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.
We show that silencing of ANGPTL3 in human hepatocytes in addition to reducing secretion of TAG-enriched VLDL upon insulin stimulation enhances glucose uptake and improves insulin response. Thus, our data provide insight into the lower insulin and glucose levels observed in humans with ANGPTL3 loss-of-function mutation.
ANGPTL3 silencing; hypolipidaemia; insulin signalling; liver; rosiglitazone; VLDL; ANGPTL, angiopoietin-like protein; CCD, coiled coil domain; FHBL2, familial combined hypolipidaemia; GLUT2, glucose transporter 2; HDL, high-density lipoprotein; IHH, immortalized human hepatocyte; IR, insulin receptor; IRS, insulin receptor substrate; LDL, low-density lipoprotein; LPL, lipoprotein lipase; NEFA, non-esterified fatty acid; PEPCK, phosphoenolpyruvate carboxykinase; PGC1α, peroxisome proliferator-activated receptor γ co-activator 1-α; PI3K, phosphoinositide 3-kinase; PL, phospholipid; PPAR, peroxisome-proliferator-activated receptor; QPCR, quantitative PCR; shRNA, small hairpin RNA; TAG, triacylglycerol; TRB3, tribbles homologue 3; VLDL, very-low-density lipoprotein
USF1 is a ubiquitous transcription factor governing the expression of numerous genes of lipid and glucose metabolism. APOA5 is a well-established candidate gene regulating triglyceride (TG) levels and has been identified as a downstream target of upstream stimulatory factor. No detailed studies about the effect of APOA5 on atherosclerotic lesion formation have been conducted, nor has its potential interaction with USF1 been examined.
Methods and Results
We analyzed allelic variants of USF1 and APOA5 in families (n=516) ascertained for atherogenic dyslipidemia and in an autopsy series of middle-aged men (n=300) with precise quantitative measurements of atherosclerotic lesions. The impact of previously associated APOA5 variants on TGs was observed in the dyslipidemic families, and variant rs3135506 was associated with size of fibrotic aortic lesions in the autopsy series. The USF1 variant rs2516839, associated previously with atherosclerotic lesions, showed an effect on TGs in members of the dyslipidemic families with documented coronary artery disease. We provide preliminary evidence of gene-gene interaction between these variants in an autopsy series with a fibrotic lesion area in the abdominal aorta (P=0.0028), with TGs in dyslipidemic coronary artery disease subjects (P=0.03), and with high-density lipoprotein cholesterol (P=0.008) in a large population cohort of coronary artery disease patients (n=1065) in which the interaction for TGs was not replicated.
Our findings in these unique samples reinforce the roles of APOA5 and USF1 variants on cardiovascular phenotypes and suggest that both genes contribute to lipid levels and aortic atherosclerosis individually and possibly through epistatic effects.
genes; USF1; APOA5; lipids; atherosclerosis; epistasis