Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition.

Collagen XVIII is an evolutionary conserved ubiquitously expressed basement membrane proteoglycan produced in three isoforms via two promoters (P). Here, we assess the function of the N-terminal, domain of unknown function/frizzled-like sequences unique to medium/long collagen XVIII by creating P-specific null mice. P2-null mice, which only produce short collagen XVIII, developed reduced bulk-adiposity, hepatic steatosis, and hypertriglyceridemia. These abnormalities did not develop in P1-null mice, which produce medium/long collagen XVIII. White adipose tissue samples from P2-null mice contain larger reserves of a cell population enriched in early adipocyte progenitors; however, their embryonic fibroblasts had ∼ 50% lower adipocyte differentiation potential. Differentiating 3T3-L1 fibroblasts into mature adipocytes produced striking increases in P2 gene-products and dramatic falls in P1-transcribed mRNA, whereas Wnt3a-induced dedifferentiation of mature adipocytes produced reciprocal changes in P1 and P2 transcript levels. P2-derived gene-products containing frizzled-like sequences bound the potent adipogenic inhibitor, Wnt10b, in vitro. Previously, we have shown that these same sequences bind Wnt3a, inhibiting Wnt3a-mediated signaling. P2-transcript levels in visceral fat were positively correlated with serum free fatty acid levels, suggesting that collagen α1 (XVIII) expression contributes to regulation of adipose tissue metabolism in visceral obesity. Medium/long collagen XVIII is deposited in the Space of Disse, and interaction between hepatic apolipoprotein E and this proteoglycan is lost in P2-null mice. These results describe a previously unidentified extracellular matrix-directed mechanism contributing to the control of the multistep adipogenic program that determines the number of precursors committing to adipocyte differentiation, the maintenance of the differentiated state, and the physiological consequences of its impairment on ectopic fat deposition.

The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

The origin, global distribution, and functional impact of the human 8p23 inversion polymorphism.

Genomic inversions are an increasingly recognized source of genetic variation. However, a lack of reliable high-throughput genotyping assays for these structures has precluded a full understanding of an inversion's phylogenetic, phenotypic, and population genetic properties. We characterize these properties for one of the largest polymorphic inversions in man (the ∼4.5-Mb 8p23.1 inversion), a structure that encompasses numerous signals of natural selection and disease association. We developed and validated a flexible bioinformatics tool that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion. This tool was applied retrospectively to diverse genome-wide data sets, revealing significant population stratification that largely follows a clinal "serial founder effect" distribution model. Phylogenetic analyses establish the inversion's ancestral origin within the Homo lineage, indicating that 8p23.1 inversion has occurred independently in the Pan lineage. The human inversion breakpoint was localized to an inverted pair of human endogenous retrovirus elements within the large, flanking low-copy repeats; experimental validation of this breakpoint confirmed these elements as the likely intermediary substrates that sponsored inversion formation. In five data sets, mRNA levels of disease-associated genes were robustly associated with inversion genotype. Moreover, a haplotype associated with systemic lupus erythematosus was restricted to the derived inversion state. We conclude that the 8p23.1 inversion is an evolutionarily dynamic structure that can now be accommodated into the understanding of human genetic and phenotypic diversity.

Evidence of a polygenic origin of extreme high-density lipoprotein cholesterol levels.

OBJECTIVE: There are several known monogenic causes of high and low high-density lipoprotein cholesterol (HDL-C) levels, but traditional sequencing studies have had limited success in identifying mutations in the majority of individuals with extreme HDL-C levels. The aim of this study was to assess the power of a targeted high-throughput sequencing strategy to elucidate the genetic basis of extreme HDL-C phenotypes. APPROACH AND RESULTS: We sequenced 195 genes with either established or implicated roles in lipid and lipoprotein metabolism plus 78 lipid-unrelated genes in patients with HDL-C <1st (n=40) or >99th (n=40) percentile values, and the results were compared with those of 498 individuals representative of the Dutch general population and 95 subjects with normal HDL-C (between 40th and 60th percentile values). The extreme HDL cohort carried more rare nonsynonymous variants in the lipid geneset than both the general population (odds ratio, 1.39; P=0.019) and normal HDL-C (odds ratio, 1.43; P=0.040) cohorts. The prevalence of such variants in the lipid-related and lipid-unrelated genesets was similar in the control groups, indicative of equal mutation rates. In the extreme HDL cohort, however, there was enrichment of rare nonsynonymous variants in the lipid versus the control geneset (odds ratio, 2.23; P<0.0001), and 70% of the lipid-related variants altered conserved nucleotides. The lipid geneset comprised 4 nonsense, 10 splice-site, and 8 coding indel variants, whereas the control geneset contained only 1 such variant. In the lipid geneset, 87% and 28% of the patients carried ≥ 2 and ≥ 5 rare variants. CONCLUSIONS: This study suggests that most extreme HDL-C phenotypes have a polygenic origin.

CDKN2B expression in adipose tissue of familial combined hyperlipidemia patients.

The purpose of this study was to determine the core biological processes perturbed in the subcutaneous adipose tissue of familial combined hyperlipidemia (FCHL) patients. Annotation of FCHL and control microarray datasets revealed a distinctive FCHL transcriptome, characterized by gene expression changes regulating five overlapping systems: the cytoskeleton, cell adhesion and extracellular matrix; vesicular trafficking; lipid homeostasis; and cell cycle and apoptosis. Expression values for the cell-cycle inhibitor CDKN2B were increased, replicating data from an independent FCHL cohort. In 3T3-L1 cells, CDKN2B knockdown induced C/EBPα expression and lipid accumulation. The minor allele at SNP site rs1063192 (C) was predicted to create a perfect seed for the human miRNA-323b-5p. A miR-323b-5p mimic significantly reduced endogenous CDKN2B protein levels and the activity of a CDKN2B 3'UTR luciferase reporter carrying the rs1063192 C allele. Although the allele displayed suggestive evidence of association with reduced CDKN2B mRNA in the MuTHER adipose tissue dataset, family studies suggest the association between increased CDKN2B expression and FCHL-lipid abnormalities is driven by factors external to this gene locus. In conclusion, from a comparative annotation analysis of two separate FCHL adipose tissue transcriptomes and a subsequent focus on CDKN2B, we propose that dysfunctional adipogenesis forms an integral part of FCHL pathogenesis.

Mutations in a Sar1 GTPase of COPII vesicles are associated with lipid absorption disorders.

Dietary fat is an important source of nutrition. Here we identify eight mutations in SARA2 that are associated with three severe disorders of fat malabsorption. The Sar1 family of proteins initiates the intracellular transport of proteins in COPII (coat protein)-coated vesicles. Our data suggest that chylomicrons, which vastly exceed the size of typical COPII vesicles, are selectively recruited by the COPII machinery for transport through the secretory pathways of the cell.

Mechanisms and genetic determinants regulating sterol absorption, circulating LDL levels, and sterol elimination: implications for classification and disease risk.

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.

Musings from the Tribbles Research and Innovation Network.

This commentary integrates historical and modern findings that underpin our understanding of the cell-specific functions of the Tribbles (TRIB) proteins that bear on tumorigenesis. We touch on the initial discovery of roles played by mammalian TRIB proteins in a diverse range of cell-types and pathologies, for example, TRIB1 in regulatory T-cells, TRIB2 in acute myeloid leukaemia and TRIB3 in gliomas; the origins and diversity of TRIB1 transcripts; microRNA-mediated (miRNA) regulation of TRIB1 transcript decay and translation; the substantial conformational changes that ensue on binding of TRIB1 to the transcription factor C/EBPα; and the unique pocket formed by TRIB1 to sequester its C-terminal motif bearing a binding site for the E3 ubiquitin ligase COP1. Unashamedly, the narrative is relayed through the perspective of the Tribbles Research and Innovation Network, and its establishment, progress and future ambitions: the growth of TRIB and COP1 research to hasten discovery of their cell-specific contributions to health and obesity-related cancers.

Family history in first degree relatives of patients with premature cardiovascular disease.

BACKGROUND: Family history (FH) of cardiovascular disease (CVD) in first degree relatives (FDR) is a major risk factor, especially for premature events. Data are sparse on FH of different manifestations of CVD among FDRs of patients with premature myocardial infarction (MI), chronic stable angina (CSA) or peripheral vascular disease (PVD). METHODS: We obtained FHs from first degree relatives (parents or siblings) of 230 consecutive patients with premature (men < 60 and women < 65 years) CVD, including 79 wth MI, 39 CSA, 51 PVD and 61 blood donors. Among 1225 parents or siblings, 421 had MI, 222 CSA, 261PVD and 321 were among blood donors. RESULTS: FH of MI were 5.6% (18/321) among blood donors, 14.0% (59/421) among patients with premature MI, 14.4% (32/222) CSA, and 8.0% (21/261) PVD. (all p < 0.05). For FH of CSA the corresponding frequencies were 3.7% 5.2%, 11.3%, and 6.9%. (all p < 0.05). For PVD, the corresponding frequencies were 2.1%, 3.4%, 0.9% and 0.7%, respectively. (p = ns). CONCLUSIONS: These data are compatible with the hypothesis that FH of MI, CSA and PVD are significantly different for patients with premature MI or CSA but not PVD.

Myeloid Tribbles 1 induces early atherosclerosis via enhanced foam cell expansion.

Macrophages drive atherosclerotic plaque progression and rupture; hence, attenuating their atherosclerosis-inducing properties holds promise for reducing coronary heart disease (CHD). Recent studies in mouse models have demonstrated that Tribbles 1 (Trib1) regulates macrophage phenotype and shows that Trib1 deficiency increases plasma cholesterol and triglyceride levels, suggesting that reduced TRIB1 expression mediates the strong genetic association between the TRIB1 locus and increased CHD risk in man. However, we report here that myeloid-specific Trib1 (mTrib1) deficiency reduces early atheroma formation and that mTrib1 transgene expression increases atherogenesis. Mechanistically, mTrib1 increased macrophage lipid accumulation and the expression of a critical receptor (OLR1), promoting oxidized low-density lipoprotein uptake and the formation of lipid-laden foam cells. As TRIB1 and OLR1 RNA levels were also strongly correlated in human macrophages, we suggest that a conserved, TRIB1-mediated mechanism drives foam cell formation in atherosclerotic plaque and that inhibiting mTRIB1 could be used therapeutically to reduce CHD.

Hepatic PGC-1beta overexpression induces combined hyperlipidemia and modulates the response to PPARalpha activation.

OBJECTIVE: Previous studies have indicated that the hyperlipidemia and gene expression changes induced by a short-term high-fat diet (HFD) are mediated through the peroxisome proliferator-activated receptor gamma coactivator (PGC)-1beta, and that in vitro both PGC-1beta and PGC -1alpha increase PPARalpha-mediated transcriptional activities. Here, we examined the in vivo effects of these two coactivators in potentiating the lipid lowering properties of the PPARalpha agonist Wy14,643 (Wy). METHODS AND RESULTS: C57BL/6 mice were fed chow or HFD and transduced with adenoviruses encoding PGC-1alpha or PGC-1beta. On chow, hepatic PGC-1beta overexpression caused severe combined hyperlipidemia including elevated plasma apolipoprotein B levels. Hepatic triglyceride secretion, DGAT1, and FAT/CD36 expression were increased whereas PPARalpha and hepatic lipase mRNA levels were reduced. PGC-1beta overexpression blunted Wy-mediated changes in expression levels of PPARalpha and downstream genes. Furthermore, PGC-1beta did not potentiate Wy-stimulated fatty acid oxidation in primary hepatocytes. PGC-1beta and PGC-1alpha overexpression did not alter SREBP-1c, SREBP-1c target gene expression, nor hepatic triglyceride content. On HFD, PGC-1beta overexpression decreased hepatic SREBP-1c, yet increased FAS and ACCalpha mRNA and plasma triglyceride levels. CONCLUSIONS: Hepatic PGC-1beta overexpression caused combined hyperlipidemia independent of SREBP-1c activation. Hepatic PGC-1beta overexpression reduced the potentially beneficial effects of PPARalpha activation on gene expression. Thus, inhibition of hepatic PGC-1beta may provide a therapy for treating combined hyperlipidemia.

Current biology of MTP: implications for selective inhibition.

The microsomal triglyceride transfer protein (MTP), along with its partner, protein disulphide isomerase, performs a wide range of lipid transport functions necessary for maintenance of whole-body lipid homeostasis. In this review, we summarize the recent deluge of comparative and functional genomic data that have forced a radical re-appraisal of the evolutionary processes that established the major lipid transport pathway in man, and the different structural and lipid transfer roles MTP plays within it. This is followed by an overview of MTP structure-function relationships, highlighting two newly identified functional roles: first, the production of small, apolipoprotein (apo)B-containing lipoprotein particles in cardiac myocytes and, second, the lipidation of a major histocompatibility complex class-I related molecule (CD1d) that presents glycolipid antigens to distinct subsets of natural killer T cells. We also discuss the interactions of MTP with proteins such as apoB and CD1d, and the complex mechanisms regulating MTP transcription in different cell types and nutritional states. The past five years has witnessed remarkable progress in teasing out the different functionalities of MTP, and the properties of the different molecules that inhibit MTP activity, data that are likely to underpin the design of the next generation of MTP/apoB inhibitors for preventing cardiovascular disease attributable to the increased production of atherogenic lipoproteins.

USF1 implicated in the aetiology of familial combined hyperlipidaemia and the metabolic syndrome.

The upstream stimulatory factor (USF) proteins are ubiquitously expressed and, as such, represent unusual candidates for involvement in disorders of carbohydrate and lipid metabolism. Nonetheless, a recent study has reported an association between specific alleles of USF1 and familial combined hyperlipidaemia, a common disorder that substantially increases the risk of premature atherosclerotic cardiovascular disease. USF1 might, therefore, also contribute to the metabolic syndrome. The use of chromatin immunoprecipitation methodologies combined with promoter microarray assays will help to define the transcriptional networks that underlie whole-body glucose and lipid homeostasis.

Confirmed locus on chromosome 11p and candidate loci on 6q and 8p for the triglyceride and cholesterol traits of combined hyperlipidemia.

UNLABELLED: Background- Combined hyperlipidemia is a common disorder characterized by a highly atherogenic lipoprotein profile and increased risk of coronary heart disease. The etiology of the lipid abnormalities (increased serum cholesterol and triglyceride or either lipid alone) is unknown. METHODS AND RESULTS: We assembled 2 large cohorts of families with familial combined hyperlipidemia (FCHL) and performed disease and quantitative trait linkage analyses to evaluate the inheritance of the lipid abnormalities. Chromosomal regions 6q16.1-q16.3, 8p23.3-p22, and 11p14.1-q12.1 produced evidence for linkage to FCHL. Chromosomes 6 and 8 are newly identified candidate loci that may respectively contribute to the triglyceride (logarithm of odds [LOD], 1.43; P=0.005) and cholesterol (LOD, 2.2; P=0.0007) components of this condition. The data for chromosome 11 readily fulfil the guidelines required for a confirmed linkage. The causative alleles may contribute to the inheritance of the cholesterol (LOD, 2.04 at 35.2 cM; P=0.0011) component of FCHL as well as the triglyceride trait (LOD, 2.7 at 48.7 cM; P=0.0002). CONCLUSIONS: Genetic analyses identify 2 potentially new loci for FCHL and provide important positional information for cloning the genes within the chromosome 11p14.1-q12.1 interval that contributes to the lipid abnormalities of this highly atherogenic disorder.

Linkage and association between distinct variants of the APOA1/C3/A4/A5 gene cluster and familial combined hyperlipidemia.

OBJECTIVE: Combined hyperlipidemia is a common disorder, characterized by a highly atherogenic lipoprotein profile and a substantially increased risk of coronary heart disease. The purpose of this study was to establish whether variations of apolipoprotein A5 (APOA5), a newly discovered gene of lipid metabolism located 30 kbp downstream of the APOA1/C3/A4 gene cluster, contributes to the transmission of familial combined hyperlipidemia (FCHL). METHODS AND RESULTS: We performed linkage and association tests on 128 families. Two independent alleles, APOA5c.56G and APOC3c.386G, of the APOA1/C3/A4/A5 gene cluster were overtransmitted in FCHL (P=0.004 and 0.007, respectively). This was paired with reduced transmission of the common APOA1/C3/A4/A5 haplotype (frequency 0.4461) to affected subjects (P=0.012). The APOA5c.56G genotype accounted for 7.3% to 13.8% of the variance in plasma triglyceride levels in probands (P<0.004). The APOC3c.386G genotypes accounted for 4.4% to 5.1% of the variance in triglyceride levels in FCHL spouses (P<0.007), suggesting that this allele marks a FCHL quantitative trait as well as representing a susceptibility locus for the condition. CONCLUSIONS: A combined linkage and association analysis establishes that variation at the APOA1/C3/A4/A5 gene cluster contributes to FCHL transmission in a substantial proportion of northern European families.

An integrated reverse functional genomic and metabolic approach to understanding orotic acid-induced fatty liver.

In functional genomics, DNA microarrays for gene expression profiling are increasingly being used to provide insights into biological function or pathology. To better understand the significance of the multiple transcriptional changes across a time period, the temporal changes in phenotype must be described. Orotic acid-induced fatty liver disease was investigated at the transcriptional and metabolic levels using microarrays and metabolic profiling in two strains of rats. High-resolution 1H-NMR spectroscopic analysis of liver tissue indicated that Kyoto rats compared with Wistar rats are predisposed to the insult. Metabolite analysis and gene expression profiling following orotic acid treatment identified perturbed metabolic pathways, including those involved in fatty acid, triglyceride, and phospholipid synthesis, beta-oxidation, altered nucleotide, methyl donor, and carbohydrate metabolism, and stress responses. Multivariate analysis and statistical bootstrapping were used to investigate co-responses with transcripts involved in metabolism and stress responses. This reverse functional genomic strategy highlighted the relationship between changes in the transcription of stearoyl-CoA desaturase 1 and those of other lipid-related transcripts with changes in NMR-derived lipid profiles. The results suggest that the integration of 1H-NMR and gene expression data sets represents a robust method for identifying a focused line of research in a complex system.

New technologies for delineating and characterizing the lipid exome: prospects for understanding familial combined hyperlipidemia.

This review summarizes the progress made in cutting through the biological and genetic complexity of the Gordian knot that is familial combined hyperlipidemia. We particularly focus on how the application of new genomic technologies, especially massively parallel sequencing and high-throughput genotyping platforms, promise to accelerate the gene discovery process in this common, highly atherogenic disorder, with important diagnostic and therapeutic implications.

The intracellular transport of chylomicrons requires the small GTPase, Sar1b.

PURPOSE OF REVIEW: The transport of lipoproteins through the secretory pathways of enterocytes and hepatocytes is pivotal for whole-body lipid homeostasis. This review focuses on the assembly and structural evolution of COPII (coat protein) transport carriers that are essential for the transport of chylomicrons from the endoplasmic reticulum to the Golgi apparatus. RECENT FINDINGS: The assembly of endoplasmic reticulum to Golgi transport carriers commences with the coating of specific areas of the endoplasmic reticulum membrane with Sar1-GTP and the Sec23/24 heterodimer. An important advance has been the crystallographic analysis of the Sar1-Sec23/24 complex. The proteins form a bow-tie shaped structure, with a concave face that seems to match the curvature of transport carriers. Mammalian cells produce two isoforms of Sar1, designated Sar1a and Sar1b, both of which are expressed in enterocytes. Sar1b is defective in chylomicron retention disease and Anderson disease, two rare recessive disorders characterized by severe fat malabsorption and a failure to thrive in infancy. Patients with chylomicron retention disease and Anderson disease selectively retain chylomicron-like particles within membrane-bound compartments. By analogy with procollagen, chylomicrons may drive the formation of endoplasmic reticulum to Golgi transport carriers from endoplasmic reticulum sites close to, but separate from, domains of the endoplasmic reticulum coated with Sar1-Sec23/24. The COPII machinery also mediates the transport of VLDL to the Golgi. SUMMARY: New insights into the role of the COPII machinery in the intracellular transport of cargo, including chylomicrons and VLDL, may suggest new drug targets for ameliorating the lipid abnormalities of the metabolic syndrome.