p-Cresyl Sulfate Promotes Insulin Resistance …jasn.asnjournals.org/content/24/1/88.full.pdfBASIC RESEARCH p-Cresyl Sulfate Promotes Insulin Resistance Associated with CKD Laetitia

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p-Cresyl Sulfate Promotes Insulin ResistanceAssociated with CKD

Laetitia Koppe,*† Nicolas J. Pillon,† Roxane E. Vella,† Marine L. Croze,† Caroline C. Pelletier,*†

Stéphane Chambert,‡ Ziad Massy,§ Griet Glorieux,| Raymond Vanholder,| Yann Dugenet,§

Hédi A. Soula,† Denis Fouque,*† and Christophe O. Soulage†

*Hospices Civils de Lyon, Department of Nephrology, Hôpital E Herriot, Lyon, France; †Université de Lyon, INSA deLyon, CarMeN, INSERM U1060, Villeurbanne, France; ‡ICBMS, Université de Lyon, Université Lyon 1, Villeurbanne,France; §Divisions of Clinical Pharmacology and Nephrology, INSERM U-1088, University of Picardie Jules Vernes,Amiens University Hospital, Amiens, France; |Nephrology Section, Department of Internal Medicine, UniversityHospital, Ghent, Belgium; and §Witaxos/BioActor b.v. BioPartner Center, Maastricht, the Netherlands

ABSTRACTThe mechanisms underlying the insulin resistance that frequently accompanies CKD are poorly under-stood, but the retention of renally excreted compounds may play a role. One such compound is p-cresylsulfate (PCS), a protein-bound uremic toxin that originates from tyrosine metabolism by intestinalmicrobes. Here, we sought to determine whether PCS contributes to CKD-associated insulin resistance.Administering PCS to mice with normal kidney function for 4 weeks triggered insulin resistance, loss of fatmass, and ectopic redistribution of lipid in muscle and liver, mimicking features associatedwith CKD.Micetreated with PCS exhibited altered insulin signaling in skeletal muscle through ERK1/2 activation. Inaddition, exposing C2C12 myotubes to concentrations of PCS observed in CKD caused insulin resistancethrough direct activation of ERK1/2. Subtotal nephrectomy led to insulin resistance and dyslipidemia inmice, and treatment with the prebiotic arabino-xylo-oligosaccharide, which reduced serum PCS bydecreasing intestinal production of p-cresol, prevented these metabolic derangements. Taken together,these data suggest that PCS contributes to insulin resistance and that targeting PCSmay be a therapeuticstrategy in CKD.

J Am Soc Nephrol 24: 88–99, 2013. doi: 10.1681/ASN.2012050503

The uremic syndrome is attributed to the pro-gressive retention of numerous compounds, whichin healthy individuals are normally excreted by thekidneys. At least 90 compounds, often referred toas uremic toxins, were described to accumulate inESRD1 and to be harmful for biologic systems. Inrecent years, the dialysis community has paid greatattention to improve clearance of water-solublemolecules, such as urea. Unfortunately, severalstudies, including the HEMO study in hemodialy-sis,2 and the ADEMEX study (Adequacy of Perito-neal Dialysis in Mexico),3 failed to significantlyimprove patient outcome. Protein-bound uremictoxins especially exert major toxic effects becauseof poor removal by the common dialysis techni-ques.4,5 p-Cresol is the mother compound of animportant group of protein-bound retention sol-utes.6 It is produced in the gut from themetabolism

of aromatic amino acids by putrefactive bacteria ofthe gut microbiota.7,8 As it crosses through the in-testinal mucosa, p-cresol is then metabolized by acytoplasmic sulfotransferase and therefore mainlycirculates in blood as its sulfate conjugate, p-cresylsulfate (PCS) (Supplemental Figure 1). PCS is

Received May 19, 2012. Accepted October 18, 2012.

D.F. and C.O.S. contributed equally to this work

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Christophe O. Soulage, INSERM U1060,Cardiovasculaire, Métabolisme, diabétologie et Nutrition (CarMeN),Bâtiment IMBL, INSA-Lyon, 20 avenue Albert Einstein 69621 Vil-leurbanne Cedex France. Email: [email protected]

Copyright © 2013 by the American Society of Nephrology

88 ISSN : 1046-6673/2401-88 J Am Soc Nephrol 24: 88–99, 2013

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excreted by the kidney mainly through proximal tubular se-cretion and therefore accumulates in serum of patients withrenal dysfunction. Of note, p-cresol/PCS has been shown to beindependently associated with mortality and cardiovasculardisease in patients with CKD.9–11 There are now accumulatingin vitro data on the harmful effects of p-cresol/PCS and relatedprotein-bound retention solutes.7,9,12–15

CKD is associated with a large range of metabolic alter-ations.16 As established by the pioneering work of DeFronzoet al., insulin resistance is a well documented feature ofCKD.17,18 Among patients with ESRD, insulin resistance is as-sociated with higher prevalence of vascular diseases.19 Althoughthe causes of CKD-associated insulin resistance remain poorlyidentified,20 several abnormalities associated with renal dys-function were reported to interfere with insulin signaling.21,22

The exactmechanisms underlying insulin resistance in patientswith CKD have not been clearly elucidated, but growing evi-dence suggests that the progressive retention of a large numberof compounds, which under normal conditions are excretedby the healthy kidneys, could play a key role. In a recent study,D’Apolito et al.23 demonstrated that increased urea levels inCKD could induce insulin resistance. However, to date nostudy has ruled out the role of protein-bound uremic toxinsin the development of insulin resistance and metabolic distur-bances in CKD.We hypothesized that increased concentrationsof PCS associated with CKD might drive insulin resistanceand metabolic disturbances associated with CKD. In this study,we demonstrate that PCS administrated to mice with normalrenal function induces insulin resistance and metabolic distur-bances mimicking those reported in CKD.We further show thatthe reduction of p-cresol intestinal production (and thus plasmaPCS) by the use of a prebiotic (arabino-xylo-oligosaccharide[AXOS]) prevented insulin resistance and dyslipidemia in amouse model of CKD.

RESULTS

Serum Concentration of PCS in PCS-Treated MiceIntraperitoneal injection of PCS (10 mg/kg) in mice with nor-mal renal function induced a transient increase in serum PCSconcentration. Serum total concentration of PCS reached5.0161.59 mg/dl 10 minutes after injection and decreasedto 0.3560.04 mg/dl 1 hour after injection (Figure 1A). Themice exhibited free PCS serum levels of 0.8260.24mg/dl at 10minutes and 0.0160.03 mg/dl at 1 hour (Figure 1B). Fourhours after injection, serum PCS concentration returned tobaseline level. The peak concentrations were in the range ofconcentrations found in sera from patients with ESRD (2–4mg/dl).24 In vehicle-treated mice, total PCS level was0.0360.01 mg/dl and free PCS remained undetectable. After4 weeks of chronic administration of PCS, no accumulationwas noticed (Supplemental Table 1). In mice, the in vivoplasma protein binding of PCS was 90.5%62.0% (Supple-mental Table 2).

PCS Mice Exhibit Hyperglycemia andHypercholesterolemiaTo evaluate the in vivo activity of PCS, mice were given dailyinjections of PCS (10 mg/kg, twice a day) or vehicle for 4weeks. Renal function was extensively evaluated in mice treatedfor 4 weeks with PCS (Table 1 and Supplemental Figure 2). It isworth noting that no difference in renal function or structurewas observed between saline- and PCS-treated mice. The fast-ing plasma glucose concentration in PCS mice was 1.5-foldhigher than in vehicle mice (P,0.001) (Table 1), suggestingthe presence of insulin resistance. Remarkably, in PCS mice,plasma cholesterol levels were increased by .50% comparedwith vehicle mice, whereas a nonsignificant trend toward in-crease was observed for plasma triacylglycerols (Table 1). Asshown in Table 1, these metabolic disturbances closely mimic

Figure 1. Intraperitoneal injection of PCS (10 mg/kg) transientlyincreases plasma PCS concentration in mice with normal renal func-tion. Serum concentrations of (A) total and (B) free PCS. Results areexpressed as mean 6 SEM for n=4 mice. Different letters betweentime points indicate a significant difference at the P,0.05 level.

J Am Soc Nephrol 24: 88–99, 2013 PCS Disrupts Insulin Signaling 89

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the profile of metabolic changes observed in CKD mice (i.e.,hyperglycemia and hypercholesterolemia).

Chronic Administration of PCS Induces InsulinResistance in Mice and Interferes with Insulin SignalingPathways in Skeletal MuscleThe results of the insulin tolerance test after 4 weeks of PCSadministration are shown in Figure 2A. Sixty minutes afterinjection of PCS, insulin administration (0.5 IU/kg)triggered a significantly (P,0.01) larger hypoglycemic re-sponse in vehicle-treated mice (change, 247%) than inPCS-treated mice (change, 219%) or CKD mice (change,214%). In vehicle-treated mice, insulin stimulationinduced a 3.2-fold increase in PKB/Akt phosphorylation onSer 473 (P,0.01). In contrast, insulin-induced PKB/Aktphosphorylation was impaired in PCS mice (P,0.01),indicating a disruption of the intracellular insulin signalingpathways (Figure 2B). Activation of stress protein kinases,such as c-Jun N-terminal kinase (JNK),25 and extracellularsignal-regulated kinase (ERK1/2)26 are described to interferewith insulin signaling pathways; we therefore studied ERKand JNK activation in muscle of PCS-treated mice. PCS-treatedmice exhibited a significant activation of ERK1/2 comparedwith vehicle-treated mice. Indeed, phosphorylation level ofERK1/2 was increased by 2.9-fold (P,0.05) compared withcontrol in the insulin-stimulated condition (Figure 2C). Incontrast, we failed to detect any phosphorylation of JNK inskeletal muscle of PCS-treated mice (data not shown). Theeffects of a single injection of PCS (10 mg/kg) on insulin sen-sitivity are shown in Supplemental Figure 3.

Chronic Treatment with PCS Decreases White AdiposeTissue Accretion and Induces Ectopic LipidRedistributionThemean daily food intake was 68.161.3 kJ/24 hours formicetreated with PCS and 69.162.1 kJ/24 hours in control animals

(n=10, nonsignificant). Biometric data for each group areshown in Table 2. Total weight and lean body mass did notsignificantly differ between PCS mice and control mice after 4weeks of treatment. PCS mice, however, exhibited a signifi-cant decrease in white adipose tissue accretion (change,251%;P,0.05) (Figure 3A). PCS treatment significantly decreasedwhite adipose tissue mass in the intraabdominal fat pads(e.g., retroperitoneal fat pads) (change, 242%; P,0.05) andthe epididymal fat pads (change,246%; P,0.05), as well as inthe subcutaneous inguinal fat pad (change, 246%; P,0.05)(Figure 3A). The effect of PCS was restricted to white adiposetissue because the mass of other organs (liver, heart, and kid-ney) remained unaltered. On the other hand, ectopic lipid con-tents were increased in muscle (83% increase; P,0.001) andliver (23% increase; P,0.05) compared with vehicle (Figure3B), suggesting lipotoxicity as a putative cause for insulin re-sistance. Of note, a similar loss of fat mass (change, 283%;P,0.05) and ectopic lipid redistribution were found in CKDmice. The distribution frequencies of adipocyte volume forsaline- or PCS-treated mice are shown in Figure 3C. A signif-icant shift leftward was observed in frequency distribution ofadipocyte volumes of mice treated with PCS compared withcontrol animals. The cellular characteristics of epididymalwhite adipose tissue in control and PCS-treatedmice are shownin Table 3. Mean adipocyte diameter was reduced by 21% inPCS-treated mice (n=8; P,0.005) resulting in a 46% decreasein adipose cell weight (n=8; P,0.01). The total number ofadipocytes per parametrial fat pad, calculated from cell weight,failed to show any difference between PCS and control mice.Thus, the reduction of white adipose tissue accretion resultedfrom a reduction in the volume of adipocytes (i.e., hypotro-phia) rather than from a decrease in the total number of adi-pocytes per fat pad (i.e., hypoplasia).

PCS Inhibits Lipogenesis and Increases Lipolysis inAdipocytesTo obtain an insight into the mechanisms of PCS action onadipose cells, we measured the lipolytic response to the b-adrenergic agonist isoproterenol and de novo lipogenesis. Tothis end, human isolated adipocytes and 3T3-L1 preadipocyteswere incubated in the presence of PCS (40 mg/ml). In 3T3-L1adipose cells, PCS decreased lipogenesis by 78% com-pared with control (Figure 4A). In the same model, incubationwith PCS did not change basal lipolysis (Figure 4B) but in-creased isoproterenol-stimulated lipolysis (26%; P,0.05). Inhuman isolated adipose cells, PCS inhibited lipogenesis byroughly 80% compared with vehicle-treated cells, thus validat-ing our results in 3T3-L1 cells (Figure 4C).

PCS Impairs Insulin-Induced Glucose Uptake andSignaling in C2C12 MyotubesSkeletal muscle accounts for a large percentage of the whole-body glucose uptake and is a major site for insulin resistancein type 2 diabetes.27,28 To get insight into the molecular mech-anisms of PCS-induced insulin resistance in skeletal muscle,

Table 1. Renal function and metabolic profile in PCS-treatedmice

Variable Control Mice PCS Mice CKD Mice

Mice (n) 8 8 5Diuresis (ml/24 hr) 0.8960.14a 0.9560.2a 2.460.4b

Water intake (ml/24 hr) 3.260.7a 2.660.5a 8.460.6b

Proteinuria (mg/24 hr) 1.0760.21a 1.1260.25a 7.162.8b

Urea (mM) 6.160.2a 5.960.3a 34.163.2b

Creatinine (mM) 9.661.1a 10.361.2a NDTriacylglycerols (mg/dl) 5165a 6567a 6763b

Total cholesterol (mg/dl) 98614a 152617b 17069c

Fasting glucose (mg/dl) 7364a 11064b 9865b

MDA (mM) 8.1961.01a 7.7660.67a NDMCP-1 (pg/ml) 21.663.1a 19.262.5a NDTNF-a (pg/ml) 18.062.4a 16.762.9a ND

Data are expressed as mean 6 SEM. ND, not determined; MDA,malondialdehyde;MCP-1, monocyte chemotactic protein-1. Different lettersindicate a significant difference at the P,0.05 level.

90 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 88–99, 2013




C2C12 myotubes were treated with PCS.No significant difference of cell viabilitywas observed between control and PCStreated cells for both tests (SupplementalTable 3). To determine whether PCScould impair glucose uptake, we studiedthe [3H]-2-deoxy-D-glucose transport inC2C12 myotubes. In control cells, stimula-tion with 100 nM insulin for 20 minutesinduced a 25% increase in [3H]-2-deoxy-D-glucose uptake (P,0.05). A 30-minutepretreatment with PCS abolished theinsulin-stimulated glucose uptake withoutaffecting the basal glucose uptake (Figure5A). To evaluate the mechanisms responsi-ble for the inhibition of glucose uptake, weanalyzed the insulin signaling pathways inthe absence or presence of PCS. Activationof the downstream kinase PKB/Akt wasestimated through the insulin-inducedphosphorylation of serine 473, which wasincreased 2.1-fold after a treatment by 100nM insulin for 20 minutes (Figure 5B).This effect was abolished after pretreat-ment with PCS from a concentration of10 to 80 mg/ml (Supplemental Figure 4).The early molecular event IRS-1 phosphor-ylation was compared in the absence orpresence of PCS. In control cells, insulintriggered a 5.6-fold increase in tyrosinephosphorylation of IRS-1 (Figure 5C). Pre-treatment with PCS did not affect the basaltyrosine phosphorylation of IRS-1 but sig-nificantly impaired its insulin-stimulatedphosphorylation. In parallel, PCS treat-ment was associated with increased inhib-

itory phosphorylation on IRS-1 on serine 636 residues (Figure5D). In good agreement, insulin stimulation induces amarkedincrease in the level of p85 protein co-immunoprecipitatedwith IRS-1 (1.7-fold compared with unstimulated cells),whereas PCS significantly decreased the level of p85 proteinco-immunoprecipitated with IRS-1 (Figure 5E). Miyamotoet al.29 recently proposed that in renal cells, PCS enters thecell via organic anion transporters (OAT), especially OAT3.We pretreated C2C12 muscle cells with probenecid (1 mM,1 hour), a potent inhibitor of OAT family, before incubationwith PCS. Pretreatment with probenecid abrogated PCS-induced disruption of insulin signaling pathways, as evidencedby the measurement of insulin-induced PKB/Akt phosphory-lation (Figure 6).

PCS Impairs Insulin Signaling in C2C12 Myotubesthrough Activation of the ERK KinasesERK can induce inhibitory phosphorylation of IRS-1 on serineresidue. To further explore the role of ERK1/2 in PCS-induced

Figure 2. PCS induces insulin resistance in mice by interfering with insulin signalingpathways. (A) For the insulin tolerance test, mice were injected intraperitoneally withinsulin (0.5 IU/kg) and blood glucose concentration was measured. Data are mean 6SEM for n=5–8 mice. NS, not significant. Baseline glucose concentrations were 7663,10365, and 9462 mg/dl for saline, PCS, and CKD mice, respectively. (B) Inhibitionof insulin-induced phosphorylation of PKB/Akt and (C) activation of phosphorylationof ERK1/2 in gastrocnemius muscle. Mice were stimulated with insulin (0.75 IU/kgintraperitoneally) before euthanasia. Data are mean 6 SEM for n=4–6 mice. Differentcharacters indicate a significant difference at P,0.05.

Table 2. Biometric data

Variable ControlMice PCS Mice CKD Mice

Mice (n) 8 8 8Initial body weight (g) 3662 3761 3263Final body weight (g) 3261 3061 2861Body length (cm) 10.260.2 10.160.1 9.760.2Lee index (3103) 31365.0 30965.0 31365.5Liver weight (mg) 1255649 1047660 1226692Heart weight (mg) 14167 15165 160612Kidney weight (mg) 338699a 338614a 2056145b

Gastrocnemius weight (mg) 16165 15368 14465Total WAT weight (mg) 17116135a 8716202b 402698c

Total WAT (mg/10 gbody weight)

534633a 277658b 141635c

Data are expressed as mean 6 SEM. Lee index was calculated as thecubic square of body weight divided by naso-anal length. WAT, whiteadipose tissue. Different letters indicate a significant difference at theP,0.05level.






muscle insulin resistance, C2C12 cells wereincubated with PCS and phosphorylationstatus of ERK1/2 was studied. C2C12 my-otubes preincubated with PCS without orwith insulin exhibited a twofold increase inERK1/2 phosphorylation (P,0.05) (Figure7A). Pretreatment of the cells with theERK1/2 inhibitor U0126 (10 mM, 1 hour)before PCS totally inhibited PCS-inducedERK1/2 activation (data not shown) andrestored insulin-induced PKB/Akt phos-phorylation (Figure 7B), pointing out thespecific role of ERK1/2 in mediating theeffects of PCS in C2C12 myotubes. In con-trast, PCS triggered only modest phos-phorylation of JNK (data not shown), butpretreatment of C2C12 with a potent JNKinhibitor (SP600125, 10 mM, 1 hour) failedto reverse the inhibitory effect of PCS oninsulin-induced phosphorylation of PKB/Akt (data not shown), thereby excluding arole of JNK in PCS-induced insulin resis-tance.

Treatment of Uremic Mice with aPrebiotic AXOS Reduces Fat MassLoss and Ectopic Lipids Associatedwith CKDBecause of its tight protein binding, PCSis poorly removed by most dialysis tech-niques. We therefore tested a prebioticstrategy aimed at reducing intestinal pro-duction of p-cresol in an attempt to pre-vent metabolic disturbances associatedwith serum PCS accumulation in CKD.AXOS are prebiotics that promote intesti-nal growth of bifidobacteria (instead offermentative bacteria), which were shownto significantly reduce the intestinal pro-duction of p-cresol.30,31 To determine invivo the effect of the PCS decrease on in-sulin resistance and glucose tolerance,CKD mice were treated for 4 weeks withthe prebiotic AXOS (5% wt/wt in diet; seeSupplemental Table 4 for diet composi-tion). CKD mice treated with AXOSexhibited a significant decrease in serumtotal PCS (change, 274%; P=0.03) com-pared with CKD mice fed the control diet(Figure 8A). After subtotal nephrectomy,mice receiving prebiotics exhibited asteady but nonsignificant improvementin weight compared with control CKDmice (P=0.09) (Supplemental Table 5).The energy intake was not significantly

Figure 3. Chronic treatment with PCS decreases white adipose tissue accretion in miceand induces ectopic lipid redistribution. (A) Weights of different pads of white adiposetissue (WAT). e, epididymal; r, retroperitoneal; sc, subcutaneous. (B) Tissue lipidcontent in gastrocnemius and liver. Note ectopic lipid redistribution in liver and gas-trocnemius muscle of PCS and CKD mice. (C) Typical frequency distribution of adi-pocyte diameters in PCS and vehicle-treated mice. Data are mean 6 SEM for n=8 mice.Different characters indicate a significant difference at P,0.05.





different between the two groups. Prebiotic treatment re-duced the loss of fat mass observed in CKD, as shown bythe weight of epididymal (16%; P=0.24), subcutaneous(31%; P=0.06), retroperitoneal (84%; P,0.05), and totalwhite adipose tissue (33%; P,0.05) (Figure 8B). The pre-biotic treatment prevented the ectopic lipid redistributionassociated with CKD (Figure 8C).

Treatment of Uremic Mice with a Prebiotic AXOSImproves Dyslipidemia, Glucose Tolerance, and InsulinResistance in CKD MicePrebiotic administration had no effect on plasma urea,adiponectin, or insulin levels in CKD mice (Table 4). In con-trast, prebiotic treatment completely prevented the expectedincrease in glycemia, total cholesterol, and triglycerides asso-ciated with CKD (Table 4). The intraperitoneal glucose toler-ance test showed significantly improved glucose control in theprebiotic group compared with the CKD group (Figure 8D).Finally, an insulin tolerance test was performed. After 3 weeks,insulin sensitivity was significantly improved in the prebioticgroup compared with the CKD group receiving standard diet(Figure 8, E and F).

DISCUSSION

To our knowledge, this study is the first to show that a majorprotein-bounduremic toxin, PCS, induces insulin resistance invivo in mice (Figure 2), as well as in vitro in C2C12 myotubes(Figure 6). The disruption of insulin signaling associated with

CKD was described in 1955,32 and the ef-fects of kidney disease on renal uptake andexcretion of insulin were reported in1970.33 Insulin resistance in CKD was evi-denced by DeFronzo et al.17 using the “goldstandard” euglycemic hyperinsulinemicclamp technique. In CKD, the decline ofrenal function is associated with the devel-opment of insulin resistance, and insulinresistance is recognized as an independentrisk factor for cardiovascular morbidity andmortality in patients with CKD.19 However,the exact causes of insulin resistance inCKD are still poorly defined.20,22,23,34,35

McCaleb et al.36 demonstrated that isolatedrat adipocytes had inhibition of insulin-stimulated glucose uptake after incubationwith serum from patients with CKD,whereas serum from obese patients or thosewith type 2 diabetes with normal renalfunction showed no effect. This suggestedthat one or several unknown circulatingmolecules unique to uremia can induce in-sulin resistance and prompted numerousfractionation studies that attempted todecipher the precise molecular nature ofthis factor. A central role for uremic solutesgenerated from protein breakdown in thegastrointestinal tract was suggested whendecreased plasma glucose concentrationsand reduced insulin requirements in ure-mic rats were reported after installmentof a low-protein diet37 and after ingestion

Table 3. Cellular characteristics of epididymal white adiposetissue in vehicle- and PCS-treated mice

Variable Control Mice PCS Mice Change (%) P Value

Mice (n) 8 8EpididymalWAT (mg)

985685 5346133 246 0.03

Cell diameter (mm) 60.762,6 48.162.2 221 0.003Cell weight (ng) 154.1621.4 81.9611.7 247 0.01No. of cells (3106) 6.8560.69 7.4561.71 9 0.76DNA content(mg/pad)

67.969.5 60.168.3 212 0.60

Data are mean 6 SEM. Data were compared using t test and, when appro-priate, Welch correction for variance inhomogeneity. Differences were con-sidered significant at the P,0.05 level. WAT, white adipose tissue.

Figure 4. PCS decreases lipogenesis and stimulates lipolysis in 3T3-L1 adipocytes andisolated human adipose cells. (A) Lipogenesis measured on 3T3-L1 adipocytes as theincorporation of [14C]-acetate into total lipids. (B) Lipolysis measured in 3T3-L1 adi-pocytes as the glycerol released in presence or absence of isoproterenol. (C) PCSinhibited lipogenesis on human isolated adipocytes. Data are mean 6 SEM for n=4mice. Different characters indicate a significant difference at P,0.05. TNL, total neu-tral lipids.



of an oral sorbent of protein degradationproducts.38 PCS is one of these products,resulting from the transformation of tyro-sine by gut bacterial fermentation. FreePCS9,10 and insulin resistance are two inde-pendent factors associated with cardiovas-cular mortality in CKD. In this study, weexplored the causal relationship betweeninsulin resistance and PCS accumulationin cellular and animal models. To ourknowledge, this study is the first to showthat PCS induces insulin resistance; as aresult, this compound can be classified asone of the major uremic toxins.

PCS chronically administered to micewith normal renal function triggered astriking insulin resistance and fat massloss. Of importance, the PCS concentrationafter administration was in the range oflevels observed in patients with ESRD.10,39

The ectopic lipid redistribution and reduc-tion of fat accretion in PCS-treated micewere the result of a direct effect of PCStreatment rather than malnutrition be-cause food intake and body weight werenot altered. Fat mass loss should be relatedto the fact that we observed a decrease inthe volume of adipocytes, which may re-flect adipocyte dysfunction. Axelssonet al. showed that an undefined circulatingfactor in patients with CKD increasedbasal lipolysis in human adipocytes invitro,40 and we indeed demonstrated thatPCS-treated adipocytes exhibit inhibited li-pogenesis and stimulated lipolysis. In PCS-treated mice, circulating lipids that couldno longer be stored into adipose tissue ac-cumulate in themuscle and liver, leading toan ectopic lipid redistribution thatmatched the one observed in CKD mice.Because intramuscular lipids are correlatedwith insulin resistance,41–43 it is likely thatlipotoxicity phenomena induced by PCScould contribute to insulin resistance, asdescribed in CKD mice. Ectopic lipids dis-rupt the insulin signaling pathway, whichinduces a reduction in tyrosine phosphor-ylation of IRS1.44 We demonstrated thatPCS in concentrations observed in ESRD(40 mg/ml)1,24,45,46 can impair glucose up-take as well as insulin pathway at the level ofIRS-1 and p-PKB/Akt in C2C12 myotubes.In type 2 diabetes, insulin resistance is re-lated to a defect in elements involved in in-sulin signal transduction, such as IRS-1,

Figure 5. PCS inhibits insulin-stimulated glucose uptake and disrupts insulin signalingpathways in C2C12 myotubes. C2C12 myotubes were incubated with PCS (40 mg/ml)for 30 minutes and stimulated by 100 nM insulin for 20 minutes. (A) Glucose uptakewas measured as the incorporation of tritiated 2-deoxy-glucose uptake into cells. Theinsulin pathway was explored by Western blotting. Also shown are the effects of PCSon (B) serine 473 phosphorylation of PKB/Akt, (C) tyrosine-phosphorylation of IRS-1,and (D) serine phosphorylation (Ser 636) of IRS-1, as well as (E) p85 subunit of PI3K co-immunoprecipitation. Data are mean 6 SEM for n=4–5 experiments. Different lettersindicate a significant difference at P,0.05.



which are modulated by several kinases (serine and tyrosinekinases).47 IRS-1 phosphorylation on serine residues impedesactivating phosphorylation on tyrosine residues and shutsdown the insulin signal transduction. Serine phosphorylationis a mechanism used by many diabetogenic factors (such asfatty acids, TNF-a, hyperinsulinemia) to inhibit insulin sig-naling. The stress signaling pathways have been shown to con-tribute to the development of insulin resistance, and IRS-1contains many potential sites of inhibitory phosphorylationby mitogen-activated protein kinase.47 We demonstrated thatin both mice and C2C12, PCS is responsible for an increase inthe mitogen-activated protein kinase–signaling ERK but notJNK. ERK1/2 involvement in the PCS noxious effects was evi-denced by the use of an ERK inhibitor (U0126), which totallyrestored the impaired insulin-induced p-AKT/PKB phosphor-ylation. Thus, PCS can directly impair the insulin signalingpathway by activating ERK. PCS has repeatedly been demon-strated (in various cell types) to exert its toxic effects by trig-gering intracellular oxidative stress. However, we failed toobserve any increase in intracellular reactive oxygen species

production or lipid peroxidation in C2C12 muscle cells(Supplemental Figure 5) excluding that oxidative stress wasresponsible for the impaired response to insulin.

Recent studies demonstrated that the bacterial flora of theintestine may play a key role in glucose homeostasis andobesity.48 Prebiotics are oligofructose or inulin polymers thatselectively stimulate growth and activity of a limited numberof beneficial bacteria in the colon.49 The prebiotics demon-strated their effectiveness to improve satiety and glycemic con-trol in patients with diabetes50 and plasma lipid profiles.51

Several studies reported a reduction in urinary p-cresol withthe use of prebiotics, such as oligo-fructose, in healthy per-sons,30 as well as in hemodialysis patients.52 Treatment withthe prebiotic AXOS decreased PCS, and prevented CKD-induced insulin resistance, the loss of adipose tissue and theaccumulation of ectopic lipids in muscle and liver. Prebioticsprobably exert pleiotropic effects that, besides suppressingprotein fermentation and blunting p-cresol production, couldalso contribute to improve metabolic status of CKD mice.

Taken together, our data demonstrate that a possible causeof insulin resistance in the chronically PCS-treated animal isadipocyte dysfunction associatedwith accumulation of ectopiclipids and that under these conditions, PCS alone is suffi-cient to induce the same degree of insulin resistance as thatreported in CKDmice. In summary, we have provided the firstin vivo and in vitro evidence that PCS induces muscle insulinresistance accompanied by a defect in the IRS/PI3K/Akt path-way, suggesting a causative link between PCS and insulin re-sistance (Figure 8). Furthermore, we showed that PCS candirectly activate ERK1/2 kinase in vivo and in vitro, leadingto disruption of the insulin pathway. Finally, we identified amechanism of lipotoxicity after long-term administration ofPCS, which reproduces the metabolic abnormalities observedin CKD mice and could be reversed by prebiotic treatment,through decreased plasmatic levels of PCS. Because insulinresistance is an important cardiovascular risk factor, noveltherapeutic approaches such as prebiotics, which could de-crease PCS more substantially than with the currently avail-able strategies, must be developed, especially because thistoxin is not very efficiently removed by hemodialysis.53

CONCISE METHODS

PCSAll in vitro experiments were performed according to the standard

approach and the recommendations for handling uremic retention

solutes published by the European Uremic Toxin Work Group

(EUTox, http://www.uremic-toxins.org).1 The potassium salt of PCS

was synthesized as described by Feigenbaum and Neuberg.54 In the

in vitro study, concentration of PCS was 40 mg/ml (212 mM), which is

the concentration found in humans with ESRD.45,46,55 Because PCS

was synthesized as a potassium salt, a solution of 35 mg/ml (200 mM)

K2SO4 in saline was chosen as control to equal the potassium concen-

tration in the K-salt of PCS. Because PCS is mainly protein-bound

Figure 6. Probenecid prevents PCS-induced disruption of insulinsignaling pathways in C2C12 myotubes. C2C12 myotubes wereincubated with probenecid (1 mM, 1 hour), a potent inhibitor oforganic anion transporters, before incubation with PCS (40 mg/ml,30 minutes) and stimulation by insulin (100 nM, 20 minutes). Ef-fect of PCS on serine 473 phosphorylation of PKB/Akt. Data aremean 6 SEM for n=6 mice. Different letters indicate a significantdifference at P,0.05.




http://www.uremic-toxins.org

in biologic systems, all in vitro experiments were performed in me-

dium supplemented with 35 g/L BSA according to the recommenda-

tions of EUTox.1

Animal ExperimentsAnimal experiments were performed under authorization no. 69–

266–0501 (CarMeN Laboratory, Direction Départementale des Ser-

vices Vétérinaires du Rhône). All experiments were carried out

according to the guidelines laid down by the French Ministère de

l’Agriculture (no. 87–848) and the European Union Council Direc-

tive for the Care and Use of Laboratory Animals of November 24,

1986 (86/609/EEC). CD1 Swiss and C57BL/6J mice were purchased

from Janvier SA (Le Genest-Saint-Isle, France) and housed in an air-

conditioned roomwith a controlled environment of 21°C60.5°C and

60%–70% humidity, under a 12-hour light/dark cycle (light on from

07:00 to 19:00) with free access to food and water.Moderate CKDwas

induced by 5/6 nephrectomy with a two-step surgical procedure.

NaHCO3 (80 mM) was added to the drinking water of CKD mice

to prevent metabolic acidosis.56 CD1 Swiss mice were randomly as-

signed to receive twice daily (8:00 a.m. and 6:00 p.m.) intraperitoneal

injections of PCS (10 mg/kg) or vehicle for control mice, sham mice,

and CKD mice, for 4 weeks.

Metabolic ChallengesAfter an overnight fast, an intraperitoneal glucose tolerance test

(glucose, 1 g/kg body weight) and insulin tolerance test (insulin, 0.50

IU/kg body weight) were performed. Blood

glucose values were determined from a drop of

blood sampled from the terminal portion of the

tail, using an automatic glucose monitor (Accu-

Check Performa, Roche, Meylan, France).

Euthanasia and Tissue DissectionTo study insulin signaling in skeletal muscle, mice

were intraperitoneally injected with insulin

(Actrapid, 0.75 IU/kg) or saline solution 60 mi-

nutes after the last administration of PCS or ve-

hicle. Thirty minutes after insulin injection mice

were anesthetized with sodium pentobarbital

(35 mg/kg intraperitoneally). Blood (750 ml)

was collected through cardiac puncture in hepa-

rinized tubes, centrifuged for 2 minutes at 3500 g

to separate plasma and stored at 280°C. Liver;

heart; kidneys; gastrocnemius muscle; and epi-

didymal, retroperitoneal, and subcutaneous in-

guinal white adipose tissue were dissected out,

weighed, and snap-frozen in liquid nitrogen.

Biochemical MeasurementsFree and total PCS were quantified in serum by

using reverse-HPLC coupled to a fluorescence

detector as previously described.15,45,57 The

plasma concentration of cholesterol, triacylglyce-

rols, adiponectin, and insulin were determined

using commercial assays.Muscle (gastrocnemius)

and liver lipids were extracted using chloroform-methanol (2:1, vol/

vol),58 and total lipid content was estimated gravimetrically.

Cellularity Study: Measurement of Adipocyte Size andNumberAdipose tissue was fixed in osmium tetroxide, and cell size was de-

termined essentially as described by Etherton et al.59 DNA content in

epididymal white adipose tissue was measured, after delipidation of

the samples, by a standard fluorometric method using bisbenzimide

and calf thymus DNA as standard.

Differentiation of C2C12 and 3T3-L1 CellsC2C12 myoblasts, from the American Type Culture Collection (LGC

Standard, Molsheim, France), were grown and differentiated to

myotubes as recommendedby the provider.Mouse 3T3-L1fibroblasts

were obtained from the American Type Culture Collection (reference

CL-173) and differentiated to adipocytes. PCS cytotoxicity was de-

terminedbymeasuring cell viability using a 3-[4,5-dimethylthiazol-2-

yl]-2,5 diphenyltetrazolium (Cell ProliferationKit I, Roche) assay and

lactate dehydrogenase activity (In Vitro Toxicology Assay Kit, Lactic

Dehydrogenase based, Sigma Aldrich).

Protein Isolation, Immunoprecipitation, andImmunoblottingC2C12 myotubes were washed and solubilized as described pre-

viously.60 Lysates were kept on ice for 20 minutes, and insoluble

Figure 7. Role of ERK1/2 in PCS-induced insulin resistance in C2C12muscle cells. C2C12myotubes were incubated with PCS (40 mg/ml) for 30 minutes and stimulated by 100 nMinsulin for 20 minutes. (A) PCS induces phosphorylation of ERK1/2. (B) ERK1/2 inhibitorU0126 (10 mM, 1 hour) reverses the effect of PCS on insulin-stimulated PKB/Akt phos-phorylation. Data are mean 6 SEM for n=6 mice. Different letters indicate a significantdifference at P,0.05.



material was removed by centrifugation at 14,000 g for 20 minutes.

Protein concentration was determined by colorimetric assay (Bio-

Rad). For immunoprecipitation and immunoblots, antibodies

were used as previously described.60 Immunoreactive proteins

were detected using horseradish peroxidase–

linked secondary antibodies and enhanced

chemiluminescence according to the manu-

facturer’s instructions. Signal intensities were

measured with Quantity One software (Bio-

Rad).

2-Deoxyglucose Uptake andLipogenesisC2C12 myotubes were incubated in serum-free

DMEM containing 0.2% (wt/vol) BSA for 12

hours and treated or not with PCS. For glucose

uptake and lipogenesis, cells were incubated and

treated as previously described.60,61 Radioactiv-

ity was counted, and samples were normalized

to protein concentration.

Adipose Tissue Collection forLipogenesis AssayHuman adipose tissue was obtained from an

ongoing study approved by the Ethical Com-

mittee (CPPLyonSud-Est IV)ofLyonUniversity

Hospital (MODAIR study, ref D-09–17). Sub-

cutaneous abdominal adipose tissue (1–2 g) was

collected from four nonobese men undergoing

elective urologic surgery (radical prostatec-

tomy). All patients gave written informed con-

sent for the study, and all procedures were in

accordance with the principles of the Declara-

tion of Helsinki. Adipocytes were isolated using

collagenase and lipogenesis was measured as

previously described.61

Statistical AnalysesData are expressed as mean6 SEM. All data were

analyzed using GraphPad Prism, version 5.0, soft-

ware (GraphPad, La Jolla, CA). Differences were

considered significant at the P,0.05 level.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. E.

Kalbacher forhumanadipose tissue collection and

Dr. F. Guebre-Egziabher for fruitful discussions.

L.K. held a fellowship from Fondation pour

la Recherche Médicale and. C.P. held a grant

from Société Française de Néphrologie. N.J.P.,

M.L.C., and R.E.V. were supported by grants

from the French Ministère de l’Education Na-

tionale, de la Recherche et de la Technologie. This work was sup-

ported by Institut National de la Santé et de la Recherche Médicale

(INSERM) and Institut National des Sciences Appliquées de Lyon

(INSA-Lyon).

Figure 8. The prebiotic AXOS reduces plasma PCS and improves metabolic measures inCKDmice. Nephrectomizedmice were fed for 4 weekswith a diet containing 5% (wt/wt) ofAXOS prebiotic as described in the Concise Methods section. (A) Effect of AXOS onplasma PCS concentration (n=4–5 mice). (B) Weight of different pads of white adiposetissue (WAT). e, epididymal; r, retroperitoneal; sc, subcutaneous. (C) Tissue lipid contentin gastrocnemius and liver (n=8). (D) Glucose tolerance test (intraperitoneal); n=6–8 mice.Baseline glucose concentrations were 8762 and 7562 mg/dl for control and prebiotic-fed CKD mice, respectively (P,0.001). AUC, area under the curve. (E and F) Insulin tol-erance test (n=5 mice). Baseline glucose concentrations were 8863 and 7462 mg/dl forcontrol and prebiotic-fed CKD mice, respectively (P,0.001). Data are mean 6 SEM.*P,0.05; **P,0.01; ***P,0.001.



DISCLOSURESNone.

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Table 4. Metabolic profile in CKD mice fed or not fed withan AXOS prebiotic–enriched diet

DietStandard

MiceAXOSMice

Change(%)

P

Value

Fasting insulin(pg/ml)

60.5611 38617 237 0.12

Fasting glucose(mg/dl)

9765 8162 216 0.002

HOMA-IR 1.860.4 1.060.2 244 0.09Triacylglycerols(mg/dl)

118612 76610 236 0.02

Total cholesterol(mg/dl)

123611 80610 235 0.01

Adiponectin (mg/ml) 2563 2661 4 0.47Urea (mmol/L) 3463 3063 213 0.29MDA (mM) 10.363.9 11.162.7 8 0.73

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