Mandal AK , Leask MP , Estiverne C , Choi HK , Merriman TR , Mount DB .

P50 AR060772 NIAMS NIH HHS

             renal absorption
             insulin metabolic process

                hyperuricemia
                type 2 diabetes mellitus

	FIGURE 1. The net excretion of urate in the human urine depends on the balance between urate reabsorption and secretion, mediated by separate sets of urate transporters in the proximal tubule. (A) Urate reabsorption: The sodium-driven SMCT1 and SMCT2 transporters increase the intracellular pool of organic anions such as nicotinate or pyrazinoate (PZA), which function in urate/anion exchange mediated by URAT1 and OAT10. OAT4 in contrast exchanges apical urate for divalent anions. The membrane potential-driven GLUT9a functions as the exclusive exit pathway for reabsorbed urate from proximal tubule into blood. (B) Urate secretion: In a sodium dependent process involving the collaboration of NADC3, urate enters at the basolateral membrane in exchange with alpha-ketoglutarate, mediated by OAT1 and OAT3. Urate is transported across the apical surface into the tubular lumen via MRP4, ABCG2, NPT1, and NPT4. (C) Regulation of urate transport by insulin signaling: Insulin binding to its receptor (INSR) leads to the activation of insulin receptor substrate (IRS), Akt, ERK1/2, p38 MAPK and PKC. The general effect of insulin on urate transport (reabsorption or secretion) is indicated. Genistein, a phosphotyrosine kinase (PTK)-specific inhibitor; LY 295002, a PI3K-specific inhibitor; PD98059, a MEK/ERK-specific inhibitor; SB 203580, a p38 MAPK-specific inhibitor; BMI, bisindolylmaleimide 1-hydrochloride, a protein kinase C (PKC)-specific inhibitor.
	FIGURE 2. Insulin stimulates [14C]-urate uptake in human renal proximal tubule epithelial cells (PTC-05) and HEK 293T cells. (A) Western blot analyses show endogenous INSR protein (IRβ; ∼96 kDa) expression in various human cell lines. (B) Western blot analyses show endogenous GLUT9 protein expression in various human cell lines in transiently transfected (GLUT9a) HEK 293T cells and Xenopus laevis oocytes. (C) Western blot analyses show endogenous ABCC4, ABCG2, OAT10 and NPT1 protein expression in various human cell lines. (D,E) RT-PCR detection of the mRNA expression of URAT1, GLUT9 isoforms (GLUT9a and GLUT9b), OAT10, ABCG2, ABCC4, OAT1, OAT3, OAT4, NPT1, NPT4 and GAPDH in human kidney, PTC-05 and HEK 293T cells. (F) Dose-response plots showing the total [14C]-urate uptake activities of endogenous urate transporters, in equal number (3 × 106) of HEK 293T or PTC-05 cells, in response to increasing concentration of insulin (0–1500 nM). (G) Bar diagram indicating the [14C]-urate uptake activities of endogenous urate transporters in HEK 293T and PTC-05 cells measured in the absence or presence of insulin (1.0 and 1.5 μM). Asterisk (“*”), P < 0.001 compared with urate uptake in the absence of insulin. (H) Time-course plot of insulin-stimulation of [14C]-urate uptake by endogenous urate transporters in HEK 293T or PTC-05 cells. Results are the average of three independent experiments ± s.e.m. (I) Western blot analyses show the effect of extracellular insulin (1.0 μM) on expression of endogenous urate transporter proteins in HEK 293T or PTC-05 cells for varying time intervals (0–90 min).
	FIGURE 3. Insulin stimulates urate uptake through activation of PTK (protein tyrosine kinase) of INSR, IRS1, PI3K, Akt, p44/42 MAPK (ERK) and/or p38 MAPK in human kidney cell lines (HEK 293T and PTC-05). (A,B) The stimulatory effect of insulin (1.0 μM) on [14C]-urate uptake by endogenous urate transporters in equal number (3x106) of PTC-05 (A) and HEK 293T (B) cells was measured in the absence and presence of 10 mM pyrazinoate (PZA), 10 mM nicotinate (Nico), 10 mM salicylate (Sal), 100 μM benzbromarone (Benz), 100 μM tranilast (Tran), or 1.0 mM probenecid (Proben) in the extracellular K+-free isotonic medium at ∼25°C. Uricosuric drugs were dissolved in dimethyl sulfoxide (DMSO). (C–E) Insulin activates IRS1, Akt, ERK and p38 MAPK in PTC-05 cells (C,D) and HEK 293T cells (C,E). (F) Insulin has no significant effect on the expression of endogenous INSR protein (IRβ; ∼96 kDa) expression. For Western blot analyses cells were treated with insulin (1.0 μM) in K+-free isotonic medium and lysates were analyzed using appropriate antibodies (see methods). (G,H) Signaling pathway-specific inhibitors differentially inhibit insulin-stimulation of [14C]-urate uptake in PTC-05 cells (G) and HEK 293T cells (H). The effect of insulin (1.0 μM) on [14C]-urate uptake was measured in K+-free isotonic uptake medium in the absence and presence of the natural protein tyrosine kinase inhibitor (genistein; 75 μM), phosphatidylinositol 3-kinase (PI3K) inhibitor (LY 294002; 50 μM), wortmanin (4 μM), p38 MAPK inhibitor (10 μM SB 203580), MEK/ERK inhibitor (20 μM PD 98059 or U0126) or protein kinase C inhibitor (10 μM bisindolylmaleimide I (BMI). Results are the average of three independent experiments ± s.e.m. Asterisk (*), P < 0.001 compared with urate uptake in the absence of uricosuric drugs, anti-uricosuric agents or in presence of insulin and DMSO.
	FIGURE 4. Insulin stimulates urate transport mediated by GLUT9, OAT10, OAT1, mOAT3 or NPT1 expressed in Xenopus oocytes. (A,B) The [14C]-urate uptake activity of human GLUT9 isoforms, URAT1, OAT10, OAT4, NPT1, OAT1, and mouse OAT3 was measured in absence and presence of insulin (1.0-1.5 μM) in K+-free isotonic medium. (C,D) The time course plot of [14C]-urate uptake mediated by human GLUT9a, GLUT9b, OAT10, NPT1 or mouse OAT3, expressed in oocytes, in the absence and presence of 1.0 μM insulin. Oocytes expressing URAT1, OAT10 or NPT1 were preloaded with 50 nl of 100 mM nicotinate (Nico) and oocytes expressing OAT4 were preloaded with maleate (Mal) with 50 nl of 100 mM maleate by microinjection 2 h before urate uptake. Asterisk (*), P < 0.001 compared with urate uptake/efflux in the absence of insulin.
	FIGURE 5. Insulin stimulates urate transport activity of GLUT9 isoforms, OAT10, NPT1, OAT3 or OAT1 in Xenopus oocytes via activation of protein tyrosine kinase (PTK) activity of INSR, phosphatidylinositol 3-kinase (PI3K), Akt, and/or p44/42 MAPK (ERK). (A) Western blot analyses of the lysates of Xenopus oocytes, treated with insulin (1.0 μM) at ∼25°C in K+-free isotonic medium using appropriate antibodies for indicated antigens. (B,C) The stimulatory effect of insulin (1.0 μM) on [14C]-urate uptake mediated by GLUT9 isoforms, OAT10, NPT1, mouse OAT3 and OAT1 in Xenopus oocytes was measured in the absence and presence of the natural PTK inhibitor (genistein; 75 μM) or PI3K inhibitor (LY 294002; 50 μM). Oocytes expressing OAT10 or NPT1 were preloaded with nicotinate (Nico) by microinjection of 50 nl of 100 mM nicotinate 2h before urate uptake. (D,E) Insulin-stimulation of urate uptake activities of human GLUT9a or GLUT9b was examined in oocytes in the absence and presence of p38 MAPK inhibitor (SB 203580; 10 μM), MEK/ERK inhibitor (PD 98059/U0126; 20 μM) or protein kinase C inhibitor (bisindolylmaleimide I hydrochloride, or BMI; 10 μM) in K+-free isotonic medium. Asterisk (*), P < 0.001 compared with urate uptake in the presence of insulin and DMSO.
	FIGURE 6. The N-terminal domain of human GLUT9 isoforms play a significant role in functional activation of by insulin. (A,B) The [14C]-urate uptake mediated by GLUT9a/GLUT9b, and its N-terminal point mutants (A) or its N-terminal deletion mutants (B) was examined in oocytes in the absence and presence of 1.0 μM insulin in K+-free isotonic medium (pH 7.4) at ∼25°C. Asterisk (*), P < 0.001 compared with the urate uptake by the normal urate transporter in the absence/presence of insulin.
	FIGURE 7. Insulin-stimulation of [14C]-urate uptake mediated by OAT10, OAT3, OAT1, NPT1 and ABCG2 in oocytes is inhibited by specific inhibitors of intracellular signaling pathways. (A–D) Insulin-stimulation of [14C]-urate uptake mediated by human OAT10 (A), mouse OAT3 (B), human OAT1 (C), or human NPT1 (D), expressed in oocytes, was measured in the absence or presence 1.0 μM insulin and p38 MAPK inhibitor (SB 203580; 10 μM), MEK/ERK inhibitor (PD 98059/U0126; 20 μM) or protein kinase C inhibitor (bisindolylmaleimide I hydrochloride, or BMI; 10 μM) in K+-free isotonic medium. Oocytes expressing NPT1 were preloaded with nicotinate (Nico) by microinjection of 50 nl of 100 mM nicotinate 2 h before urate uptake. (E–F) Insulin-stimulation of [14C]-urate efflux mediated by ABCG2 or ABCG2 mutant (Q141K) (E), expressed in oocytes, was measured in the absence or presence 1.0 μM insulin and/or genistein (75 μM) or LY 294002 (50 μM) (F), in K+-free isotonic medium (pH 7.4) at ∼25°C. Asterisk (*), P < 0.001 compared with the urate uptake by urate transporters in the absence/presence of insulin.

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