Human CIDEC transgene improves lipid metabolism and protects against high-fat diet-induced glucose intolerance in mice.

Cell death-inducing DNA fragmentation factor-like effector C (CIDEC) expression in adipose tissue positively correlates with insulin sensitivity in obese humans. Further, E186X, a single-nucleotide CIDEC variant is associated with lipodystrophy, hypertriglyceridemia, and insulin resistance. To establish the unknown mechanistic link between CIDEC and maintenance of systemic glucose homeostasis, we generated transgenic mouse models expressing CIDEC (Ad-CIDECtg) and CIDEC E186X variant (Ad-CIDECmut) transgene specifically in the adipose tissue. We found that Ad-CIDECtg but not Ad-CIDECmut mice were protected against high-fat diet-induced glucose intolerance. Furthermore, we revealed the role of CIDEC in lipid metabolism using transcriptomics and lipidomics. Serum triglycerides, cholesterol, and low-density lipoproteins were lower in high-fat diet-fed Ad-CIDECtg mice compared to their littermate controls. Mechanistically, we demonstrated that CIDEC regulates the enzymatic activity of adipose triglyceride lipase via interacting with its activator, CGI-58, to reduce free fatty acid release and lipotoxicity. In addition, we confirmed that CIDEC is indeed a vital regulator of lipolysis in adipose tissue of obese humans, and treatment with recombinant CIDEC decreased triglyceride breakdown in visceral human adipose tissue. Our study unravels a central pathway whereby adipocyte-specific CIDEC plays a pivotal role in regulating adipose lipid metabolism and whole-body glucose homeostasis. In summary, our findings identify human CIDEC as a potential 'drug' or a 'druggable' target to reverse obesity-induced lipotoxicity and glucose intolerance.

Mouse Fat-Specific Protein 27 (FSP27) expressed in plant cells localizes to lipid droplets and promotes lipid droplet accumulation and fusion.

Fat-Specific Protein 27 (FSP27) belongs to a small group of vertebrate proteins containing a Cell-death Inducing DNA fragmentation factor-α-like Effector (CIDE)-C domain and is involved in lipid droplet (LD) accumulation and energy homeostasis. FSP27 is predominantly expressed in white and brown adipose tissues, as well as liver, and plays a key role in mediating LD-LD fusion. No orthologs have been identified in invertebrates or plants. In this study, we tested the function of mouse FSP27 in stably-transformed Arabidopsis thaliana leaves and seeds, as well as through transient expression in Nicotiana tabacum suspension-cultured cells and N. benthamiana leaves. Confocal microscopic analysis of plant cells revealed that, similar to ectopic expression in mammalian cells, FSP27 produced in plants 1) correctly localized to LDs, 2) accumulated at LD-LD contact sites, and 3) induced an increase in the number and size of LDs and also promoted LD clustering and fusion. Furthermore, FSP27 increased oil content in transgenic A. thaliana seeds. Given that plant oils have uses in human and animal nutrition as well as industrial uses such as biofuels and bioplastics, our results suggest that ectopic expression of FSP27 in plants represents a potential strategy for increasing oil content and energy density in bioenergy or oilseed crops.

Understanding gene expression in coronary artery disease through global profiling, network analysis and independent validation of key candidate genes.

Molecular mechanism underlying the patho-physiology of coronary artery disease (CAD) is complex. We used global expression profiling combined with analysis of biological network to dissect out potential genes and pathways associated with CAD in a representative case-control Asian Indian cohort. We initially performed blood transcriptomics profiling in 20 subjects, including 10 CAD patients and 10 healthy controls on the Agilent microarray platform. Data was analysed with Gene Spring Gx12.5, followed by network analysis using David v 6.7 and Reactome databases. The most significant differentially expressed genes from microarray were independently validated by real time PCR in 97 cases and 97 controls. A total of 190 gene transcripts showed significant differential expression (fold change>2,P<0.05) between the cases and the controls of which 142 genes were upregulated and 48 genes were downregulated. Genes associated with inflammation, immune response, cell regulation, proliferation and apoptotic pathways were enriched, while inflammatory and immune response genes were displayed as hubs in the network, having greater number of interactions with the neighbouring genes. Expression of EGR1/2/3, IL8, CXCL1, PTGS2, CD69, IFNG, FASLG, CCL4, CDC42, DDX58, NFKBID and NR4A2 genes were independently validated; EGR1/2/3 and IL8 showed >8-fold higher expression in cases relative to the controls implying their important role in CAD. In conclusion, global gene expression profiling combined with network analysis can help in identifying key genes and pathways for CAD.

CELSR2-PSRC1-SORT1 gene expression and association with coronary artery disease and plasma lipid levels in an Asian Indian cohort.

BACKGROUND: Genetic regulation of plasma lipids has been shown to influence the risk of coronary artery disease (CAD). We analyzed the relationship between rs599839 and rs646776 single nucleotide polymorphisms (SNPs) present in the CELSR2-PSRC1-SORT1 gene cluster, candidate gene expression, and their association with CAD and circulating lipid levels in a representative cohort of Asian Indians selected from the Indian Atherosclerosis Research Study. METHODS: SNPs rs599839 and rs646776 were genotyped by Taqman assay in 1034 CAD patients (cases) and 1034 age- and gender-matched controls. Expression of CELSR2, PSRC1, and SORT1 genes was measured in 100 cases and 100 controls. Plasma levels of total cholesterol (TC), triglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol (LDL-c) were measured by enzymatic assay. RESULTS: Both rs646776 and rs599839 were in strong linkage disequilibrium (r = 0.98) and showed significant protective association with CAD (OR = 0.315, 95% CI 0.136-0.728, p<0.007 and OR = 0.422, 95% CI 0.181-0.981, p = 0.045, respectively). Haplotype TA showed 72% frequency and was associated with CAD (OR 0.77, 95% CI 0.67-0.88, p = 0.0002). PSRC1 gene expression was lower in the cases than in the controls (0.75 ± 0.405 versus 1.04 ± 0.622, p = 2.26 × 10(-4)). The homozygous variant and heterozygous genotypes showed 30% and 15% higher PSRC1 expression, respectively. Correspondingly, the minor alleles were associated with lower plasma TC and LDL-c levels. CONCLUSION: PSRC1 in the cholesterol gene cluster shows a significant association with CAD by virtue of the two SNPs, rs646776 and rs599839 that also regulate plasma cholesterol levels.

Genetic analysis of the 9p21.3 CAD risk locus in Asian Indians.

The 9p21.3 locus is the best replicated region to date for coronary artery disease (CAD). We investigated the association of 9p21.3 common variants with CAD, candidate gene expression including ANRIL, a non-coding RNA, followed by in vitro validation. Five variants, rs10757278, rs10757274, rs2383206, rs1333049 and rs4977574 were genotyped in 1,034 cases and 1,034 controls. Gene expression of C9orf5, MTAP1, MTAP 2, p16INK4a, p14ARF, p15INK4b and two ANRIL splice variants, NR_003529 and EU741058, were measured in 100 cases and 100 controls. Human aortic smooth muscle cells (HuAoSMCs) were transfected with siRNA targeting ANRIL exon 19 (siRNA-1) or exon 2 (siRNA-2) and consequent effect determined. rs2383206 showed the highest association with CAD (odds ratio [OR] 2.02, 95% confidence interval [CI] 1.56 -2.62) and an adjusted OR of 2.55, 1.33-2.88 along with rs10757278. Conventional risk factors (conventional RFs), rs2383206 and rs10757278 variants together yielded a higher c index (OR 0.790, 95% CI 0.770 -0.810) as compared to conventional RFs (OR 0.783, 95% CI 0.763-0.803) or genetic variants (OR 0.561, 95% CI 0.536-0.586) alone. GAAAA haplotype showed significant protective association with CAD compared to CGGGG risk haplotype (OR 0.45, 95% CI 0.27-0.77). Expression of p16INK4a, p14ARF and p15INK4b as well as plasma CDKN2A levels were lower in cases than controls. GG genotype was associated with higher EU741058 expression and lower p16INK4a expression. HuAoSMCs transfected with siRNA-1 showed lower NR_003529, p16INK4aand p14ARFexpression. Our study provides further evidence on the significance of 9p21.3 locus for CAD wherein the risk allele regulate the expression of ANRIL and adjacent tumour suppressor genes which in turn alter smooth muscle proliferation, a fundamental process in atherosclerosis.

Expression analysis of leukotriene-inflammatory gene interaction network in patients with coronary artery disease.

AIM: Leukotrienes are important lipid inflammatory mediators that play a pivotal role in the pathogenesis of atherosclerosis. We aimed to construct a network of interactions between leukotrienes and inflammatory biomarkers and evaluate the expression of key members of the leukotriene pathway and leukotriene-induced inflammatory molecules in patients with coronary artery disease (CAD) and healthy controls. METHODS: Leukotrienes and their regulatory inflammatory molecules reported in the literature were used to construct a biological network employing Gene spring GX v12.5. Key leukotriene genes and their closely interacting members were selected for expression study in 64 patients and 64 matched controls. Four single nucleotide polymorphisms(SNPs) (rs6538697, rs2660898, rs17525495 and rs1978331) in the leukotriene A4 hydrolase(LTA4H) gene were genotyped using SYBR green method, and plasma leukotriene B4 (LTB4) levels were measured using ELISA. RESULTS: The expression levels of arachidonate 5-lipoxygenase(ALOX5), LTA4H, tumor necrosis factor (TNF) and interleukin-8 (IL-8) genes were significantly higher in patients than in the controls(p＜0.05). IL-8(r=0.35-0.47) and TNF (r=0.42-0.53) expression levels exhibited strong correlations with the leukotriene genes. The SNPs rs17525495 and rs1978331 were associated with LTA4H mRNA expression, while LTA4H and IL-8 levels were associated with CAD. The addition of these two markers to the conventional risk factors improved the c-statistics(area under the receiver operating characteristic(ROC) curve) from 0.75 to 0.93(p＜0.01), with a Net Reclassification Index of 0.45(p＜0.01) and Integrated Discrimination Improvement of 0.26(p＜0.01). CONCLUSIONS: Leukotrienes and inflammatory genes, in particular, LTA4H and IL-8, exhibit close association in subjects with cardiovascular disease. Assessing these markers may provide incremental value for predicting cardiovascular risk beyond that obtained with classical risk factors.

FSP27 promotes lipid droplet clustering and then fusion to regulate triglyceride accumulation.

Fat Specific Protein 27 (FSP27), a lipid droplet (LD) associated protein in adipocytes, regulates triglyceride (TG) storage. In the present study we demonstrate that FSP27 plays a key role in LD morphology to accumulate TGs. We show here that FSP27 promotes clustering of the LDs which is followed by their fusion into fewer and enlarged droplets. To map the domains of FSP27 responsible for these events, we generated GFP-fusion constructs of deletion mutants of FSP27. Microscopic analysis revealed that amino acids 173-220 of FSP27 are necessary and sufficient for both the targeting of FSP27 to LDs and the initial clustering of the droplets. Amino acids 120-140 are essential but not sufficient for LD enlargement, whereas amino acids 120-210 are necessary and sufficient for both clustering and fusion of LDs to form enlarged droplets. In addition, we found that FSP27-mediated enlargement of LDs, but not their clustering, is associated with triglyceride accumulation. These results suggest a model in which FSP27 facilitates LD clustering and then promotes their fusion to form enlarged droplets in two discrete, sequential steps, and a subsequent triglyceride accumulation.

Sumoylation of the zinc finger protein ZXDC enhances the function of its transcriptional activation domain.

The transcription of major histocompatibility complex class II (MHC II) genes is dependent on the co-activator protein class II trans-activator (CIITA). We have recently identified a protein known as zinc finger X-linked duplicated family member C (ZXDC) that, along with its binding partner ZXDA, forms a complex that interacts with CIITA and regulates MHC II transcription. Western blot analysis with anti-ZXDC antibodies identified two species of the ZXDC protein, one migrating near its predicted molecular mass and one with slower electrophoretic mobility. We report here that the slower migrating form is the result of sumoylation at a single lysine residue within the transcriptional activation domain of ZXDC. Three SUMO proteins (SUMO-1, -2 and -3) can modify the ZXDC protein. Multiple SUMO E3 ligase enzymes and HDAC4 can facilitate ZXDC sumoylation, and one ligase, PIASy, interacts with a specific region of the ZXDC protein. We found that sumoylation does not appear to disrupt or modulate the interaction of ZXDC with its binding partners. Rather, sumoylation of ZXDC is required for full activity of the transcriptional activation domain. Our findings suggest that sumoylation is an important regulator of ZXDC.

The zinc finger proteins ZXDA and ZXDC form a complex that binds CIITA and regulates MHC II gene transcription.

The transcription of major histocompatibility complex class II (MHC II) genes depends critically upon the activity of the class II trans-activator (CIITA) protein. We previously described a novel CIITA-binding protein named zinc finger X-linked duplicated family member C (ZXDC) that contributes to the activity of CIITA and the transcription of MHC II genes. Here, we examined the contribution of a closely related family member of ZXDC, the ZXDA protein, to MHC II gene transcription. ZXDA has a domain organization similar to ZXDC, containing ten zinc fingers and a transcriptional activation domain. Knockdown and overexpression of ZXDA demonstrated its importance in the transcriptional activation of MHC II genes. We found that ZXDA and ZXDC can self-associate, and also form a complex with each other. The regions of the two proteins that contain zinc fingers mediate these interactions. Importantly, we found that the ZXDA-ZXDC complex interacts with CIITA, rather than either protein alone. Given our additional finding that ZXDC is present at MHC II promoters in HeLa cells, prior to and after treatment with IFN-gamma, it appears that ZXDA and ZXDC form an important regulatory complex for MHC II gene transcription.

ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription.

The class II trans-activator (CIITA) is recognized as the master regulator of major histocompatibility complex (MHC) class II gene transcription and contributes to the transcription of MHC class I genes. To better understand the function of CIITA, we performed yeast two-hybrid with the C-terminal 807 amino acids of CIITA, and cloned a novel human cDNA named zinc finger, X-linked, duplicated family member C (ZXDC). The 858 amino acid ZXDC protein contains 10 zinc fingers and a transcriptional activation domain, and was found to interact with the region of CIITA containing leucine-rich repeats. Over-expression of ZXDC in human cell lines resulted in super-activation of MHC class I and class II promoters by CIITA. Conversely, silencing of ZXDC expression reduced the ability of CIITA to activate transcription of MHC class II genes. Given the specific interaction between the ZXDC and CIITA proteins, as well as the effect of ZXDC on MHC gene transcription, it appears that ZXDC is an important regulator of both MHC class I and class II transcription.