Metabolic changes in WT and <i>db/db</i> mice treated with TO901317 (TO) or DMSO (control).

<p>The WT and <i>db/db</i> mice were fed a standard laboratory diet for 9 weeks. Body weight and the fasting glucose at the basal level <b>(A)</b>, intraperitoneal insulin tolerance test (IPITT) curves before treatment <b>(B)</b> were measured. Then the mice were given a daily intraperitoneal injection of TO or DMSO respectively. Effect of TO and DMSO on fasting glucose <b>(C)</b>, fasting insulin and HOMA-IR level <b>(D)</b>, body weight <b>(E)</b> and fasting FFA <b>(F)</b> were measured after 2 weeks treatment. Results are presented as mean ± SD, *^{, #}P<0.05. 10–15 mice were used in each group.</p

Activation of the Liver X Receptor by Agonist TO901317 Improves Hepatic Insulin Resistance via Suppressing Reactive Oxygen Species and JNK Pathway

<div><p>Activation of Liver X receptors (LXRs), key transcriptional regulators of glucose metabolism, normalizes glycemia and improves insulin sensitivity in rodent models with insulin resistance. However, the molecular mechanism is unclear. This study is aimed to elucidate the mechanism of LXRs-mediated liver glucose metabolic regulation <i>in vitro</i> and <i>in vivo</i>. <i>Db/db</i> mice were used as an <i>in vivo</i> model of diabetes; palmitate (PA)-stimulated HepG2 cells were used as an <i>in vitro</i> cell model with impairment of insulin signaling. TO901317 (TO) was chosen as the LXRs agonist. We demonstrated that TO treatment for 14 days potently improved the hepatic glucose metabolism in <i>db/db</i> mice, including fasting blood glucose, fasting insulin level, and HOMA-IR. TO had no effect on the glucose metabolism in normal WT mice. TO-mediated activation of hepatic LXRs led to strong inhibition of ROS production accompanied by inactivation of JNK pathway and re-activation of Akt pathway. TO also suppressed the expression of gluconeogenic genes such as PEPCK and G-6-pase in <i>db/db</i> mice, but not in WT mice. In HepG2 cells, TO almost completely restored PA-induced Akt inactivation, and suppressed PA-stimulated ROS production and JNK activation. Interestingly, basal level of ROS was also inhibited by TO in HepG2 cells. TO significantly inhibited PA-stimulated expressions of gluconeogenic genes. Finally, we found that anti-oxidative genes, such as Nrf2, were up-regulated after LXRs activation by TO. These results strongly support the notion that activation of LXRs is critical in suppression of liver gluconeogenesis and improvement of insulin sensitivity in diabetic individuals. At molecular levels, the mode of action appears to be as fellows: under diabetic condition, ROS production is increased, JNK is activated, and Akt activity is inhibited; TO-mediated LXR activation potently inhibits ROS production, increases anti-oxidative gene expressions, suppresses JNK activation, and restores Akt activity. Our data provide new evidence to support LXRs as promising therapeutic targets for anti-diabetic drug development.</p></div

TO901317 (TO) suppressed both ROS production and JNK phosphorylation in the liver of <i>db/db</i> mice.

<p><b>(A)</b> After 2 weeks treatment of TO or DMSO (control), ROS production in the liver was assessed by confocal microscope with <i>in situ</i> DHE stain. ROS generation was dramatically reduced in the TO-treated <i>db/db</i> mice, compared with that in DMSO controls. But there were no difference between the WT mice. <b>(B)</b> TO treatment (2 weeks) significantly reduced MDA levels in both plasma and liver of <i>db/db</i> mice, but not WT mice. <b>(C)</b> After 2 weeks treatment of TO or DMSO, mice liver tissues were collected for the WB analyses of JNK. JNK phosphorylation was significantly decreased in <i>db/db</i> mice with TO treatment, compared with that in DMSO controls, while the levels of total JNK protein were unaltered. No significant change was observed in either phosphorylation or total JNK protein levels in WT mice. Results in (<b>B</b>) are presented as mean ± SD, *P<0.05. Representatives of 10–15 mice in each group were shown.</p

Sequences of the primers used in real-time PCR.

<p>Sequences of the primers used in real-time PCR.</p

TO901317 (TO) suppressed the expressions of PEPCK and G-6-Pase and increased the Akt phosphorylation in <i>db/db</i> mice.

<p><b>(A&B)</b> Expressions of PEPCK <b>(A)</b> and G-6-Pase <b>(B)</b> were significantly decreased in TO-treated <i>db/db</i> mice, compared with the DMSO-treated controls, but no significant differences between TO and DMSO-treated WT mice. <b>(C)</b> SREBP-1c expression was significantly increased in both WT and TO-treated <i>db/db</i> mice, compared with the respective DMSO-treated controls. <b>(D)</b> Insulin-stimulated phosphorylation of Akt was significantly increased in TO-treated <i>db/db</i> mice while the total Akt protein levels remained the same, but there was no change in either phosphorylation or total protein of IRS1, compared with DMSO-treated controls. TO had no effect on the phosphorylation and total protein of either Akt or IRS1 in WT mice. Results in (<b>A</b>), (<b>B</b>), and (<b>C</b>) are presented as mean ± SD, *P<0.05. 10–15 mice were used in each group.</p

Effect of TO901317 on palmitate-induced alterations in insulin-stimulated phosphorylation of AKT and the expressions of PEPCK and G-6-Pase in HepG2 cells.

<p><b>(A)</b> HepG2 cells were incubated in the presence or absence of palmitate (PA) [0 (control), 100, 250, 500, 750 μM] for 16 hr prior to 15 min stimulation with insulin (0, 1, 10, 100 nM). Total cell lysates were collected for WB analyses. Insulin activated Akt phosphorylation in HepG2 cells with a dose-dependent manner, but it was effectively suppressed by PA in a dose dependent manner, with almost a completely suppression at 750 μM. <b>(B)</b> HepG2 cells were incubated with either TO (1.0 μM) or PA (500 μM) for various periods of time (0, 4, 8, 12, 16, 24 hr) respectively. Total cell lysates were collected for the real-time PCR. PA treatment increased the expressions of PEPCK and G-6-Pase gradually over time, and peaked at 16hr, while there was no significant change in TO-treated cells. <b>(C &D)</b> HepG2 cells were incubated in the presence or absence of PA (500 μM) or PA (500 μM) + TO (1.0 μM) for 16 hr. Total cell lysates were collected for the real-time PCR and WB analyses. TO potently suppressed PA-stimulated expression of PEPCK and G-6-Pase <b>(C)</b>. Furthermore, TO reversed PA-suppressed Akt activation/phosphorylation without affecting total Akt expression <b>(D)</b>. Results in (<b>B</b>) &(<b>C</b>) are presented as mean ± SD, *^{, #}P<0.05. 10–15 mice were used in each group.</p

TO901317 (TO) improves insulin sensitivity in <i>db/db</i> mice but also increases the lipid accumulation in the liver.

<p>IPITT were performed on WT and <i>db/db</i> mice following 14 days of treatment with TO or DMSO (controls). 5 hours after last TO injection on the morning of day 14, mice already fasted for 12 hours were subjected to intraperitoneal insulin challenge (0.75 U/kg for WT and 1.5 U/kg for <i>db/db</i>). IPITT curves showed significant differences between the TO and the DMSO-treated <i>db/db</i> mice <b>(A)</b>, but no significant differences between the TO and the DMSO-treated WT mice <b>(B)</b>. H&E staining of the liver showed TO treatment increased the lipid accumulation, compared with the DMSO controls in <i>db/db</i> mice <b>(C)</b>, but no significant difference was observed in WT mice (<b>D</b>). Results in (<b>A</b>) and (<b>B</b>) are presented as mean ± SD, *P<0.05. 10–15 mice were used in each group.</p

Unique 5′-P recognition and basis for dG:dGTP misincorporation of ASFV DNA polymerase X

<div><p>African swine fever virus (ASFV) can cause highly lethal disease in pigs and is becoming a global threat. ASFV DNA Polymerase X (<i>Asfv</i>PolX) is the most distinctive DNA polymerase identified to date; it lacks two DNA-binding domains (the thumb domain and 8-KD domain) conserved in the homologous proteins. <i>Asfv</i>PolX catalyzes the gap-filling reaction during the DNA repair process of the ASFV virus genome; it is highly error prone and plays an important role during the strategic mutagenesis of the viral genome. The structural basis underlying the natural substrate binding and the most frequent dG:dGTP misincorporation of <i>Asfv</i>PolX remain poorly understood. Here, we report eight <i>Asfv</i>PolX complex structures; our structures demonstrate that <i>Asfv</i>PolX has one unique 5′-phosphate (5′-P) binding pocket, which can favor the productive catalytic complex assembly and enhance the dGTP misincorporation efficiency. In combination with mutagenesis and in vitro catalytic assays, our study also reveals the functional roles of the platform His115-Arg127 and the hydrophobic residues Val120 and Leu123 in dG:dGTP misincorporation and can provide information for rational drug design to help combat ASFV in the future.</p></div

The impacts of the H115-Arg127 platform on the dG:dGTP misincorporation.

<p>The dC:dGTP and dG:dGTP base pairs observed in (<b>A</b>) <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP structure and (<b>B</b>) <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure, respectively. The 2F_o-F_c maps are contoured at 1.5 σ level. (<b>C</b>) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT <i>Asfv</i>PolX, H115D, H115E, H115F, R127A, and H115F/R127A mutants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). The data represent the mean of three independent experiments with SD values indicated by error bars. The dG:dGTP and dT:ddA base pairs observed at the insertion and postinsertion sites of (<b>D</b>) H115F/R127A:1nt-gap(P) DNA6:dGTP and (<b>E</b>) H115F:1nt-gap(P) DNA6:dGTP, respectively. (<b>F</b>) Structural comparison showing the conformational differences between <i>Asfv</i>PolX:1nt-DNA(P) DNA6:dGTP and H115F:1nt-gap(P) DNA6:dGTP. For clarity, the insertion site dG:dGTP base pairs and the <i>Asfv</i>PolX protein in <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure are omitted. The C atoms of Phe115, Arg127, and the postinsertion site dT:ddA of H115F:1nt-gap(P) DNA6:dGTP are colored green in both (<b>E</b>) and (<b>F</b>), whereas, the C atoms are colored white for His115, Arg127, and for the postinsertion site dT:ddA of <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP in (<b>F</b>).</p

5′-P of downstream oligo facilitates the productive complex assembly.

<p>(<b>A</b>) Sequence of 1nt-gap DNA4 and the overall structure of <i>Asfv</i>PolX:1nt-gap DNA4 complex. <i>Asfv</i>PolX is shown as a cartoon with the palm and finger domains colored in cyan and white, respectively. The template strand, primer, and downstream oligo are shown as stick with the C atoms colored in green, yellow, and yellow, respectively. The template residue (G8) is indicated with arrow. (<b>B</b>) Sequences of 2nt-gap(P) DNA5 and 1nt-gap(P) DNA5. (<b>C</b>) Overall structure of <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP. <i>Asfv</i>PolX is shown as cartoon with palm and finger domains colored in cyan and white, respectively. DNA is shown as sticks with the C atoms colored in yellow, green, and green, for the template strand, primer, and downstream oligo, respectively. dGTP is also shown as sticks, Mn²⁺ ions are shown as red spheres.</p