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      Dose-Ranging Effects of Canagliflozin, a Sodium-Glucose Cotransporter 2 Inhibitor, as Add-On to Metformin in Subjects With Type 2 Diabetes

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          Abstract

          OBJECTIVE

          To evaluate the effects of canagliflozin, a sodium-glucose cotransporter 2 inhibitor, in type 2 diabetes mellitus inadequately controlled with metformin monotherapy.

          RESEARCH DESIGN AND METHODS

          This was a double-blind, placebo-controlled, parallel-group, multicenter, dose-ranging study in 451 subjects randomized to canagliflozin 50, 100, 200, or 300 mg once daily (QD) or 300 mg twice daily (BID), sitagliptin 100 mg QD, or placebo. Primary end point was change in A1C from baseline through week 12. Secondary end points included change in fasting plasma glucose (FPG), body weight, and overnight urinary glucose-to-creatinine ratio. Safety and tolerability were also assessed.

          RESULTS

          Canagliflozin was associated with significant reductions in A1C from baseline (7.6–8.0%) to week 12: −0.79, −0.76, −0.70, −0.92, and −0.95% for canagliflozin 50, 100, 200, 300 mg QD and 300 mg BID, respectively, versus −0.22% for placebo (all P < 0.001) and −0.74% for sitagliptin. FPG was reduced by −16 to −27 mg/dL, and body weight was reduced by −2.3 to −3.4%, with significant increases in urinary glucose-to-creatinine ratio. Adverse events were transient, mild to moderate, and balanced across arms except for a non–dose-dependent increase in symptomatic genital infections with canagliflozin (3–8%) versus placebo and sitagliptin (2%). Urinary tract infections were reported without dose dependency in 3–9% of canagliflozin, 6% of placebo, and 2% of sitagliptin arms. Overall incidence of hypoglycemia was low.

          CONCLUSIONS

          Canagliflozin added onto metformin significantly improved glycemic control in type 2 diabetes and was associated with low incidence of hypoglycemia and significant weight loss. The safety/tolerability profile of canagliflozin was favorable except for increased frequency of genital infections in females.

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          Most cited references 12

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          Active sugar transport in health and disease.

          Secondary active glucose transport occurs by at least four members of the SLC5 gene family. This review considers the structure and function of two premier members, SGLT1 and SGLT2, and their role in intestinal glucose absorption and renal glucose reabsorption. Genetics disorders of SGLTs include Glucose-Galactose Malabsorption, and Familial Renal Glucosuria. SGLT1 plays a central role in Oral Rehydration Therapy used so effectively to treat secretory diarrhoea such as cholera. Increasing attention is being focused on SGLTs as drug targets for the therapy of diabetes.
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            SLC2A9 Is a High-Capacity Urate Transporter in Humans

            Introduction Elevated serum urate levels are associated with important common disorders such as gout, metabolic syndrome, diabetes, hypertension, and cardiovascular morbidity and mortality [1–4]. Uric acid is principally derived from the breakdown of dietary and cellular purines. Humans and great apes are exposed to higher urate levels than other mammalian species because of the inactivation of hepatic uricase [5]. In humans the kidney has a pivotal role in urate handling, with secretory mechanisms balanced against efficient reabsorption resulting in only 10% of the filtered load actually being excreted in the urine [5]. The established urate transporter systems in the proximal nephron includes; the urate anion transporter (URAT1), which is a target of uricosuric drugs, multiple organic anion transporters (OATs 1–4), the urate transporter (UAT), and a voltage dependent organic anion transporter (OATv1) [5]. It is possible that genetic variation in either enzymatic breakdown of purines or renal transporters of uric acid might elevate serum levels and account for the long-standing association with blood pressure and common cardiovascular phenotypes [1–5]. Recently, two separate genome-wide association scans identified and replicated association of serum urate level with common variants within the glucose transporter SLC2A9 (GLUT9) gene region on Chromosome 4 [6,7]. Interestingly, this member of the facilitative glucose transporter family has two splice variants most strongly expressed in the apical and basolateral membranes of the proximal tubular epithelial cells of the kidney [8,9]. SLC2A9 is not as efficient a glucose transporter as GLUT1 and GLUT4 [8], so we set out to test whether SLC2A9a and SLC2A9b splice variants act as urate transporters. We used data from the Olivetti Heart Study to test association of SLC2A9 variants and fractional urate excretion, and then tested for association with blood pressure and hypertension in additional cohorts. Methods Reagents and Antibodies All chemicals were obtained from Sigma-Aldrich unless otherwise noted. Two polyclonal antibodies against N-terminal peptides of both splice variants of human SLC2A9 were raised in rabbits (Rockland Immunochemicals). The SLC2A9a antibody was raised against DTSHARPPGPGRALLEC and the SLC2A9b was raised against KSRGEDEESDSAKKC. Both antibodies were peptide purified before use. Two polyclonal antibodies against N-terminal peptides of both splice variants of mouse SLC2A9 were also raised in rabbits (Rockland Immunochemicals). The mSLC2A9a antibody was raised against MDSRELALASLMC and the mSLC2A9b antibody was raised against MKLSEKNSAETKESC, and both antibodies were peptide purified before use. mRNA Preparation and Xenopus laevis Oocyte Microinjection Plasmids containing genes encoding the wild-type human SLC2A9a or SLC2A9b isoform were linearized with Nhe I and transcribed in vitro with T7 polymerase mMESSAGE mMACHINE (Ambion). Adult female X. laevis oocytes (prepared as described in [9]) were injected with 20 nl (1 ng/nl) SLC2A9a or SLC2A9b synthetic mRNA transcript and incubated for 5 d at 16–18 °C prior to functional uptake assays. The concentration of RNA prior to injection was determined using Bio-Rad SmartSpec 3000 machine. Determination of Functional Activity by Radiotracer Flux Assays The influx experiments were performed at 22 °C using 10–12 oocytes for each condition and 14C-labelled urate at a specific activity of 54 mCi/mmole, 250 μCi/ml. Oocytes were washed with ice-cold modified Barth's medium (MBM) to stop the uptake, and then individual oocytes were placed in vials and dissolved in 0.5 ml of 5% SDS for 30 min. Finally, scintillation fluid (5 ml) was added to each vial and radioactivity measured using a Beckman LS6500 liquid scintillation counter. All experiments were performed three to six times and the results were corrected for the flux values obtained with non-injected oocytes obtained from the same frog. Efflux experiments were performed by injecting oocytes with 40 nl of 14C-urate just prior to the flux measurements. Eggs were then incubated in batches of 20 in 1 ml of medium for 20 min at 22 °C. Samples of 20 μl of incubation medium were taken every 2 min to measure the appearance of urate in the outside solution. The incubation volume was kept constant by sequential addition of medium after the removal of each sample. The activity remaining in the eggs at the end of the incubation was measured by solubilising the oocytes in 1 ml of 5% SDS overnight. Incubation medium consisted of MBM supplemented with 5 mM D-glucose, D-fructose, uric acid, or L-glucose (control). Efflux data were then plotted as a function of the fraction of total urate remaining in the eggs at each incubation time point. This was expressed as the log (remaining urate/initial injected urate × 100) for the SLC2A9a-expressing eggs. Net SLC2A9a-mediated efflux was determined by subtracting urate efflux observed in non-injected oocytes from total urate efflux from SLC2A9a oocytes at each time point. Thin-Layer Chromatography To determine if urate was metabolized within the Xenopus oocytes we microinjected 40 nl of 14C- labelled 2 mM urate and collected the efflux medium from both water-injected eggs and those expressing SLC2A9a. We then loaded 100 μl of the efflux media onto a silica-coated glass chromatography plate and eluted with a 80:15:5 n-propanol:ammonium hydroxide (25%):water solution. Standard urate and allantoin solutions were used for comparison. After drying the plate, a vanillin spray was used to visualize the urate and allantoin. The R f value (distance travelled from solvent front) for urate was 0.0103 and that for allantoin 0.238. Kinetic Analysis Urate transport into oocytes expressing SLC2A9a or SLC2A9b was measured over a range of concentrations from 0.001 to 2 mM using 20 min incubations, which had been determined to be within the linear component of uptake. Uptake was corrected for nonspecific entry using non-mRNA injected eggs from the same batch in each experiment. SIGMAPLOT 6 software was used to determine the transport kinetics for the SLC2A9 mediated urate uptake by nonlinear regression analysis. The analysis assumed standard simple Michaelis-Menten kinetics for a facilitative transporter with no cooperativity. In addition to the regression analysis, an Eadie-Hofstee linear transformation was included, the results of which could imply that a more complex kinetic model is required to fully describe the characteristics of this transporter. Inhibitors were used at concentrations similar to previous studies of other glucose or urate transporters, e.g., phloretin, benzbromarone, and furosemide, and which corresponded with effective pharmacological doses, e.g., probenecid [10]. HEK293 Cells and Urate Uptake HEK293 cells were stably transfected with human SLC2A9a or SLC2A9b as shown previously [8]. These cells were grown to 90% confluency in T75 flasks. The cells were then washed with EDTA, trypsinised, decanted into 15 ml conical tubes, and centrifuged for 4 min at 1,100 rpm at 4 °C. The supernatant was removed and the cells resuspended in 4 ml of Dulbecco's modified Eagle medium (DMEM) with 2% serum. After incubation for 2 h at 37 °C while shaking at 120 rpm, the cells were centrifuged for 4 min at 1,100 rpm at 4 °C and the supernatant removed. Next, the cells were washed twice in Kreb's Ringer phosphate (KRP) buffer, centrifuged for 4 min at 1,100 rpm at 4 °C and resuspended in 0.450 ml of KRP buffer with 50 μl of 1.2 mM 14C-labeled urate (Moravek Biochemicals). The cells were then incubated at 37 °C with shaking at 120 RPM for 6 min, after which they were placed on ice for 5 min. Finally, the supernatant was removed after centrifugation and the pellet washed twice with cold KRP buffer, before lysis. After incubation in lysis media for 30 min on ice, a final centrifugation step at 1,100 rpm for 1 min was performed, a 500 μl aliquot of the supernatant was added to scintillation fluid (5 ml) and the radioactivity measured using a Beckman LS6500 liquid scintillation counter. The remainder of the supernatant was used for protein quantitation and Western immunoblot. All experiments were done at least three times. Western Immunoblot Analysis Cell lysates were solubilised in Laemmli buffer and subjected to SDS-PAGE, transferred onto nitrocellulose membranes, blocked with 5% dry milk in Tris-buffered saline/Tween 20 (TBS-T), and probed with an antibody raised against the human SLC2A9a N terminus (1.5 μg/ml in 1% dry milk/TBS-T), or the human SLC2A9b N terminus (1.0 μg/ml in 1% dry milk/TBS-T). For the murine insulinoma cell (MIN6) lysates, polyclonal antibodies to the mouse SLC2A9a and SLC2A9b, previously described, were used [11]. Blots were then probed with a horseradish peroxidase-coupled goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) and detection was performed using the SuperSignal Dura Western kit (Pierce Biotechnology). Blot quantitation after scanning was carried out using NIH Image. Culture of Mouse Insulinoma Cells (MIN6) MIN6 [12] cells were maintained in DMEM containing 25 mM glucose, supplemented with 15% heat-inactivated foetal bovine serum, 50 μM β-mercaptoethanol, 100 U/ml penicillin,100 μg/ml streptomycin, 100 μg/ml L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate, in 5% CO2, 95% air at 37 °C. MIN6 cells were used between passages 20 and 30. The radiolabelled urate uptake assays were conducted as described above for the HEK293 cells. For the cold urate competition assays, 50 μl of cold uric acid stock at a concentration of 1 mM dissolved in Tris buffer (pH 8.1) was added to the KRP buffer with labelled urate to give a final concentration of 1 mM unlabeled urate. The remainder of the assay was performed as described above. SLC2A9 siRNA Transfection The polyamine transfection reagent, Trans IT- TKO (Mirus) was used to transfect the insulinoma cells according to the manufacturer's instructions. The mouse SLC2A9 siRNA and scrambled siRNA (negative control) were obtained from Ambion. Cells were transfected for 72 h for protein isolation. Radiolabelled urate uptake assays were conducted as described above. Association Studies with Serum Urate, Fractional Urate Excretion, Blood Pressure, and Hypertension: Participants The Olivetti Heart Study. The Olivetti Heart Study population is derived from the male work force of the Olivetti factories of Pozzuoli (Naples) and Marcianise (Caserta), Italy. The general characteristics of the study and its methodological procedures have been described [12,13]. The local ethics committee approved the study protocol, and informed consent was obtained from all participants. A total of 1,085 individuals aged 25–74 y were examined in 1994–1995: of these, 907 (83.6%) were seen again in 2002–2004, and of these, 868 individuals had DNA available for genotyping at both time points [14]. We analysed individuals at both time points for association with serum urate and fractional excretion of uric acid and the 1994–1995 dataset only for association with blood pressure and hypertension. Whitehall II study. After ethical clearance the Whitehall study enrolled 10,308 subjects (3,413 women) aged 35–55 y working in the London offices of 20 civil service departments between 1985–1988. In this longitudinal study, blood pressure was recorded at phase 1 (1985–1988), phase 3 (1991–1993), phase 5 (1997–1999), and phase 7 (2003–2004). DNA was stored from phase 7 of the study. For association testing with blood pressure and hypertension we selected individuals from phase 5 as diabetes ascertainment and blood pressure medication records were most complete from this phase. For case–control analyses, hypertensive participants were selected using the following criteria: blood pressure recordings of ≥ 145/95 mm Hg, prescribed antihypertensive medication, or a physician diagnosis of hypertension. Normotensive control participants were selected on the basis of blood pressure recordings ≤ 130/85 mmHg and not taking any antihypertensive medications. English Longitudinal Study of Ageing (ELSA). After ethical approval the participants were drawn from around 12,000 respondents to the Health Survey for England (HSE) over three separate years (1998, 1999, and 2001) to provide a representative sample of the English population aged 50 y and over. Each individual had a mean of three blood pressure measures taken when the participant was seated, and antihypertensive medications were recorded, DNA was extracted from 5,672 participants in wave 2 (2004). For association testing with blood pressure and hypertension we selected individuals from wave 2. Cases and normotensive controls were defined using the same criteria as the Whitehall II study. BRIGHT study. The MRC BRIGHT study (http://www.brightstudy.ac.uk/) comprises 2,500 hypertensive participants and 2,000 normotensive controls of white European ancestry. Case ascertainment and phenotyping has been described previously [15]. Briefly, cases were included if they had blood pressure readings ≥ 150/100 mm Hg based on one reading or ≥ 145/95 mm Hg based on the mean of three readings. Healthy age- and sex-matched normotensive controls ( 35 and those with diabetes were excluded from all analyses. Inverse-variance weighted meta-analysis was performed using the “metan” [21] procedure in Stata 10.0. Results Human SLC2A9 Is a High-Capacity Urate Transporter To test whether SLC2A9 splice variants act as urate transporters we separately microinjected synthetic human SLC2A9a and SLC2A9b mRNA transcripts into X. laevis oocytes and measured uptake or efflux of radiolabelled urate. Both proteins were readily expressed in the oocyte plasma membrane within a couple of days as confirmed by immunohistochemistry (Figure S1). However, we studied the oocytes 4 d after mRNA injection so that urate fluxes could be determined under the same conditions as previously published for SLC2A9 mediated hexose transport. We found that human SLC2A9a and SLC2A9b mediated very rapid urate fluxes which necessitated incubation for only 20 min in subsequent kinetic and inhibition experiments (Figure 1A). In contrast, uptake of urate into non-injected eggs was very slow, indicating negligible endogenous transport activity for this substrate. Transport was then measured over a range of urate concentrations, which bracketed the normal human physiological plasma concentrations (200–500 μM). Both human SLC2A9a- and SLC2A9b-mediated urate fluxes showed saturation and were identical, so data for the two splice variants were combined for kinetic analysis. Figure 1B shows the averaged data from six such experiments and nonlinear regression analysis was used to fit a Michaelis-Menten function with a K m of 981 μM and a V max of 304 pmol/oocyte/20 min. The insert shows the Eadie-Hofstee plot for the same data. In contrast, we were unable to detect any urate flux (100 μM) mediated by either human GLUT1 (SLC2A1) or GLUT2 (SLC2A2), both class I facilitative glucose transporters (Figure 1C). Figure 1 Characterisation of Urate Fluxes and Kinetics Mediated by Human SLC2A9 Expressed in Xenopus laevis Oocytes and the Effect of Short-Chain Fatty Acids and Uricosurics on Urate Transport (A) Time course of SLC2A9a-mediated urate uptake into Xenopus oocytes. Oocytes were injected with SLC2A9a cRNA or water and 4 d later incubated with 10 μM urate at 22 °C for the time periods indicated. Symbols represent the average uptake into ten oocytes per time point, with error bars representing the standard error of the mean (SEM). Solid circles show total uptake into oocytes expressing SLC2A9a cRNA, open circles urate uptake into water-injected oocytes, and inverted triangles the net uptake obtained by subtracting the water data from the total uptake for each time point. (B) Kinetics of human SLC2A9-mediated urate uptake in Xenopus oocytes. Symbols represent the mean data from six separate experiments each using ten oocytes per substrate concentration. Uptake was measured in oocytes injected with either SLC2A9a or SLC2A9b cRNA 4 d prior and corrected for uptake into water-injected oocytes. The curve was fitted by nonlinear regression analysis. The K m = 981 μM and the V max = 304 pmol/oocyte/20 min. Insert shows an Eadie-Hofstee plot of the same data. (C) Effect of uricosemics and short chain fatty acids on human SLC2A9a mediated urate uptake. Xenopus oocytes were injected with water or SLC2A9a cRNA and urate uptake measured 4 d later. Oocytes were incubated with 10 μM urate for 20 min, and bars represent net uptake determined by subtracting uptake into water-injected eggs from total uptake into SLC2A9a expressing oocytes, error bars are the SEM. Note: at no time was the urate uptake into the water-injected eggs more than 11% of the total uptake into SLC2A9-expressing oocytes. Compounds used were: no additional reagent (Cont), lactate 1 mM (Lac), pyruvate 1 mM (Pyr), butyrate 1 mM (But), acetate 1 mM (Ace), probenecid 1 mM (Prob), furosemide 100 μM (Furo), and phloretin 1 mM (Phlo). For comparison, a separate series of oocytes were injected with SLC2A1 (GLUT1) or SLC2A2 (GLUT2) cRNA and 100 μM urate uptake measured 4 d later using 30 min incubation times. Bars represent net uptake corrected for uptake into water-injected oocytes. *Significant inhibition at p ≤ 0.05. (D) Dose-dependent effect of benzbromarone on SLC2A9a-mediated urate uptake. Oocytes were injected with SLC2A9a cRNA or water and 4 d later were incubated with 10 μM urate for 20 min in the presence of 0, 1, 10, or 100 μM benzbromarone. Points represent the mean net uptake into ten oocytes corrected for uptake into water-injected eggs. Error bars were smaller than the data points. Transport of Urate by SLC2A9 Shows Limited Sensitivity to Uricosurics A number of compounds known to promote urate loss in the urine via inhibition of other renal urate transporters such as URAT1 were tested to determine if they reduced SLC2A9 mediated urate transport. Benzbromarone showed a dose-responsive inhibition with 10 μM reducing urate uptake by 34% and 100 μM by 80% (Figure 1D). Additional kinetic analyses using two concentrations of urate (10 and 100 μM) and a Dixon analysis showed that the K i for benzbromarone inhibition of urate transport mediated by SLC2A9a was 27 μM. However, probenecid at a concentration of 1 mM had no significant effect on urate uptake, p > 0.05 (Figure 1C). Similarly, furosemide had no significant effect at 100 μM, and, of the short chain fatty acids lactate, pyruvate, butyrate, and acetate at a concentration of 1 mM, only lactate induced a small and just significant inhibition, p 0.05 (Figure 1C). SLC2A9 Mediates Exchange of Urate for Glucose or Fructose Because SLC2A9a and SLC2A9b have previously been characterized as high-affinity, low-capacity glucose and fructose transporters, we tested the ability of these two hexoses to inhibit urate fluxes and, conversely, for urate to inhibit hexose uptake. Surprisingly, concentrations of glucose up to 1 mM had no effect on the transport of 10 μM urate (Figure 2B), and urate concentrations of up to 2 mM had no effect on the uptake of 50 μM D-glucose or D-fructose (Figure 2A and 2C). Note that the rates of urate transport were significantly greater than those for glucose. Urate fluxes were measured at 5 or 10 μM for only 20 min, whereas glucose rates were determined using 50 μM substrate for 30 min. Thus, converting flux rates to the same time period and equivalent concentrations suggest that urate is transported by SLC2A9 at rates 45- to 60-fold faster. The lack of significant competition between the hexoses and urate suggests that urate binds to a site on SLC2A9 that is different from the binding site for the hexoses. However, it could be argued that the injection of SLC2A9 mRNA was inducing expression of an endogenous protein that could separately mediate urate uptake. Therefore, we attempted to determine whether SLC2A9 could exchange hexoses with urate, providing further support for a single pathway mechanism. Since glucose and fructose are both rapidly phosphorylated upon entry into the oocyte, only urate efflux could be used to give a meaningful estimate of exchange rates. Oocytes were injected with radiolabelled urate and the efflux determined in the presence of 5 mM extracellular D-glucose, D-fructose, L-glucose, or 2 mM urate. Urate efflux could be described by a single exponential curve over a period of up to 20 min, and the presence of extracellular D-glucose greatly accelerated urate movement, while fructose did so to a lesser degree (7-fold versus 3-fold, respectively, Figures 3A and 3B). Extracellular cold urate accelerated the efflux of radiolabelled urate to the greatest degree, while extracellular L-glucose had no effect. Thin-layer chromatography was used to analyze the efflux medium, and only urate and no allantoin were detected on the plate for both water-injected eggs and those expressing SCL2A9a. Thus, SLC2A9 exhibits classical exchange trans-stimulation between urate and glucose or fructose. The rate of loss of injected radiolabelled L-glucose into the efflux medium was negligible, indicating that the injection protocol did not result in a nonspecific leakage of the substrate from the oocytes (Figure 3A). Figure 2 Interaction between Hexoses and Human SLC2A9a-Mediated Urate Uptake in Xenopus Oocytes Oocytes were injected with SLC2A9a cRNA 4 d prior to uptake experiments. Symbols represent mean net substrate uptake and error bars the SEM for measurements made in 6–10 oocytes per condition. Total uptake of substrates was measured into SLC2A9a cRNA-injected oocytes and then corrected for the uptake measured under identical conditions using water injected oocytes from the same batch of eggs. (A) Uptake over 30 min of 50 μM glucose in the presence of increasing concentrations of urate. (B) Uptake over 20 min of 5 μM urate in the presence of increasing concentrations of glucose. (C) Uptake over 30 min of 50 μM fructose in the presence of increasing concentrations of urate. (D) Human SLC2A9a-mediated glucose and urate exchange. Oocytes injected with SLC2A9a cRNA or water, 4 d prior, were incubated with non-radiolabelled 2 mM L-glucose or 2 mM urate for 1 h. Oocytes were then washed and then incubated at 22 °C for 30 min in 14C-labeled 10 μM D-glucose. Bars represent the average total uptake into 20 eggs expressing SLC2A9a or water-injected eggs, and the difference between the two, the net uptake. Error bars represent the SEM. Figure 3 Human SLC2A9a-Mediated Urate Efflux from Xenopus Oocytes (A) Comparison of urate and L-glucose efflux from SLC2A9a-expressing oocytes. Oocytes injected with SLC2A9a cRNA 4 d prior (triangles) or water-injected eggs (circles or squares) were injected with 14C-labelled urate (circles, triangles, or diamonds) or 14C-labelled L-glucose (filled squares) to give an estimated initial intracellular concentration of 200 μM. 20 oocytes per condition were incubated at 22 °C in efflux medium, which was sampled every 2 min. Efflux media contained 5 mM D-glucose (open circles), 5 mM L-glucose (filled squares, circles or triangles), or 2 mM urate (filled diamonds). Data points represent the log percentage of urate or L-glucose remaining in the oocytes for each time point. Lines were fitted by linear regression. (B) Acceleration of SLC2A9a mediated urate efflux by extracellular D-glucose or D-fructose. Oocytes injected with SLC2A9a cRNA 4 d prior (triangles) or water-injected eggs were injected with 14C-labelled urate to give an estimated intracellular concentration of 200 μM. 20 oocytes per condition were incubated at 22 °C in efflux medium, which was sampled every 2 min. Efflux media contained 5 mM D-glucose (filled circles), 5 mM L-glucose (filled triangles) or 5 mM D-fructose (open circles). Data points represent the log percentage of urate remaining in the oocytes for each time point corrected for the efflux of urate from the water-injected eggs under the same conditions. Lines were fitted by linear regression. The exchange of urate for glucose was confirmed by preloading the oocytes with 2 mM cold urate for 1 h and then, after washing, to remove extracellular urate, measuring the uptake of 100 μM D-glucose. The influx of D-glucose was increased 3-fold by intracellular urate in SLC2A9a-expressing eggs compared to preincubation with 2 mM L-glucose (Figure 2D). These data confirm that SLC2A9a can exchange extracellular glucose for intracellular urate. Increased Urate Uptake by Kidney Cells Overexpressing SLC2A9 Isoforms Both overexpressed proteins appear as a smear of glycosylated bands between 37 and 50 kDa, as previously reported using a different polyclonal antibody to the C terminus that was unable to distinguish SLC2A9a from SLC2A9b [8,21]. Previous studies had also already demonstrated plasma membrane expression of human (h) SLC2A9 in these cell lines [8]. In order to check the specificity of each antibody, total protein from cells overexpressing the different splice variants were loaded side by side in polyacrylamide gels and immunoblotted with the two antibodies separately. Each individual antibody detected the correct overexpressed protein but not the alternative splice variants. Both SLC2A9a and SLC2A9b were expressed at low levels endogenously in HEK293 cells as seen by the multiple bands in lanes 1 and 3 of Figure 4A and lanes 1 and 2 of Figure 4B. In prior studies we have demonstrated that these multiple bands in HEK cells reduce to one band at 37 kDa upon deglycosylation [8]. In the presence of preimmune sera (unpublished data) no specific bands were detected. Figure 4 Specificity of Human SLC2A9a and SLC2A9b Antibodies and Urate Uptake into SLC2A9 Transfected Human Embryonic Kidney Cells (A) Western blotting of hSLC2A9 expressed in HEK cells. Expression of hSLC2A9a was detected as a broad band at approximately 50 kDa by a polyclonal antibody raised against the N terminus of SLC2A9a in HEK293 cells overexpressing SLC2A9a (middle lane, Slc2a9a) but not detected in cells overexpressing SLC2A9b (right lane, Slc2a9b), or nontransfected HEK cells (left lane, HEK). (B) Expression of hSLC2A9b in transfected HEK293 cells. hSLC2A9b was detected by a polyclonal antibody raised against the N terminus of hSLC2A9b in HEK293 cells overexpressing hSLC2A9b (right lane, Slc2a9b), but not detected in cells overexpressing hSLC2A9a (middle lane, Slc2a9a). (C) Increase in urate uptake in HEK293 cells overexpressing either hSLC2A9a or hSLC2A9b. Radiolabelled urate uptake was measured in human embryonic kidney cells, which were stably overexpressing SLC2A9a or SLC2A9b as compared to their respective nontransfected controls. Uptakes of 120 μM urate were measured over 6 min at 37 °C. *p 0.6). Table 4 Results of SNP rs13113918 of SLC2A9 within Individual Hypertensive Case–Control Participants from All Resources and in Meta-analysis Analysed under an Additive Model Testing for Influence on Hypertension Discussion Recent genome-wide association scans identified and replicated association between SNPs at the SLC2A9 gene locus and serum urate and with gout [6,7,25,26]. In this study we have shown that both human SLC2A9 splice variants, SLC2A9a and SLC2A9b, can mediate urate fluxes at a very high rate and significantly faster than their facilitated transport of either glucose or fructose. The kinetics indicate that the transporter's apparent capacity for substrate, or K m value (∼ 1 mM), is above the basal, physiologic plasma concentrations of urate. Moreover, the V max value indicates a high-capacity transporter. All of these data suggest that this membrane protein plays an important role in the handling of urate in the proximal nephron, which completely fits with the findings from genome-wide scans of common allelic variation elevating urate by 20 μmol/l per allele [7]. In the context of everyday clinical practice this genetic influence on urate is equivalent to 5%–10% of the normal range of serum urate (180 μmol/l to 420 μmol/l), which is not trivial. These findings are confirmed by complementary functional studies on SLC2A9 in relation to urate handling and gout [25–27]. Current models of urate handling in the proximal convoluted tubule indicate that several types of transporter are involved in the fluxes of urate across both the apical and the basolateral membranes of the epithelial cells. In the apical membrane, these transporters include URAT1, a urate/lactate exchanger that mediates urate movement from urine to epithelium [28]; OATv1, a putative voltage dependent organic anion transporter [29]; MRP4, an ATP driven pump [30]; and UAT, a postulated urate channel [31]. At the other pole of the cells two of the organic anion exchangers, OAT1 and OAT2, present in basolateral membrane, are thought to be able to handle urate, but their physiological role remains to be confirmed [32,33]. Therefore at present there is a well-defined absorptive route across the apical membrane via UAT1 and a secretory route via MRP4, while the means by which urate can either leave the renal epithelium and enter the blood or move in the opposite direction across the basolateral membrane remains to be confirmed. Recently, it has been proposed that both SLC2A9 proteins are high-affinity glucose/fructose transporters. However, when compared with the principal members of the SLC2A9 gene family their transport capacity (V max) is very low [11,34]. We now have evidence that urate is a preferred substrate for both human SLC2A9 variants and for the mouse orthologue. The ability of SLC2A9 to exchange urate with glucose in the absence of competition between these two substrates when present on the same side of the membrane indicates that the protein has separate binding sites. This is not a unique phenomenon in exchange proteins, as the glycerol-6-phosphate transporter exchanges glycerol-6-phosphate for inorganic phosphate [35]. This ability of SLC2A9 to exchange glucose and, to a lesser degree, fructose for urate may be physiologically important. The renal proximal nephron plays a major role in the reabsorption of glucose from the urine using a combination of sodium-coupled hexose transporters, SGLT1 and SGLT2, and members of the SLC2A family, GLUTs 2, 5, and possibly 9 [36–40]. Furthermore, the proximal convoluted tubular epithelium is a major site of gluconeogenesis, converting pyruvate to glucose, which is then released across the basolateral membrane into the blood [41–43]. Our data showing that SLC2A9a can exchange glucose for urate suggest that this protein might play an important role in the secretion of urate from the blood into the urine. Glucose in the urine could exchange for urate in the proximal convoluted tubule epithelial cells across the apical membrane, resulting in the release of urate back into the urine. In addition, glucose in the epithelial cells resulting both from reabsorption and neogenesis could exchange for plasma urate across the basolateral membrane, promoting the accumulation of urate in the cells. Furthermore, such a mechanism could explain the known correlation between the glycosuria seen in diabetes and the reduction in plasma urate levels. The increased glucose in the urine could accelerate the SLC2A9-mediated urate efflux across the apical membrane of the proximal convoluted tubule, but further work is needed to confirm this hypothesis. Therefore, our findings, in combination with epidemiologic data showing correlation of elevated serum urate with diabetes, metabolic syndrome, obesity, and hyperinsulinaemia, provide a potential mechanism for these associations that warrants further investigation [44–46]. In this context it is of particular interest that recent data from the Health and Nutrition Survey shows correlation between consumption of sugar-based soft drinks with serum urate levels and gout, which might be partly explained by sugar-facilitated uptake of urate by SLC2A9 isoforms [47]. Probenecid and benzbromarone are uricosuric drugs that inhibit renal uptake of urate via URAT 1 on the apical proximal nephron membrane [5]. We found that probenecid had no effect on urate uptake into SLC2A9a-expressing oocytes, whereas benzbromarone showed a dose-dependent inhibition. The significant inhibition of urate transport by 10 and 100 μM benzbromarone implies that there are common features in the binding sites for urate in both URAT1 and SLC2A9a. When in clinical use benzbromarone promoted the loss of urate in the urine; this is believed to be a consequence of an inhibition of uptake across the apical membrane mediated by URAT1 [2]. In contrast, the high K i of 27 μM for benzbromarone action on SLC2A9 suggests that at therapeutic doses this drug would have a minimal effect on urate transport mediated by this protein and so does not affect urate secretion. The Olivetti Heart Study has previously found a strong longitudinal relationship of serum urate levels with blood pressure [48]. The authors also reported a correlation of urate with reduced fractional clearance of lithium as an index of sodium reabsorption in the proximal nephron. This finding could reflect sodium retention and offer a mechanism for previously reported associations of urate and blood pressure [12]. The two SNPs genotyped in the Olivetti cohort validate association of the SLC2A9 locus with serum urate and demonstrate the association of both SNPs with reduced urinary urate excretion at two time points 8 y apart. This provides additional support for our hypothesis that SLC2A9 variants reduce urinary urate loss and is further confirmed by recent cross-sectional findings for this gene in gout [25–27]. In view of the additive influence of alleles of SLC2A9 on serum uric acid and urinary urate excretion, we explored a relationship between SLC2A9 SNPs with systolic and diastolic blood pressure in 11,629 individuals under an additive model. This analysis showed no significant association between SNP rs13113918 and blood pressure. Furthermore, we did not find any evaluated association of the same SLC2A9 SNP with hypertension under an additive model in 5,249 hypertensive cases and 6,648 normotensive controls drawn from all the studies. In the context of this large-scale case–control and population-based cohorts we conclude that there is no support for an association with blood pressure using the most biologically plausible genetic model. Limitations of This Study We have not defined precisely the causative variant of SLC2A9 responsible for elevated serum urate and reduced urinary urate clearance. There are several known SNPs within the gene region that might influence function of the SLC2A9 protein. Such studies will be facilitated by detailed re-sequencing of the SLC2A9 gene to establish a comprehensive inventory of genetic variation across this locus. In addition to dietary and metabolic influences on uric acid levels there will be other, as-yet unidentified genetic influences on serum urate level that may contribute to epidemiologic correlations with metabolic syndrome, diabetes, gout, and cardiovascular disease. Conclusion In this paper we have translated the genetic association of the SLC2A9 locus with serum urate derived from genome-wide scanning into a functional confirmation that SLC2A9 splice variants acts as a high-capacity urate transporter that can be facilitated by exchange with hexoses and inhibited by high concentrations of some uricosurics and siRNA technology. These findings offer novel potential pathogenic mechanisms and new drug targets for diseases such as gout. Supporting Information Figure S1 Expression of SLC2A9a or 9b in Xenopus Oocyte Plasma Membrane after cRNA Injection Oocytes were injected with 50 nl (1 ng/nl) SLC2A9a or SLC2A9b cRNA or water and incubated at 18 °C for 4 d. The expression of SLC2A9a and SLC2A9b was visualized using a primary antibody to the same C-terminal peptide sequence of both proteins, a fluorescent secondary antibody, and confocal microscopy. All three images show the x, y, and z within the same focal plane. (A) Water-injected oocyte. (B) SLC2A9a. (C) SLC2A9b. (204 KB PDF) Click here for additional data file.
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              Diabetes prevalence and therapeutic target achievement in the United States, 1999 to 2006.

              Changes in the prevalence, treatment, and management of diabetes in the United States from 1999 to 2006 were studied using data from the National Health and Nutrition Examination Survey. Data on 17,306 participants aged 20 years or more were analyzed. Glycemic, blood pressure, and cholesterol targets were glycosylated hemoglobin less than 7.0%, blood pressure less than 130/80 mm Hg, and low-density lipoprotein (LDL) cholesterol less than 100 mg/dL, respectively. The prevalence of diagnosed diabetes was 6.5% from 1999 to 2002 and 7.8% from 2003 to 2006 (P < .05) and increased significantly in women, non-Hispanic whites, and obese people. Although there were no significant changes in the pattern of antidiabetic treatment, the age-adjusted percentage of people with diagnosed diabetes achieving glycemic and LDL targets increased from 43.1% to 57.1% (P < .05) and from 36.1% to 46.5% (P < .05), respectively. Glycosylated hemoglobin decreased from 7.62% to 7.15% during this period (P < .05). The age-adjusted percentage achieving all 3 targets increased insignificantly from 7.0% to 12.2%. The prevalence of diagnosed diabetes increased significantly from 1999 to 2006. The proportion of people with diagnosed diabetes achieving glycemic and LDL targets also increased. However, there is a need to achieve glycemic, blood pressure, and LDL targets simultaneously.
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                Author and article information

                Journal
                Diabetes Care
                Diabetes Care
                diacare
                dcare
                Diabetes Care
                Diabetes Care
                American Diabetes Association
                0149-5992
                1935-5548
                June 2012
                11 May 2012
                : 35
                : 6
                : 1232-1238
                Affiliations
                1Dallas Diabetes and Endocrine Center at Medical City, Dallas, Texas
                2Aggarwal & Associates Ltd., Brampton, Ontario, Canada
                3Janssen Global Services, LLC, La Jolla, California
                4Janssen Global Services, LLC, Raritan, New Jersey
                Author notes
                Corresponding author: Julio Rosenstock, juliorosenstock@ 123456dallasdiabetes.com .
                Article
                1926
                10.2337/dc11-1926
                3357223
                22492586
                © 2012 by the American Diabetes Association.

                Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

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                Categories
                Original Research
                Clinical Care/Education/Nutrition/Psychosocial Research

                Endocrinology & Diabetes

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