PPAR-γ induces adipocyte differentiation and genes involved in fat deposition

<p><b>Copyright information:</b></p><p>Taken from "PPAR-γ: a thrifty transcription factor"</p><p>Nuclear Receptor Signaling 2003;1():-.</p><p>Published online 27 Jun 2003</p><p>PMCID:PMC1402226.</p><p>Copyright © 2003, Auwerx et al. This is an open-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercial use distribution and reproduction in any medium, provided the original work is properly cited. </p> Plasma-derived fatty acids are directed to adipose tissue at the expense of skeletal muscle, which increases glucose uptake and utilization in the muscle. Direct effects of PPAR-γ activation have also been observed in liver, including decreased gluconeogenesis and increased fat uptake and storage. Additionally, PPAR-γ activation results in increased cholesterol efflux in macrophages via upregulation of ABCA1, but also increased uptake of proatherogenic oxidized LDL particles via upregulation of CD36

Evolutionary analysis links <i>Ahr</i> to movement.

<p>(<b>A</b>) Phylogenetic BLAST analysis of mouse <i>Ahr</i> showed that the gene is highly conserved down to simple multicellular animals such as <i>C. elegans</i>, the gene likely has conserved basic metabolic functions. (<b>B</b>) Sequence analysis of the three missense mutations of <i>Ahr</i> between B6 and D2 known to have an impact oh AHR activity (375, 471, 805). (<b>C</b>) B6 mice with the humanized AHR allele are nearly twice as active as controls. The humanized AHR allele is similar to the D2 allele in many tests of enzymatic activity, with a ∼90%+ reduction in activity compared to the B6 allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004673#pgen.1004673-Connor1" target="_blank">[24]</a>. (<b>D</b>) <i>D. melanogaster</i> with a heterozygous deletion allele of the <i>Ahr</i> ortholog <i>ss</i> are also significantly more active than controls. The 50% reduced expression appears to increase movement by about 20% in both males and females. Each comparison is a separate Welch's <i>t</i>-test with <i>p</i><0.001. Females are ∼30% less active than males in both instances (<i>p</i><0.001). (<b>E</b>) <i>C. elegans</i> treated from early development with RNAi for <i>ahr-1</i> are nearly twice as active as worms treated with a control vector. Reduced doses of RNAi have intermediary effects on activity. <i>p</i> = 2.9e-6 for 100% vs. empty vector (ev).</p

Increased pSmad2/3 expression and activation of TGFβ signaling in LRP⁻ mouse aorta.

<p>Longitudinal sections of abdominal aorta from SM22Cre⁺;LRP^flox/flox;LDLR^−/− (LRP⁻) and LRP^flox/flox;LDLR^−/− (LRP⁺) mice were stained with anti-TSP1, anti-TGFβ1, anti-pSmad2/3 and anti-pSmad1 antibodies. Reduced LRP1 expression results in greatly enhanced expression of pSmad2/3 and its target gene, TSP1. By contrast, TGFβ1 levels were slightly reduced, pSmad1 levels did not change. Bar in a indicates 20 µm.</p

Identification and validation of a movement QTL.

<p>(<b>A</b>) Nighttime rearing and ambulatory activity for all 68 males and 68 females phenotyped across 22 (male) and 19 (female) strains. These 19 strains were phenotyped in both sexes. Females are slightly more active. (<b>B</b>) Despite moving somewhat more, female and male activity are strongly correlated by strain. (<b>C</b>) Nighttime rearing and ambulatory activities for all 196 animals across 43 strains. Each strain has ∼5 biological replicates. (<b>D</b>) Ambulatory and rearing activity are tightly correlated, though the measurements are technically independent. (<b>E</b>) Nighttime rearing activity for all 43 strains, ordered by value. Activity varies by 3.9 fold across the population. The strong heritability (h²) of 0.68 indicates that the majority of this variance can be attributed to genetic factors. (<b>F</b>) Body weight (Left) and food intake (Right) have no effect on ambulatory or rearing activity, suggesting movement is largely independent of the weight or the need to eat or drink. Animals must rear to reach the food basket or drink. (<b>G</b>) Rearing and ambulatory movement mapped to a common narrow 2 Mb locus on chromosome 12. (<b>H</b>) The target locus (chromosome 12 from 35.5–37.6 Mb) explains ∼40% of variance (r²) in rearing activity and ∼25% of variance in ambulatory activity.</p

<i>C</i>. <i>elegans</i> immobilization protocol using a hydrogel-microbead matrix.

<p>(A) Worms are transferred from an agar plate into a droplet of liquid S Medium on a glass slide. (B) Precooled (~4°C) liquid Pluronic-microbead suspension is applied on the glass slide around the S Medium (red trace) and on a coverslip (not shown here). (C) The coverslip is positioned upside down and worms are immobilized in the gel-microbead matrix (red) in between the two glass substrates after thermalization (<i>T</i> ≈ 25°C). (D) Schematics of the worm immobilization technique with microbead spacers.</p

Schematic of the visOCM setup for <i>C. elegans</i> imaging.

<p>Light from a laser source with a broad spectrum in the visible range (<b>A</b>, inset) is collimated by lens L1 and split by beam-splitter BS1 into a sample (green) and reference (blue) arm. In the sample arm, the axicon lens generates a Bessel-like illumination beam which is then guided to the tube lens (TL) and objective by the X-Y galvo-scanner unit. The back-reflected light (red) from the sample (<b>B</b>, inset) is recombined with the reference arm by beam-splitter BS2 and focused by L2 into the detection fiber. Finally, the spectrometer (<b>C</b>, inset), records the interference pattern which is processed to yield a depth profile of the <i>C. elegans</i> structure. The data processing steps are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181676#pone.0181676.s001" target="_blank">S1 Fig</a>. Scale bars: 25 μm.</p

Specific inhibition of ATG5 and ATG7 decrease low glucose-induced LC3-II accumulation.

<p>Lentiviruses expressing specific shRNA were used to decrease the expression of ATG5 and ATG7 as described in material & methods, then each clonal cell colony was treated and cultured as mentioned in material & methods. <b>A)</b> Representative western blot analysis of ATG7, and quantification. Results are expressed as mean ± SEM of 3 experiments performed in triplicate, *p<0.0001. <b>B)</b> Quantification of <i>Atg5</i> mRNA expression was performed by PCR and results expressed as mean ± SEM of 2 experiments performed in duplicate, *p<0.002. <b>C)</b> Representative western blot analysis of LC3-II expression in specific ATG7 and ATG5 knockdown cells cultured at 1 mM and 25 mM glucose during 48 h and quantification. Results are expressed as mean ± SEM of 3–4 experiments performed in triplicate, #p<0.0001.</p

3-MA chemical inhibition of autophagy increases low glucose-induced cell death and caspase 3 activity.

<p>661W cells were cultured as mentioned in material & methods, and then incubated at low (1 mM) or high (25 mM) glucose for different periods of time (8 or 48 h). <b>A)</b> TUNEL assay was performed in absence (a, d) or in presence (b, c and e, f) of 600 µM 3-MA, at 1 mM (a, b, c) or 25 mM (d, e, f) glucose concentrations, for different periods of time as indicated to the left. White arrows show TUNEL positive cells. Quantification of TUNEL positive cells was performed in three different experiments, *p<0.05; #p<0.0001 and **p<0.002 <b>B)</b> Measures of Caspase 3 activity, results are expressed as mean ± SEM of 3 experiments (n = 13), *p<0.04 and #p<0.0002, and immunostaining of cleaved Caspase 3 in 661W cells incubated at 1 mM (a and b) or 25 mM (c and d) glucose concentrations in absence (a and c) or presence of 600 µM 3-MA (b and d).</p

Activation of TGFβ and PDGF signaling in LRP⁻ mouse aortas are both prevented upon rosiglitazone treatment.

<p>Mice had been cholesterol-fed for 5 weeks in the absence (−Rosi) or presence (+Rosi) of rosiglitazone (GlaxoSmithKline, 25 mg/kg/day) before analysis. Mouse aortas expressing (LRP⁺) or not expressing (LRP⁻) LRP in VSMC were analyzed by western blot (Panel A) and immunohistochemistry (Panel B) for expression of PDGFRβ (d–f), and for activation of Smad2/3 (pSmad2/3, a–c), and Erk1/2 (pErk1/2, g–i). Panel C shows elastic staining of corresponding sections and gaps in elastic fiber continuity (arrows). Bar indicates 40 µm, insert scale bar in B,a indicates 10 µm.</p

Anatomy of the <i>C. elegans</i> as revealed by visOCM.

<p><b>(A, B)</b><i>En face</i> projections at two different depths, and <b>(C)</b> side view at the location highlighted in <b>(B)</b>. Scale bars indicate 50 μm. <b>(D)</b> Top: A 3D rendered model of the head with the pharynx highlighted in green. Bottom: Maximum-intensity projection through the entire animal’s head. <b>(E)</b> <i>En face</i> view (top) and corresponding transverse sections (bottom), with the lumen of the intestine highlighted in yellow. <b>(F)</b> Zoom regions of the reproductive system showing germ cells, oocytes, spermatheca, embryos and the vulva. The 3D sub-micrometer resolution and the intrinsic contrast of our technique enable a clear and detailed visualization of tissue structures down to the sub-cellular level (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181676#pone.0181676.s003" target="_blank">S2 Video</a>).</p