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Blood lipid levels are heritable, treatable risk factors for cardiovascular disease. We systematically assessed genome-wide coding variation to identify novel lipid genes, fine-map known lipid loci, and evaluate whether low frequency variants with large effect exist. Using an exome array, we genotyped 80,137 coding variants in 5,643 Norwegians. We followed up 18 variants in 4,666 Norwegians to identify 10 loci with coding variants associated with a lipid trait (P < 5×10−8). One coding variant in TM6SF2 (p.Glu167Lys), residing in a GWAS locus for lipid levels, modifies total cholesterol levels and is associated with myocardial infarction. Transient overexpression and knockdown of TM6SF2 in mouse produces alteration in serum lipid profiles consistent with the association observed in humans, identifying TM6SF2 as the functional gene at a large GWAS locus previously known as NCAN/CILP2/PBX4 or 19p13. This study demonstrates that systematic assessment of coding variation can quickly point to a candidate causal gene.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily. Originally cloned in 1990, PPARs were found to be a mediator of pharmacological agents that induce hepatocyte peroxisome proliferation. PPARs are also expressed in cells of the cardiovascular system. PPARγ appears to be highly expressed during atherosclerotic lesion formation, suggesting that increased PPARγ expression may be a vascular compensatory response. Also, ligand-activated PPARγ decreases the inflammatory response in cardiovascular cells, particularly in endothelial cells. PPARα, similar to PPARγ, also has pleiotropic effects in the cardiovascular system, including anti-inflammatory and anti-atherosclerotic properties. PPARα activation inhibits vascular smooth muscle pro-inflammatory responses, attenuating the development of atherosclerosis. However, PPARδ overexpression may lead to elevated macrophage inflammation and atherosclerosis. On the other hand, PPARδ ligands are shown to attenuate the pathogenesis of atherosclerosis by improving endothelial cell proliferation and survival while decreasing endothelial cell inflammation and vascular smooth muscle cell proliferation. Furthermore, the administration of PPAR ligands in the form of TZDs and fibrates has been disappointing in terms of markedly reducing cardiovascular events in the clinical setting. Therefore, a better understanding of PPAR-dependent and -independent signaling will provide the foundation for future research on the role of PPARs in human cardiovascular biology.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (AGXT1). Genetic (Agxt1−/− mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid β-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus Clostridium sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFβ/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.

Objective-Endothelial activation is implicated in atherogenesis and diabetes. The role of peroxisome proliferator-activated receptor-␦ (PPAR-␦) in endothelial activation remains poorly understood. In this study, we investigated the anti-inflammatory effect of PPAR-␦ and the mechanism involved. Methods and Results-In human umbilical vein endothelial cells (HUVECs), the synthetic PPAR-␦ ligands GW0742 and GW501516 significantly inhibited tumor necrosis factor (TNF)-␣-induced expression of vascular cell adhesion molecule-1 and E-selectin (assayed by real-time RT-PCR and Northern blotting), as well as the ensuing endothelialleukocyte adhesion. Activation of PPAR-␦ upregulated the expression of antioxidant genes superoxide dismutase 1, catalase, and thioredoxin and decreased reactive oxygen species production in ECs. Chromatin immunoprecipitation assays showed that GW0742 switched the association of BCL-6, a transcription repressor, from PPAR-␦ to the vascular cell adhesion molecule (VCAM)-1 promoter. Small interfering RNA reduced endogenous PPAR-␦ expression but potentiated the suppressive effect of GW0742 on EC activation, which suggests that the nonliganded PPAR-␦ may have an opposite effect. Conclusions-We have demonstrated that ligand activation of PPAR-␦ in ECs has a potent antiinflammatory effect, probably via a binary mechanism involving the induction of antioxidative genes and the release of nuclear corepressors. PPAR-␦ agonists may have a potential for treating inflammatory diseases such as atherosclerosis and diabetes.

Klotho is an anti-aging gene whose expression is regulated by many stimuli. Here we examined the transcriptional regulation of the klotho gene by peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The PPAR-gamma agonists thiazolidinediones increased both klotho mRNA and protein expression in HEK293 cells and several renal epithelial cell lines. The induction was blocked by PPAR-gamma antagonists or small-interfering RNA-mediated gene silencing of PPAR-gamma, suggesting a PPAR-gamma-dependent mechanism. Chromatin immuno-precipitation and gel shift assays found a noncanonical PPAR-responsive element within the 5'-flanking region of the human klotho gene with promoter-reporter assays further confirming transcriptional functionality. Moreover, thiazolidinediones or adenovirus-mediated overexpression of PPAR-gamma increased klotho expression in mouse kidneys while renal klotho expression was attenuated in mice treated with PPAR-gamma antagonists. These results demonstrate that klotho is a target gene of PPAR-gamma.

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Peroxisome proliferator-activated receptor gamma (PPARγ) is emerging as a major regulator in neurological diseases. However, the role of (PPARγ) and its co-regulators in cerebrovascular endothelial dysfunction after stroke is unclear. Here, we have demonstrated that (PPARγ) activation by pioglitazone significantly inhibited both oxygen-glucose deprivation-induced cerebral vascular endothelial cell death and middle cerebral artery occlusion-triggered cerebrovascular damage. Consistent with this finding, selective (PPARγ) genetic deletion in vascular endothelial cells resulted in increased cerebrovascular permeability and brain infarction in mice after focal ischaemia. Moreover, we screened for (PPARγ) co-regulators using a genome-wide and high-throughput co-activation system and revealed KLF11 as a novel (PPARγ) co-regulator, which interacted with (PPARγ) and regulated its function in mouse cerebral vascular endothelial cell cultures. Interestingly, KLF11 was also found as a direct transcriptional target of (PPARγ). Furthermore, KLF11 genetic deficiency effectively abolished pioglitazone cytoprotection in mouse cerebral vascular endothelial cell cultures after oxygen-glucose deprivation, as well as pioglitazone-mediated cerebrovascular protection in a mouse middle cerebral artery occlusion model. Mechanistically, we demonstrated that KLF11 enhanced (PPARγ) transcriptional suppression of the pro-apoptotic microRNA-15a (miR-15a) gene, resulting in endothelial protection in cerebral vascular endothelial cell cultures and cerebral microvasculature after ischaemic stimuli. Taken together, our data demonstrate that recruitment of KLF11 as a novel (PPARγ) co-regulator plays a critical role in the cerebrovascular protection after ischaemic insults. It is anticipated that elucidating the coordinated actions of KLF11 and (PPARγ) will provide new insights into understanding the molecular mechanisms underlying (PPARγ) function in the cerebral vasculature and help to develop a novel therapeutic strategy for the treatment of stroke.