Early-onset metabolic syndrome in mice lacking the intestinal uric acid transporter SLC2A9

Abstract

Excess circulating uric acid, a product of hepatic glycolysis and purine metabolism, often accompanies metabolic syndrome. However, whether hyperuricaemia contributes to the development of metabolic syndrome or is merely a by-product of other processes that cause this disorder has not been resolved. In addition, how uric acid is cleared from the circulation is incompletely understood. Here we present a genetic model of spontaneous, early-onset metabolic syndrome in mice lacking the enterocyte urate transporter Glut9 (encoded by the SLC2A9 gene). Glut9-deficient mice develop impaired enterocyte uric acid transport kinetics, hyperuricaemia, hyperuricosuria, spontaneous hypertension, dyslipidaemia and elevated body fat. Allopurinol, a xanthine oxidase inhibitor, can reverse the hypertension and hypercholesterolaemia. These data provide evidence that hyperuricaemia per se could have deleterious metabolic sequelae. Moreover, these findings suggest that enterocytes may regulate whole-body metabolism, and that enterocyte urate metabolism could potentially be targeted to modulate or prevent metabolic syndrome.

ARTICLE

Received 13 Jan 2014 | Accepted 10 Jul 2014 | Published 7 Aug 2014

DOI: 10.1038/ncomms5642

Early-onset metabolic syndrome in mice lacking the intestinal uric acid transporter SLC2A9

Brian J. DeBosch1, Oliver Kluth2, Hideji Fujiwara1, Annette Schrmann2 & Kelle Moley1

Excess circulating uric acid, a product of hepatic glycolysis and purine metabolism, often accompanies metabolic syndrome. However, whether hyperuricaemia contributes to the development of metabolic syndrome or is merely a by-product of other processes that cause this disorder has not been resolved. In addition, how uric acid is cleared from the circulation is incompletely understood. Here we present a genetic model of spontaneous, early-onset metabolic syndrome in mice lacking the enterocyte urate transporter Glut9 (encoded by the SLC2A9 gene). Glut9-decient mice develop impaired enterocyte uric acid transport kinetics, hyperuricaemia, hyperuricosuria, spontaneous hypertension, dyslipidaemia and elevated body fat. Allopurinol, a xanthine oxidase inhibitor, can reverse the hypertension and hypercholesterolaemia. These data provide evidence that hyperuricaemia per se could have deleterious metabolic sequelae. Moreover, these ndings suggest that enterocytes may regulate whole-body metabolism, and that enterocyte urate metabolism could potentially be targeted to modulate or prevent metabolic syndrome.

1 BJC Institute of Health, Washington University School of Medicine, 10th Floor 425 S., Euclid Avenue Campus, Box 8064, St Louis, MO 63110, USA.

2 Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal 14558, Germany. Correspondence and requests for materials should be addressed to K.M. (email: mailto:[email protected]

Web End [email protected] ).

NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 1

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5642

Metabolic syndrome is a complex multifactorial disease that aficts two in ve adults in the U.S. alone1, and scores more worldwide. Observations over the prior

two decades demonstrated increased metabolic syndrome, type 2 diabetes mellitus and cardiovascular morbidity in hyperuricaemic subjects2,3, and that serum uric acid levels independently predict diabetes, fatty liver and metabolic syndrome2. Work in rodent models further mechanistically implicated uric acid in fructose-induced metabolic syndrome4, although some controversy over the exact physiological function of urate (the predominant form of uric acid at physiological pH) remains5,6. Elucidating how urate is removed from the circulation may nevertheless have broad individual and public health implications.

Approximately 6070% of circulating uric acid clearance occurs in the kidney and the other 3040% is cleared via intestinal enterocytes7, although the enterocyte may become the primary excretory pathway in renally insufcient patients8 (for example, in patients with diabetes, hypertension or cardiorenal disease). Recent data suggest that defective extrarenal clearance is a common cause of hyperuricemia9, yet few studies address enterocyte urate-handling mechanisms911, and none address endogenous urate clearance mechanisms as effectors of mammalian metabolism. The putative enterocyte urate transportersBCRP/ABCG2 (and potentially SLC17A4/NPT5) are apical transporters10,11, whereas a basolateral transporter has not been identied. Therefore, it was unresolved how uric acid ux occurred down its gradient from the blood into the enterocyte cytoplasm before excretion in the stool.

Recent data in humans and in rodents identied GLUT9/Glut9 as a high-capacity urate transporter12,13, the deletion of which alters urate homeostasis in a tissue-specic manner14,15. Furthermore, Glut9 is a basolateral and apical membrane transporter in other polarized epithelial cell types12. Thus, we examined GLUT9 localization, cellular function and role in urate homeostasis in the murine intestine.

Here we show that Glut9 is localized to the apical and basolateral enterocyte membranes, and that enterocyte-specic Glut9 deciency impairs enterocyte urate transport. Concomitant with these urate clearance defects, enterocyte Glut9-decient mice develop hyperuricaemia, hyperuricosuria and early-onset metabolic syndromehypertension, dyslipidaemia, hyperinsulinaemia and hepatic fat depositionwhich is partly mitigated by ad lib administration of the xanthine oxidase inhibitor, allopurinol. These results suggest that Glut9 regulates enterocyte urate clearance, and that enterocyte Glut9 deciency may have deleterious metabolic sequelae.

ResultsExpression and localization of enterocyte Glut9. Immunoblot analysis demonstrated that Glut9 was abundantly expressed in intestine (Fig. 1a), highly in the jejunum and ileum (Fig. 1b), the segments of the intestine that perform the majority of urate excretion8,10. Confocal immunouorescence microscopy of xed mouse intestine revealed Glut9 localized predominantly to the basolateral enterocyte membrane with some apical staining (Fig. 1c).

Generation of mice lacking enterocyte Glut9. Given that Glut9 is the only reported basolateral urate transporter, we hypothesized that mice lacking Glut9 in enterocytes would have impaired enteric urate handling. We thus generated enterocyte-specic Glut9-decient mice by crossing mice harbouring a oxed SLC2A9 allele with mice overexpressing cre recombinase driven by the enterocyte-specic villin promoter (Fig. 1d,e). vil-Cre/ SLC2A9/ (G9EKO) mice were live-born in Mendelian ratios,

were fertile and had no gross morphologic defects (Table 1). Immunoblotting conrmed deciency of total Glut9A and total Glut9 protein in G9EKO small-bowel lysates (Fig. 1f and Supplementary Figs 1 and 2). Isolated villous enterocytes from G9EKO mice exhibited an B65% [14C]-urate uptake defect versus wild-type (WT) enterocytes when compared with mice expressing vil-Cre with WT SLC2A9 alleles (hereafter referenced as WT mice) (Fig. 1g). Congruent with these ndings, liquid chromatographymass spectrometry (LC/MS) of stool extracts revealed signicantly decreased stool urate concentrations in G9EKO mice versus WT mice (Fig. 1h). Together, these results suggested impaired enterocyte uptake and efux into stool in G9EKO animals.

Physiological consequences of impaired enterocyte urate clearance. We next examined physiological consequences of impaired enterocyte urate efux in mice fed standard rodent chow (Picolab #5053: 25% protein, 13% fat and 62% carbohydrate). Uricase-based urate determinations further revealed signicantly higher uric acid concentrations in both serum and urine in G9EKO mice (Fig. 2a,b). Body composition analysis by EchoMRI revealed signicantly elevated body fat mass and fat percentage (Fig. 2c,d) despite higher resting energy expenditure as determined by indirect calorimetry in G9EKO mice (Table 2). Plasma analysis revealed G9EKO mice had signicantly elevated total cholesterol, free fatty acids and triglycerides (Fig. 2eg). Some evidence of early insulin resistance was observed, with an approximately 40% elevation in fasting plasma insulin in G9EKO mice (Fig. 2h) in context of normal fasting blood glucose (Fig. 2i) and insulin tolerance testing (Fig. 2j). Tail-cuff plethysmography in unanaesthetized G9EKO mice demonstrated signicant baseline hypertension when compared with WT mice (Fig. 2k).

We next assessed whether blocking uric acid production could reverse any of these effects of impaired urate clearance. WT and G9EKO mice were thus exposed to 8-week allopurinol (150 mg l 1) ad libitum in sterilized drinking water approximately 4 weeks after weaning4. Plasma uric acid was signicantly decreased in G9EKO mice following allopurinol treatment without signicant effects on urine urate concentrations (Fig. 3a,b). Strikingly, in comparison with untreated G9EKO mice, allopurinol treatment also lowered blood pressure and total cholesterol (Fig. 3c,d) without signicant effects on free fatty acids and triglycerides suggesting at least partial allopurinol reversibility of the spontaneous metabolic syndrome in G9EKO miceeither through xanthine oxidase inhibition or through direct effects on renal uric acid transporters by allopurinol or by its active metabolite, oxypurinol. Fasting free fatty acid and triglyceride were not signicantly affected by allopurinol (Fig. 3e,f).

We then looked at well-described secondary complications of hyperuricaemia in G9EKO mice, including non-alcoholic fatty liver disease and cardiovascular disease. Consistent with ndings of increased steatosis/non-alcoholic fatty liver disease in patients with hyperuricaemia16, hepatic triglycerides and free fatty acids were elevated in G9EKO livers versus WT livers (Fig. 4a,b). In addition, mRNA for sterol regulatory element-binding protein, a marker of cholesterol metabolism, was signicantly elevated in the livers of G9EKO mice, and there was a trend toward elevated collagen type 1 alpha and fatty acid synthase, markers of brosis and fat synthesis.

The increased incidence of cardiovascular morbidity in hyperuricaemic patients prompted us to survey the cardiac phenotype of G9EKO mice. Basal heart rate observed by echocardiography was signicantly elevated in G9EKO mice

2 NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5642 ARTICLE

a

b

75 kDa 50 kDa

75 kDa

Glut9

GAPDH

50 kDa

Glut9

GAPDH

50 kDa

Liver

Kidney

Lung

Spleen

Intestine

Testis

WAT

Duodenum

Jejunum

Ileum

Colon

c

Pre-immune serum

Preimmune

serum

anti-Glut9

serum

75 kDa 50 kDa

Kidney

Liver

Liver

Kidney

Duodenum

5 Homologus sequence 3 Homologus sequence

4 5 6 N E O 7

Exon

Jejunum Jejunum

d

loxP-site

for rev

e

f g

+/+

loxP/+

loxP/loxP

WT

G9EKO

50 kDa

50 kDa

75 kDa

Total Glut9

Glut9A

100

14 C]-uric acid uptake

(nmol*mg1 *min1 )

75 50 25

*

Actin

720 bp

Total Glut9

Glut9A

GAPDH

554 bp

WT

G9EKO

WT

G9EKO

G9EKO

WT

0[

Intestine Liver Kidney

h

WT

G9EKO

Uric acid

1.00e58.00e46.00e44.00e42.00e40.00

1.00e58.00e46.00e44.00e42.00e40.00

1.00e58.00e46.00e44.00e42.00e40.00

0.95

4.33

4.25

4.053.84

3.0 4.0 5.0 6.0 7.0

4.12 4.19

4.26

2.17

0.97 1.18

0.78 2.00 2.23 2.813.20

3.86

1.0 2.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0

1.00e58.00e46.00e44.00e42.00e40.00

Internal standard

1.53

1.0 2.0 3.0 4.0 5.0 6.0 7.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Figure 1 | Characterization of intestinal Glut9 and genetic deletion of enterocyte SLC2A9. (a) Immunoblotting against full-length Glut9 in murine tissue. (b) Small-bowel segment-specic Glut9 immunoblotting. (c) Left, confocal immunouorescence microscopy demonstrating basolateral and apical Glut9 localization in duodenum and jejunum. Middle, jejunal immunostaining with pre-immune serum. Scale bar, 10 mm. Right, specicity of total Glut9 antiserum versus pre-immune serum by liver and kidney lysate immunoblotting (d) Glut9-targeting construct used to generate mice harbouring oxed SLC2A9 anking exons 5 and 6. Forward and reverse (for/rev) genotyping primers ank the ox site adjacent to exon 5. (e) PCR bands depicting larger oxed (720 bp) and wild-type (WT) (554 bp) sequences. (f) Immunoblot of Glut9A in WT and G9EKO mouse whole intestine, liver and kidney lysate. (g) [14C]-uric acid uptake in puried villous enterocyte fractions from WT and G9EKO mice. n 5 per group. In vitro experiment replicated thrice.

*Po0.05 versus WT (two-tailed t-testing). Error bars represent s.e.m. (h) LC/MS analysis of stool from WT and G9EKO mice. Upper spectrauric acid (arrows) elution. Lower spectra (arrowheads)1-methyluric acid internal standard. Experiment replicated thrice.

NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 3

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5642

(Fig. 4d). Echocardiogram also revealed evidence of cardiac hypertrophic remodelling, (Fig. 4e,f)decreased diastolic left ventricular internal diameter with increased relative wall thicknessa common nding in patients with hyperuriceamia17. However, although allopurinol treatment signicantly lowered body weight (Fig. 5a) and hepatic triglyceride content (Fig. 5c) associated with a trend toward lower body fat mass in G9EKO mice (Fig. 1b), allopurinol treatment did not signicantly reverse the G9EKO-associated changes in heart rate, left ventricular internal diameter or relative wall thickness (Fig. 5df).

DiscussionMice lacking enterocyte Glut9 thus develop early-onset spontaneous hyperuricaemic metabolic syndrome that is partially mitigated by allopurinol, a xanthine oxidase inhibitor. Not all

deleterious ndings in the G9EKO model were reversed by allopurinol, suggesting either (a) some metabolic syndrome components occur independently of hyperuricaemia, (b) some metabolic syndrome components caused by hyperuricaemia are irreversible once clinically apparent or (c) uric acid is unrelated to any ndings, and the allopurinol effects are derived specically from blocking oxidant production via xanthine oxidase inhibition per se. It should be noted, however, that the key advantage to this model of impaired urate excretion in delineating the specic

Table 2 | Indirect calorimetric data from 8- and 16-week-old wild type and G9EKO mice.

n VO2(ml g 1 h 1)

VCO2 (ml g 1 h 1)

RER (ratio)

Heat (kcal h 1)

Wild type (8 week)

9 9.14 7.55 0.83 0.81

Table 1 | Basic morphometric parameters in wild-type (WT) and G9EKO mice.

n BW LW HW LW/BW HW/BW Wild type 19 24.9 952.5 132.2 39.3 5.5

G9EKO 10 26.1 922.2 129.9 40.1 5.7 P-value 0.116 0.585 0.741 0.634 0.439

BW, body weight; HW, heart weight; LW, liver weight.

P-values are derived from two-tailed t-tests.

200

9EKO(8 week)

12 10.24* 7.44 0.74* 1.17*

Wild type (16 week)

10 8.61 7.32 0.86 0.99

9EKO(16 week)

12 9.07* 7.27 0.81 0.97

RER, respiratory exchange ratio; VCO , volume of expired carbon dioxide; VO2, volume of

inspired oxygen.

*Po0.05 versus age-matched wild-type control, as determined by two-tailed t-tests.

a b c d

**

**

*** **

Serum urate (M)

150

100

500

Urine urate (M)

5,000 8.0

6.0

4.0

2.0

0.0

25 20 15 10

5 0

4,000 3,000 2,000 1,000

0

Body fat (g)

Body fat (%)

WT

G9EKO

0

e f g h

5

1.0

* *** ** *

1.2

1.5

4

0.8

1.0

Blood glucose (% T 0) Serum TG (mM)

Cholesterol (nM)

3

0.8

0.6

1

FFA (mM)

0.6

Insulin (nM)

2

0.4

0.4

0.5

1

0.2

0.2

0

125

100

75

50

25

0

0.0

0.0 0

i j k

140 ***

Blood glucose (mg dl1 )

Blood pressure (mm Hg)

120 100

80 60 40 20

0

120

806040 WT

9E KO

*** ***

100

20

0

0 15 30

Time (min)

45 60 120

SBP DBP MBP

Figure 2 | Physiological consequences of enterocyte Glut9 deciency. (a,b) Serum (n 9, 16) and urine (n 13, 12) uric acid concentration in

fasting 68-week-old mice. (c,d) EchoMRI analysis of fat mass and percentage fat (n 19, 10). (ei) Plasma cholesterol (n 6, 5), triglycerides,

free fatty acids, insulin (n 5, 4) and glucose in fasting WTand G9EKO mice. (j) Insulin tolerance testing in 4-h fasting mice (n 6, 22). (k) Systolic (SBP),

diastolic (DBP) and mean (MBP) blood pressure in non-fasting WT mice and G9EKO (n 9, 17). *Po0.05, **Po0.01 and ***Po0.001 versus WT

(two-tailed t-testing). Error bars represent s.e.m.

4 NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5642 ARTICLE

a b c d e f

140 120 100

80 60 40 20

0

*

*

120 100

80 60 40 20

#

*

Plasma urate (M)

120 100

80 60 40 20

0

*

* *

*

# #

#

#

Urine urate (M)

2,500 2,000 1,500 1,000

500 0

Pressure (mm Hg)

FFA (mM)

10.80.60.40.2 0

TG (mM)

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0 SBP DBP MBP

Cholestrol (mg dl1)

WT G9EKO G9EKO + Allopurinol

Figure 3 | Allopurinol mitigates G9EKO hyperuricaemia, hypertension and dyslipidaemia. (a,b) Serum (n 8, 18, 8) and urine uric acid (n 4, 7, 7)

in WT and G9EKO fed sterile water or water containing allopurinol. (c) Systolic (SBP), diastolic (DBP) and mean (MBP) blood pressure in unmedicated or in allopurinol-treated G9EKO and WT mice (n 5, 9, 7). (df) Fasting cholesterol (n 6, 5, 8), free fatty acid (FFA) (n 5, 5, 5) and triglyceride

(TG) (n 5, 5, 10) in WT and G9EKO mice. *Po0.05 versus WT. #Po0.05 versus untreated G9EKO mice (two-tailed t-testing with BonferroniDunn

post hoc correction for data sets on which multiple comparisons are made). Error bars represent s.e.m.

12 10

8 6 4 2 0

a b c d e f

2.52.01.51.00.50.0

***

***

Hepatic TG

25 20 15 10

5 0

*

*

*

Hepatic FFA

(nmol mg1)

*

(nmol mg1)

Fold expression

700

600

500

400

300

200

100

0

LVIDd (mm)

5.04.03.02.01.00.0

Rel. wall thickness

(mm)

0.6

0.5

0.4

0.3

0.2

0.1

0.0

COL1A FAS SREBP

Heart rate (b.p.m)

WT G9EKO

Figure 4 | Early-onset non-alcoholic fatty liver disease (NAFLD) and cardiac remodeling in G9EKO mice. (a,b) Hepatic free fatty acid (FFA)(n 6, 6) and triglyceride (TG) (n 6, 6) in WT and G9EKO liver homogenates. (c) Relative mRNA abundance of collagen type 1a, fatty acid synthase

and sterol response element-binding protein-1 by quantitative reverse transcriptase (qRTPCR) analysis. (df) Echocardiographic analysis of heart rate, diastolic left ventricular internal diameter and relative wall thickness in WT and G9EKO mice (n 5, 5). *Po0.05 versus WT. ***Po0.001 versus WT

(two-tailed t-testing). Error bars represent s.e.m.

effects of hyperuricaemia is that it does not rely on xanthine oxidase activity to produce hyperuricaemia as in some of the diet-induced models (for example, fructose- or purine-rich diets). Nevertheless, while further insight into the effects of hyperuricaemia on its host are required, the G9EKO model suggests that attenuated enterocyte urate clearance could be a critical, initiating step in metabolic syndrome pathogenesis. Interdicting circulating urate accumulation by focusing on gut urate reuptake and clearance could represent a novel treatment paradigm to block or modulate hyperuricaemia and its complications, including metabolic syndrome. We expect this model can now be used to probe mechanistically into the role of urate in the complications and sequelae of metabolic syndrome.

Methods

Generation of G9EKO Mice. Enterocyte-specic Glut9-decient mice were generated by homologous recombination of the 18.9 kb targeting vector harbouring loxP sites anking exons 5 and 6 and a neomycin resistance cassette 30 of exon 6.

Modied embryonic stem cells from 129Sv mice carrying this targeting allele were introduced into C57BL/6 blastocysts. Chimeric offspring were twice backcrossed into the C57BL/6 line to generate N2 mice heterozygous for the targeting allele (genotype Slc2a9/ ). N2 mice were intercrossed to generate homozygous (Slc2a9/) mice. Genotyping primers were as follows (listed 5030): forward primer50-TGGTGCTACTCTGTGGTGCTA-30; reverse primer50-CACAGCG GTGAAAGTAACGA-30. Mice harbouring homozygous oxed Slc2a9 alleles were crossed with mice expressing Cre recombinase under the mouse villin 1 promoter on a C57/BL6 background (Jackson Laboratories, stock number 004586) to generate G9EKO mice. Mice expressing villin promoter-driven Cre with WT Slc2a9 alleles were used as control mice throughout these studies. For allopurinol treatment experiments, 8-week-old mice were maintained on 150 mg l 1 allopurinol4

for 8 weeks before metabolic characterization. All animal procedures were approved by the Washington University School of Medicine Animal Studies Committee.

Calorimetry body composition and blood pressure determination. Indirect calorimetry, echoMRI and blood pressure analyses on unanaesthetized mice were performed via the Mouse Diabetic Models Phenotyping Core Facility at Washington University using an Oxymax Indirect Calorimeter (Columbus Instruments, Columbus, OH, USA), an EchoMRI 31 (Echo Medical Systems, Houston, TX, USA18,19 and a Columbus Instruments NIBP (Columbus) tail-cuff and plethysmograph. Food consumed in mice was measured in 24-h increments during each calorimetry run.

Radiolabelled uric acid uptake measurements. Apical villous enterocytes rst were enriched by calcium chelation and mechanical disruption20. Briey, sections of distal jejunum (56 cm) were removed, ushed with ice-cold phosphate-buffered saline and opened longitudinally before immersion in ice-cold phosphate-buffered saline. The bowel was transferred into 5 ml ice-cold balanced salt solution (BSS) buffer (1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate, 8 mm KH2PO4, 5.6 mM

Na2HPO4, 15 mM EDTA, and 1 mM dithiothreitol) and vortexed at 4 C at maximum speed for 20 min. Following this step, almost all epithelial cells were recovered in the BSS solution. The BSS solution (containing crypts and villi) was ltered through a 70-mm cell strainer (BD Biosciences, San Jose, CA, USA). The larger size villi were captured in the cell strainer whereas crypts were discarded in the efuent. Crypt fractions were then spun down at 1,000g for 10 min at 4 C and washed once with Tris buffer (150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM Tris-HCl, pH 8.0) then with ice-cold DMEM before assay. To assay uptake, cells were resuspended in pre-warmed, 37 C DMEM containing 1 mCi ml 1 [14C]-uric acid (American Radiolabeled Chemicals, St Louis, MO, USA) for 3 min before quenching and three wash steps in ice-cold Hanks balanced salt solution. Samples were subjected to liquid scintillation counting and counts were normalized to sample protein, as determined by bicinchoninic acid assay kit (Pierce, Rockford, IL, USA).

NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 5

& 2014 Macmillan Publishers Limited. All rights reserved.

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5642

a

30

b

6

###

###

*

NS

NS

NS

Body weight (g)

Hepatic TG

(nmol mg1)

10

0 30

4

Body fat (g)

20

4

2 0

c d

800

0.8

* *

20 10

0

Heart rate (b.p.m.)

600

400

200

0

e f

*

NS

3

*

LVIDd (mm)

2

1

0

Rel. wall thicknes

(mm)

0.6

0.4

0.2

0.0

WT

G9EKO G9EKO + Allopurinol

Figure 5 | Allopurinol reduces body weight and hepatic triglycerides in G9EKO mice. Shown is the effect of B8-week allopurinol treatment(150 mg l 1 fed ad libitum in water) on (a) body weight and (b) body fat (n 21, 19, 24) and (c) hepatic triglycerides (triglyceride (TG), n 11, 9, 7).

Panels d,e,f, demonstrate the effect of allopurinol on echocardiographic parameters (n 12, 6, 12). HR, heart rate, LVIDd, internal left ventricular

diameter in diastole, RWT, relative wall thickness. *Po0.05 versus WT control by two-tailed t-test after BonferroniDunn post hoc correction for multiple comparisons. ###Po0.001 versus untreated G9EKO mice. NS, not statistically signicantly different versus G9EKO untreated mice (two-tailed t-test after BonferroniDunn post hoc correction). Error bars represent s.e.m.

Western Blotting and immunostaining. Western blotting was performedusing 50100 mg protein subjected to 10% SDSPAGE, followed by transfer to nitrocellulose membrane and overnight primary antibody incubation21,22 (1:1,000 dilutions at 4 C) with the following antisera: total Glut9, glyceraldehyde 3-phosphate dehydrogenase (clone 14C10, Cell Signaling Technologies, Beverly, MA, USA), Glut9A12 Actin (clone C4, EMD Millipore, Billerica, MA, USA). Fixed (4% paraformaldehyde), parafn-embedded intestinal sections were sectioned, permeablized in 0.1% Triton X-100 in phosphate-buffered saline and blocked with 2% bovine serum albumin (Sigma-Aldrich, St Louis, MO, USA) before immunostaining using pre-immune serum or GLUT9-specic antibody (Generated in the laboratory of Dr Annette Schrmann) at a 1:200 dilution.

LC and tandem mass spectrometry. Uric acid analysis in stool was performed by the Washington University Metabolomics Facility. Uric acid was extracted from

B20 mg dried, pulverized, sieved mouse stool using water containing 1-methyluric acid (Sigma) for LC/MS/MS using a modied US analysis method22. 1-methyluric acid was chosen as the internal standard and a Thermo betasil LC column was used for negative ion electrospray LC/MS/MS analysis.

Hepatic lipid extraction. Hepatic lipids were extracted by homogenizing 100 mg of liver tissue in 2 ml (2:1) chloroform:methanol23. Remaining tissue was pelleted by centrifugation and 1020 ml supernatant aliquots were dried for 2 h before resuspension in assay reagent. Values were normalized to tissue input mass.

Biochemical analyses. Serum and urine uric acid, serum and hepatic cholesterol, triglyceride, free fatty acid and serum insulin were measured using the following kits precisely per the manufacturers instructions18,19,23: Amplex Red Uric Acid Assay Kit (Invitrogen, Carlsbad, CA, USA), Innity Cholesterol Assay Kit, (Thermo Scientic, Waltham, MA, USA), Innity triglyceride assay kit (Thermo Scientic, Waltham, MA, USA), NEFA free fatty acid determination kit (Wako Diagnostics, Richmond, VA, USA) and rat/mouse insulin ELISA assay kit (Millipore, Billerica, MA, USA).

Echocardiography. Echocardiography was performed by the Washington University Mouse Cardiovascular Phenotyping Core Facility21. Non-invasive

transthoracic cardiac ultrasound exams were performed under light general anaesthesia using a VisualSonics Vevo 2100 cardiac echocardiography machine with a 15-MHz linear transducer.

Statistical analysis. All values are expressed as the means.e.m. Two-tailed t-tests were applied with BonnferroniDunn post hoc correction for multiple comparisons. Po0.05 was considered to be statistically signicant between groups.

References

1. Alberti, K. G. et al. Harmonizing the metabolic syndrome: a joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society; and International Association for the Study of Obesity. Circulation 120, 1640 (2009).

2. Johnson, R. J. et al. Sugar, uric acid, and the etiology of diabetes and obesity. Diabetes 62, 3307 (2013).

3. Feig, D. I., Kang, D. H. & Johnson, R. J. Uric acid and cardiovascular risk.N. Engl. J. Med. 359, 1811 (2008).4. Nakagawa, T. et al. A causal role for uric acid in fructose-induced Metabolic Syndrome. Am. J. Physiol. Renal. Physiol. 290, F625 (2006).

5. Palmer, T. M. et al. Association of plasma uric acid with ischaemic heart disease and blood pressure: mendelian randomisation analysis of two large cohorts. BMJ 347, f4262 (2013).

6. Hughes, K. et al. Mendelian randomization analysis associates increased serum urate, due to genetic variation in uric acid transporters, with improved renal function. Kidney Int. 85, 344 (2014).

7. So, A. & Thorens, B. Uric acid transport and disease. J. Clin. Invest. 120, 1791 (2010).

8. Sorensen, L. B. Role of the intestinal tract in the elimination of uric acid. Arthritis Rheum. 8, 694 (1965).

9. Ichida, K. et al. Extra-renal urate excretion is a common cause of hyperuricemia. Nat. Commun. 3, 764 (2012).

10. Hosomi, A., Nakanishi, T., Fujita, T. & Tamai, I. Extra-renal elimination of uric acid via intestinal efux transporter BCRP/ABCG2. PLoS ONE 7, e30456 (2012).

11. Anzai, N. & Endou, H. Urate transporters: an evolving eld. Semin. Nephrol. 31, 400 (2011).

12. Augustin, R. et al. Identication and characterization of human glucose transporter-like protein-9 (Glut9): alternative splicing alters trafcking. J. Biol. Chem. 279, 16229 (2004).

13. Cauleld, M. J. et al. SLC2A9 is a high-capacity urate transporter in humans. PLoS Med. 5, e197 (2008).

14. Preitner, F. et al. Glut9 is a major regulator of urate homeostasis and its genetic inactivation induces hyperuricosuria and urate nephropathy. Proc. Natl Acad. Sci.USA 106, 15501 (2009).

15. Preitner, F. et al. Urate-induced acute renal failure and chronic inammation in liver-specic Glut9 knockout mice. Am. J. Physiol. Renal. Physiol. 305, F786 (2013).

16. Katsiki, N., Athyros, V. G., Karagiannis, A. & Mikhailidis, D. P. Hyperuricaemia and non-alcoholic fatty liver disease (NAFLD): a relationship with implications for vascular risk? Curr. Vasc. Pharmacol. 9, 698 (2011).

17. Kurata, A., Shigamatsu, Y. & Hikagi, J. Sex-related differences in relations of uric acid to left ventricular hypertrophy and remodeling in Japanese hypertensive patients. Hypertens. Res. 28, 133 (2005).

18. DeBosch, B. J., Chi, M. & Moley, K. H. Glucose transporter 8 (GLUT8) regulates enterocyte fructose transport and global mammalian fructose utilization. Endocrinology 153, 4181 (2012).

19. DeBosch, B. J., Chen, Z., Finck, B. N., Chi, M. & Moley, K. H. Glucose transporter 8 (GLUT8) mediates glucose intolerance and dyslipidemia in high-fructose diet-fed male mice. Mol. Endocrinol. 27, 1887 (2013).

20. Sun, R. C., Choi, P. M., Guo, J., Erwin, C. R. & Warner, B. W. Insulin-like growth factor 2 and its enterocyte receptor are not required for adaptation in response to massive small bowel resection. J. Ped. Surg. 49, 966 (2014).

21. DeBosch, B. et al. Akt1 is required for physiological cardiac growth. Circulation 113, 2097 (2006).

22. Kim, K. M. et al. A sensitive and specic liquid chromatography-tandem mass spectrometry method for the determination of intracellular and extracellular uric acid. J. Chromatogr. B Analyst. Technol. Biomed. Life Sci. 877, 20322038 (2009).

23. DeBosch, B. J., Chen, Z., Saben, J. L., Finck, B. N. & Moley, K. H. Glucose transporter 8 (GLUT8) mediates fructose-induced de novo lipogenesis and macrosteatosis. J. Biol. Chem. 289, 10989 (2014).

Acknowledgements

We thank Nicole Junicke for technical work in generating the targeting vector used to

produce the G9EKO mouse line. This work was supported by the Pediatric Scientist

Development Program (PSDP) NIH grant 5K12HD000850-27 (to B.J.D.), DDRCC grant

6 NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

& 2014 Macmillan Publishers Limited. All rights reserved.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5642 ARTICLE

P30DK52574 (to B.J.D.) and R01HD040390-07 (K.M.). The German Ministry

of Education and Research (BMBF: DZD, 01GI0922), and the State of Brandenburg

(to A.S. and O.K.). The Washington University Mouse Diabetes Model Phenotyping

Core is supported by DRTC grant P60 DK020579. Mass spectrometry was performed

in the Metabolomics Facility at Washington University (P30 DK020579).

Author contributions

B.J.D. designed, executed and interpreted experiments, and wrote the manuscript. O.K.

and A.S. designed, executed and interpreted experiments and generated the oxed Glut9

model. H.F. designed, executed and interpreted the LC/MS experiments. K.M. designed

and interpreted experiments and critically reviewed the paper.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Web End =http://www.nature.com/

http://www.nature.com/naturecommunications

Web End =naturecommunications Competing nancial interests: The authors declare no competing nancial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

Web End =http://npg.nature.com/

http://npg.nature.com/reprintsandpermissions/

Web End =reprintsandpermissions/

How to cite this article: DeBosch, B. J. et al. Early-onset metabolic syndrome

in mice lacking the intestinal uric acid transporter SLC2A9. Nat. Commun. 5:4642

doi: 10.1038/ncomms5642 (2014).

NATURE COMMUNICATIONS | 5:4642 | DOI: 10.1038/ncomms5642 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications 7

& 2014 Macmillan Publishers Limited. All rights reserved.