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

TROUBLE SHOOTING DURING BIOANALYTICAL ESTIMATION OF DRUG AND METABOLITES USING LC-MS/MS: A REVIEW

Bioanalysis frequently involves the measurement of very low analyte concentrations in complex and potentially variable matrices. An initial attempt has been made to apply a risk management tool to the bioanalytical method development like selection of spiked plasma volume, selection of internal standard to minimize processing error, selection of medium and extraction procedure, setting of mobile phase and pH, determination of chromatographic conditions etc. and to minimize instrumental error like; ion suppression and matrix effect

Purification and functional reconstitution of intact ral-binding GTPase activating protein, RLIP76, in artificial liposomes.

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39, 9327, 2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 μM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity

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