Differential effect of gemfibrozil in RLIP76^{<b>+<b>/</b>+</b>} and RLIP76^−/− mice.

<p><b><u>Panel A</u>:</b> Effect of RLIP76 depletion by RLIP76 antisense on triglycerides level in RLIP76^+/+ mice. p<0.02, when compared to scrambled antisense treatment. <b><u>Panel B</u>:</b> triglycerides level was measured prior to and 24 h after a single oral dose of gemfibrozil (100 mg/kg b.w.) by gavage in RLIP76^+/+ and RLIP76^−/− mice. p<0.001, when compared between RLIP76^+/+ and RLIP76^−/− mice, and p<0.02 when compared to gemfibrozil treatment in RLIP76^+/+ mice. In panels A & B, 5 mice per group were used. <b><u>Panel C</u>:</b> Effect of gemfibrozil on PPARα expression by Western blot in mouse liver tissue lysates, and developed bands were quantified by scanning densitometry. GAPDH expression was used as loading control. WT, wild-type; KO, RLIP76-knockout; Gemf, gemfibrozil; <b><u>Panel D</u>:</b> Effect of gemfibrozil on PPARα expression in paraffin embedded RLIP76^+/+ and RLIP76^−/− mouse liver tissues section by immuno-histochemistry using ABC staining kit (Vector). Immuno-reactivity is evident as a dark brown stain, whereas non-reactive areas display only the background color. Sections were counter-stained with Hematoxylin (blue). Photographs at 40× magnification were acquired using Olympus Provis AX70 microscope. Percent staining was determined by measuring positive immuno-reactivity per unit area. Arrows represent the area for positive staining for an antigen. The intensity of antigen staining was quantified by digital image analysis. Bars represent mean ± S.E. (n = 5); * p<0.002 compared with control.</p

The activity of gluconeogenesis enzymes.

<p>The activity of PEPCK, F-1, 6-BPase, and G6Pase was tested in un-dialyzed and dialyzed liver homogenates of control and metformin treated RLIP76^+/+ and RLIP76^−/− mice (n = 3) as protocols standardized by us <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Awasthi1" target="_blank">[1]</a>. *p<0.001, when compared to RLIP76^+/+, and **p<0.005, when compared with metformin treatment in RLIP76^+/+. The enzyme PEPCK, catalyze the conversion of phosphoenolpyruvate to fructose 1,6-biphosphate in a series of steps involving oxidation of NADH to NAD. In this assay, the loss of NADH was determined spectrophotometrically by measuring absorbance at 340 nm, based on the method of Opie and Newsholme <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Opie1" target="_blank">[28]</a>. To detect F-1, 6-BPase activity, a spectrophotometric coupled enzyme assay was used by a method of Taketa and Pogell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Taketa1" target="_blank">[29]</a>. F-1, 6-BPase activity was coupled with phosphoglucose isomerase and NADP dependent glucose 6-phosphate dehydrogenase, and NADPH formation was measured at 340 nm. G6Pase activity was determined spectrophotometrically using the method of Gierow and Jergil <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024688#pone.0024688-Gierow1" target="_blank">[30]</a>. The method is based on a coupled enzyme reaction in which glucose formed is reacted with glucose oxidase and peroxidase and the quinoneimine formed is a colored product and its formation can be followed spectrophotometrically at 510 nm.</p

Differential effect of metformin in RLIP76^{<b>+<b>/</b>+</b>} and RLIP76^−/− mice.

<p><b><u>Panel A</u>:</b> BG was measured prior to and after a single oral dose of metformin (250 mg/kg b.w.) by gavage at various time points (n = 6 mice/group). p<0.001, when compared between RLIP76^+/+ and RLIP76^−/− mice, and metformin treatment in RLIP76^+/+ mice. <b><u>Panel B</u>:</b> Effect of metformin on RLIP76, pAkt, pJNK, PPARγ, and pAMPK expression by Western blot in RLIP76^+/+ and RLIP76^−/− control and metformin treated mouse liver tissue lysates, and developed bands were quantified by scanning densitometry. GAPDH expression was used as loading control. <b><u>Panel C</u>:</b> Inhibition of the transport activity of purified rec-RLIP76 towards ³H-GSHNE by metformin. The experiment was repeated twice and similar results were obtained. WT, wild-type (RLIP76^+/+); KO, RLIP76-knockout (RLIP76^−/−); Met, metformin.</p

Differential effect of rosiglitazone in RLIP76^{<b>+<b>/</b>+</b>}<i>vs.</i> RLIP76^−/− mice.

<p><b><u>Panel A</u>:</b> Effect of RLIP76 depletion by RLIP76 antisense on BG in RLIP76^+/+ mice. p<0.01, when compared to scrambled antisense treatment. <b><u>Panel B</u>:</b> BG was measured prior to and after a single oral dose of rosiglitazone (10 mg/kg b.w.) by gavage at various time points. p<0.001, when compared between RLIP76^+/+ and RLIP76^−/− mice, and rosiglitazone treatment in RLIP76^+/+ mice. <b><u>Panel C</u>:</b> Effect of rosiglitazone on RLIP76 expression (by QRT-PCR), RLIP76 protein content, and PPARγ protein content (by Western blot) in mouse liver tissue lysates. GAPDH expression was used as loading control. <b><u>Panel D</u>:</b> Inhibition of the transport activity of purified recombinant human RLIP76 towards physiological substrate ³H-GSHNE by rosiglitazone. In panels A & B, 5 mice per group were used. These experiments were repeated three times and similar results were obtained.</p