correlation structure of 130 metabolites measured by nuclear magnetic resonance, we identified 11 metabolic networks and performed a multivariate genome-wide association analysis. We identified 34 genomic loci at genome-wide significance, of which 7 are novel. In comparison to univariate tests, multivariate association analysis identified nearly twice as many significant associations in total. Multi-tissue gene expression studies identified variants in our top loci, SERPINA1 and AQP9, as eQTLs and showed that SERPINA1 and AQP9 expression in human blood was associated with metabolites from their corresponding metabolic networks. Finally, liver expression of AQP9 was associated with atherosclerotic lesion area in mice, and in human arterial tissue both SERPINA1 and AQP9 were shown to be upregulated (6.3-fold and 4.6-fold, respectively) in atherosclerotic plaques. Our study illustrates the power of multi-phenotype GWAS and highlights candidate genes for atherosclerosis.