FAM13A affects body fat distribution and adipocyte function.

Genetic variation in the FAM13A (Family with Sequence Similarity 13 Member A) locus has been associated with several glycemic and metabolic traits in genome-wide association studies (GWAS). Here, we demonstrate that in humans, FAM13A alleles are associated with increased FAM13A expression in subcutaneous adipose tissue (SAT) and an insulin resistance-related phenotype (e.g. higher waist-to-hip ratio and fasting insulin levels, but lower body fat). In human adipocyte models, knockdown of FAM13A in preadipocytes accelerates adipocyte differentiation. In mice, Fam13a knockout (KO) have a lower visceral to subcutaneous fat (VAT/SAT) ratio after high-fat diet challenge, in comparison to their wild-type counterparts. Subcutaneous adipocytes in KO mice show a size distribution shift toward an increased number of smaller adipocytes, along with an improved adipogenic potential. Our results indicate that GWAS-associated variants within the FAM13A locus alter adipose FAM13A expression, which in turn, regulates adipocyte differentiation and contribute to changes in body fat distribution.

Nat1 Deficiency Is Associated with Mitochondrial Dysfunction and Exercise Intolerance in Mice.

We recently identified human N-acetyltransferase 2 (NAT2) as an insulin resistance (IR) gene. Here, we examine the cellular mechanism linking NAT2 to IR and find that Nat1 (mouse ortholog of NAT2) is co-regulated with key mitochondrial genes. RNAi-mediated silencing of Nat1 led to mitochondrial dysfunction characterized by increased intracellular reactive oxygen species and mitochondrial fragmentation as well as decreased mitochondrial membrane potential, biogenesis, mass, cellular respiration, and ATP generation. These effects were consistent in 3T3-L1 adipocytes, C2C12 myoblasts, and in tissues from Nat1-deficient mice, including white adipose tissue, heart, and skeletal muscle. Nat1-deficient mice had changes in plasma metabolites and lipids consistent with a decreased ability to utilize fats for energy and a decrease in basal metabolic rate and exercise capacity without altered thermogenesis. Collectively, our results suggest that Nat1 deficiency results in mitochondrial dysfunction, which may constitute a mechanistic link between this gene and IR.

Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

miR-206 controls LXRα expression and promotes LXR-mediated cholesterol efflux in macrophages.

Liver X receptors (LXRα and LXRß) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRß. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.

Nicotinic acid inhibits hepatic APOA gene expression: studies in humans and in transgenic mice.

Elevated plasma lipoprotein(a) (LPA) levels are recognized as an independent risk factor for cardiovascular diseases. Our knowledge on LPA metabolism is incomplete, which makes it difficult to develop LPA-lowering medications. Nicotinic acid (NA) is the main drug recommended for the treatment of patients with increased plasma LPA concentrations. The mechanism of NA in lowering LPA is virtually unknown. To study this mechanism, we treated transgenic (tg) APOA mice with NA and measured plasma APOA and hepatic mRNA levels. In addition, mouse and human primary hepatocytes were incubated with NA, and the expression of APOA was followed. Feeding 1% NA reduced plasma APOA and hepatic expression of APOA in tg-APOA mice. Experiments with cultured human and mouse primary hepatocytes in addition to reporter assays performed in HepG2 cells revealed that NA suppresses APOA transcription. The region between -1446 and -857 of the human APOA promoter harboring several cAMP response element binding sites conferred the negative effect of NA. In accordance, cAMP stimulated APOA transcription, and NA reduced hepatic cAMP levels. It is suggested that cAMP signaling might be involved in reducing APOA transcription, which leads to the lowering of plasma LPA.

FGF19 signaling cascade suppresses APOA gene expression.

OBJECTIVE: Lipoprotein(a) is a highly atherogenic lipoprotein, whose metabolism is poorly understood. Currently no safe drugs exists that lower elevated plasma lipoprotein(a) concentrations. We therefore focused on molecular mechanisms that influence apolipoprotein(a) (APOA) biosynthesis. METHODS AND RESULTS: Transgenic human APOA mice (tg-APO mice) were injected with 1 mg/kg of recombinant human fibroblast growth factor 19 (FGF19). This led to a significant reduction of plasma APOA and hepatic expression of APOA. Incubation of primary hepatocytes of tg-APOA mice with FGF19 induced ERK1/2 phosphorylation and, in turn, downregulated APOA expression. Repression of APOA by FGF19 was abrogated by specific ERK1/2 phosphorylation inhibitors. The FGF19 effect on APOA was attenuated by transfection of primary hepatocytes with siRNA against the FGF19 receptor 4 (FGFR4). Using promoter reporter assays, mutation analysis, gel shift, and chromatin immune-precipitation assays, an Ets-1 binding element was identified at -1630/-1615bp region in the human APOA promoter. This element functions as an Elk-1 binding site that mediates repression of APOA transcription by FGF19. CONCLUSIONS: These findings provide mechanistic insights into the transcriptional regulation of human APOA by FGF19. Further studies in the human system are required to substantiate our findings and to design therapeutics for hyper lipoprotein(a).

Farnesoid X receptor represses hepatic human APOA gene expression.

High plasma concentrations of lipoprotein(a) [Lp(a), which is encoded by the APOA gene] increase an individual's risk of developing diseases, such as coronary artery diseases, restenosis, and stroke. Unfortunately, increased Lp(a) levels are minimally influenced by dietary changes or drug treatment. Further, the development of Lp(a)-specific medications has been hampered by limited knowledge of Lp(a) metabolism. In this study, we identified patients suffering from biliary obstructions with very low plasma Lp(a) concentrations that rise substantially after surgical intervention. Consistent with this, common bile duct ligation in mice transgenic for human APOA (tg-APOA mice) lowered plasma concentrations and hepatic expression of APOA. To test whether farnesoid X receptor (FXR), which is activated by bile acids, was responsible for the low plasma Lp(a) levels in cholestatic patients and mice, we treated tg-APOA and tg-APOA/Fxr-/- mice with cholic acid. FXR activation markedly reduced plasma concentrations and hepatic expression of human APOA in tg-APOA mice but not in tg-APOA/Fxr-/- mice. Incubation of primary hepatocytes from tg-APOA mice with bile acids dose dependently downregulated APOA expression. Further analysis determined that the direct repeat 1 element between nucleotides -826 and -814 of the APOA promoter functioned as a negative FXR response element. This motif is also bound by hepatocyte nuclear factor 4α (HNF4α), which promotes APOA transcription, and FXR was shown to compete with HNF4α for binding to this motif. These findings may have important implications in the development of Lp(a)-lowering medications.