RLIP76, a Glutathione-Conjugate Transporter, Plays a Major Role in the Pathogenesis of Metabolic Syndrome

PURPOSE: Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76⁻/⁻ mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76⁻/⁻ mice. RESEARCH DESIGN AND METHODS: Blood glucose (BG) and lipid measurements were performed in RLIP76⁺/⁺ and RLIP76⁻/⁻ mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining. RESULTS: The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76⁻/⁻ mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK. CONCLUSIONS/SIGNIFICANCE: All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76⁻/⁻ mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome

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 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

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

Major mechanisms contributing to RLIP76-mediated anti-cancer effects and chemo-radiotherapy resistance in pancreatic cancer.

<p>RLIP76 depletion leads to predominant inhibition of PI3K/Akt pathway as revealed by the inhibition of PI3K as well as decrease in the levels and phosphorylation of Akt. The associated downstream proteins that regulate differentiation, proliferation and apoptosis like E-cadherin, Bim, Bcl2 and CDK4 are also differentially regulated due to RLIP76 targeted interventions. The collective impact of RLIP76 depletion on the regulation of cellular signaling pathways as well as detoxification of glutathione-conjugates (GS-Es) of lipid peroxidation and chemotherapy drugs represents precious opportunities for the development of novel and effective therapeutic interventions for pancreatic cancers. <i>Green arrow</i>: Up regulation following RLIP76 depletion; <i>Red arrow</i>: down regulation following RLIP76 depletion; <i>Blue arrow</i>: Normal signal transduction.</p

RLIP76 mediated DOX transport and radiation-protection in pancreatic cancer cells.

<p>The crude membrane vesicles (inside-out vesicles, IOV) from BxPC3 cells were separately coated with 40 µg/ml final concentration of either anti-RLIP76 IgG, anti-MRP1 IgG, or anti-Pgp IgG and used to measure the ATP-dependent uptake of ¹⁴C-DOX. ATP-dependent uptake of ¹⁴C-DOX was determined by subtracting the radio-activity (cpm) of the controls without ATP from that of the experimental groups containing ATP (<b>panel A</b>). Approximately, 2.5×10³ BxPC3 cells grown in RPMI-1640 medium, were treated with control and RLIP76-liposomes (50 µg/ml final concentration) for 24 h prior to radiation at 100, 200, 500 and 1000 cGY (6 MeV photons). After 7 days, cells were stained with methylene-blue and the colonies were counted using Alpha Imager HP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Nagaprashantha1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Nagaprashantha2" target="_blank">[33]</a> (<b>panel B</b>) * p<0.05, ** p<0.01 compared to controls. The fold change in the radiation-resistance of RLIP76 <i>vs.</i> control-liposome treated pancreatic cancer cells at each dose of irradiation is represented in the bar diagram (<b>panel C</b>). The results presented are the mean and s.d. from three separate experiments (n = 9) * p<0.05, ** p<0.01 compared to control.</p

RLIP76 protein expression in human normal and pancreatic cancer cells.

<p>Cell lines were cultured in respective medium and homogenate was prepared from 10⁸ cells. RLIP76 was purified from total crude membrane fraction using DNP-SG affinity column chromatography and quantified by ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Awasthi4" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Singhal6" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034582#pone.0034582-Singhal7" target="_blank">[31]</a>. Values represent mean ± s.d. from three separate determinations.</p