Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α.     

Coordinate induction of PPARα and SREBP2 in multifunctional protein 2 deficient mice

Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal β-oxidation, develop multiple pathologies in diverse tissues already starting in the postnatal period. Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPARα responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPARα target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, in lactating and in adult MFP2 knockout mice. In accordance, the rate of cholesterol biosynthesis was significantly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In MFP2/PPARα double knockout mice, up-regulations of SREBP2 and HMGCR were markedly attenuated. These data demonstrate a tight interrelationship between induction of PPARα by endogenous ligands and up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2.     

Activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase during high fat diet feeding

The liver plays a central role in regulating cholesterol homeostasis. High fat diets have been shown to induce obesity and hyperlipidemia. Despite considerable advances in our understanding of cholesterol metabolism, the regulation of liver cholesterol biosynthesis in response to high fat diet feeding has not been fully addressed. The aim of the present study was to investigate mechanisms by which a high fat diet caused activation of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) leading to increased cholesterol biosynthesis. Mice were fed a high fat diet (60% kcal fat) for 5 weeks. High fat diet feeding induced weight gain and elevated lipid levels (total cholesterol and triglyceride) in both the liver and serum. Despite cholesterol accumulation in the liver, there was a significant increase in hepatic HMG-CoA reductase mRNA and protein expression as well as enzyme activity. The DNA binding activity of sterol regulatory element binding protein (SREBP)-2 and specific protein 1 (Sp1) were also increased in the liver of mice fed a high fat diet. To validate the in vivo findings, HepG2 cells were treated with palmitic acid. Such a treatment activated SREBP-2 as well as increased the mRNA and enzyme activity of HMG-CoA reductase leading to intracellular cholesterol accumulation. Inhibition of Sp1 by siRNA transfection abolished palmitic acid-induced SREBP-2 and HMG-CoA reductase mRNA expression. These results suggest that Sp1-mediated SREBP-2 activation contributes to high fat diet induced HMG-CoA reductase activation and increased cholesterol biosynthesis. This may play a role in liver cholesterol accumulation and hypercholesterolemia.     

Luteolin Inhibits Human Prostate Tumor Growth by Suppressing Vascular Endothelial Growth Factor Receptor 2-Mediated Angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.