uciferase reporter gene. Thereafter, cells had been EAI-045 cost incubated with 7 M of AA in presence or absence of apoD. At this concentration, AA will not induce any cellular toxicity [53] but a slight lower in apoD expression (Fig 6A). Over-expression of apoD increased PPAR transcriptional activity (three.7 fold, Fig 6B). Addition of AA alone improved PPAR transcriptional activity to a related extent (three.9-fold). Pretty interestingly, a combination of AA and apoD over-expression showed an extremely strong synergistic transactivation effect on PPAR transcriptional activity (about 9-fold) (Fig 6B).
Lipogenesis inside the liver of H-apoD Tg mice. Western blot evaluation of total and phospho-AMPK (A), total and phospho-ACC (B) and FAS (C) protein expression in the liver of WT and H-apoD Tg mice. The graphs represent the levels of protein expressions standardized by amidoblack staining. Representative gels are presented. D- Semi-quantitative RT-PCR analysis of ACC, SCD1, DGAT and LXR mRNA expression. The graph represents 
the level of mRNA normalized by HPRT. Representative gels are presented. E- In vivo lipogenesis measured in 1 year old mice. The values represent the quantity of 3H2O incorporated into triglycerides. Values are expressed fairly to the WT mice and will be the means SD of four mice per group. P0.05, P0.01 vs WT mice.
Evaluation of genes involved in -oxidation inside the liver of H-apoD Tg mice. A- Western blot analysis of PPAR protein expression. The graph represents the level of PPAR protein expression standardized by amidoblack staining. A representative gel is presented. Semi-quantitative RT-PCR analysis of PGC-1 (B) and CPT1 (C) expression in liver of WT and H-apoD Tg mice. PGC1 and CPT1 gene expression was normalized by HPRT. For each graph, the H-apoD Tg values have been normalized by the WT values and will be the suggests SD of four mice per group. P0.05 and P0.01 vs WT mice.
To support our hypothesis on the function of apoD in AA transport and the 23200243 consequence on PPAR activation, we evaluated the total concentration of AA (totally free and bound to TG and PL) in plasma and liver of WT and H-apoD Tg mice employing quantitative isotope dilution gas chromatography-mass spectrometry. Our information show that the absolute AA concentration is significantly decreased within the plasma of H-apoD Tg compared to WT mice. Consequently, we also observed a important enrichment in hepatic AA within the total fatty acid pool (Fig 7). On the other hand, the prostaglandin E2 concentration is related in between WT and H-apoD Tg mice (S2 Fig) Taken collectively, our study shows that overexpression of H-apoD leads to increased hepatic PPAR expression and subsequent activation of the proteins involved in LD formation. This impact is also related to an elevation of fatty acid uptake whilst lipogenesis remains unaffected. Since the metabolic syndrome is mild, the slight boost inside the mitochondrial – oxidation is almost certainly a compensatory mechanism. Experiments performed with HepG2 cells recommend that the hepatic steatosis is often a outcome of an enhanced AA by apoD in the liver as confirmed by the enrichment of hepatic AA in H-apoD mice. This leads to PPAR transcriptional activation and downstream effects which include the mild metabolic syndrome along with the linked insulin resistance.
PPAR transcriptional activity in presence of AA and/or apoD. A- HepG2 cells were either non transfected (NT) or transfected with a myc-Tag apoD-cDNA or empty vector (EV) construct and incubated with BSA or arachidonic acid (AA). The amount of H-apoD expression
