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Butyrate modulates TGF- b1 generation and function:Potential renal benefit for Acacia(sen) SUPERGUM TM

(gum arabic)?N Matsumoto 1 , S Riley1 , D Fraser1 , S Al-Assaf 2 , E Ishimura3 , T Wolever4 , GO Phillips2 and AO Phillips1

1 Institute of Nephrology, Cardiff University School of Medicine, Heath Park, Cardiff, UK;2 North East Wales Institute, Phillips HydrocolloidsResearch Centre, Wrexham, UK; 3 Department of Nephrology, Osaka City University, Osaka, Japan and 4 Division of Endocrinology and Metabolism, St Michaels Hospital, Toronto, Ontario, Canada

Anecdotal evidence suggests that high fibresupplementation of dietary intake may have health benefits

in renal disease related to alterations in circulating levels of short-chain fatty acids. The aim of the study was to examinethe hypothesis that dietary manipulation may increase serumbutyrate and thus have potential beneficial effects in renaldisease. We examined the effect of dietary supplementationwith a gum arabic sample of standardized molecularcharacteristics, Acacia(sen) SUPERGUM TM EM2(SUPERGUMTM ), on systemic levels of butyrate in normalhuman subjects. In an in vitro study, we also examined thepotential role of butyrate in modifying the generation of the profibrotic cytokine transforming growth factor-beta(TGF-b1) by renal epithelial cells. Following 8 weeks of dietarysupplementation with 25 g/day of SUPERGUM TM , there was atwo-fold increase in serum butyrate ( n 7, P 0.03). In vitrowork demonstrated that exposure of renal epithelial cells toelevated concentrations of butyrate suppressed both basaland stimulated TGF- b1 synthesis. The action of butyrate wasmediated by suppression of the extracellular signal-regulatedkinase/mitogen-activated protein kinase signalling pathway.In addition, butyrate exposures reduced the response of renal epithelial cells to TGF- b1 as assessed by luciferaseactivity of a TGF- b-responsive reporter construct. Attenuationof TGF-b1 signalling was associated with reducedphosphorylation of Smad 3 and decreased trafficking of TGF-b1 receptors into signalling, non-lipid raft-associatedmembrane fractions. In conclusion, the data demonstratethat dietary supplementation with SUPERGUM TM increasedserum butyrate, which at least in vitro has beneficial effectson renal pro-fibrotic cytokine generation.Kidney International (2006) 69, 257265. doi:10.1038/sj.ki.5000028

KEYWORDS: TGF-b ; renal; butyrate; SUPERGUM

Epidemiological evidence suggests that a high intake of dietary bre is associated with numerous health benets. 1 To

date, much of the research interest has focused on their rolein protection against colorectal cancer and inammatory bowel disease. However, there is now evidence that alteredsystemic levels of short-chain fatty acids (SCFA), resultantfrom colonic bacterial fermentation of bre, may also offerhealth benet beyond the gastrointestinal tract. Studies overthe last decade have linked dietary bre intake andcardiovascular risk and also epithelial cell carcinogenesis innumerous organs including the kidney. These health benetsof high-bre diets are thought to be specically linked to thegeneration of the SCFA butyrate, which displays a host of biologically important effects in vitro, including modulation

of cell proliferation, apoptosis, regulation of angiogenesis andinammation. 2

Gum arabic is a tree gum exudate, which has been animportant food additive since ancient times. Supplementa-tion of the diet with gum arabic has been shown to increasefaecal nitrogen excretion and lower serum urea nitrogenconcentration in patients with chronic renal failure. 3 Thisis dependent on an increase in bacterial growth and activity in the gut. Colonic bacteria produce ureases that hydrolyseurea to ammonia and CO 2 . The resultant ammonia canthen be incorporated into bacterial proteins, which aresubsequently excreted in the bacterial mass fraction of thefaeces. The net result is increased N excretion in the faeces.More recent studies, in rat models of acute renal failure, 4suggest that gum arabic may also improve renal functionindependently of its action on faecal bacterial ammoniametabolism.

It is now clear that in renal disease, regardless of itsaetiology, the prognosis best correlates with the degree of renal interstitial brosis. As a result, interest has focused onthe role of cells, such as the epithelial cells of the proximaltubule, in the initiation of the brotic response. There is now overwhelming evidence that the pro-brotic cytokine trans-forming growth factor-beta (TGF- b1) is a key mediator of progressive renal injury. Much of our research work has

http://www.kidney-international.org o r i g i n a l a r t i c l e& 2006 International Society of Nephrology

Received 15 June 2005; revised 15 August 2005; accepted 15 September2005

Correspondence: AO Phillips, Institute of Nephrology, School of Medicine,Cardiff University, Heath Park, Cardiff CF14 4XN, UK.E-mail: [email protected]

Kidney International (2006) 69 , 257265 257


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focused on the generation and function of this cytokine by proximal tubule.

The aim of this study was to examine the potential use of dietary supplementation with gum arabic in progressive renaldisease. More specically, we have examined the relationshipbetween ingestion of gum arabic and systemic levels of SCFAin human subjects, and examined the effects of the SCFAbutyrate and propionate on the generation of, and responseto, the pro-brotic cytokine TGF- b1 in vitro in proximaltubular epithelial cells.

A weakness associated with previous dietary bre studiesinvolving gum arabic has been the inherent variable nature of this natural product. 5 This study comprising 75 commercialsamples showed that their average molecular weight variedfrom 4.6 to 10.2 105 . The traditional gum arabic used in thefood industry is the exudate from the tree Acacia senegal .There are two variety forms available commercially, namely A. senegal var. senegal and A. senegal var. karensis. Both areacceptable as food additives and conform to the specicationnow approved by the FAO Joint Expert Committee on FoodAdditives and the Codex Alimenarius Commission. Here, wehave used an A. senegal var. senegal sample, which has beenmatured to give a standardized and reproducible testmaterial, ensuring that the work can be accurately repeatedand that any future product will be able to reproduce theeffects reported.

RESULTSDietary supplementation increases serum butyrateThe SUPERGUMTM was well tolerated, with only minorsymptoms of bloating being reported. All volunteers

completed the 10-week course of bre supplementation.There was a signicant increase in serum butyrate levelsfrom a baseline value of 0.497 0.08 to 0.717 0.11 mmol/l after10 weeks (Figure 1a). Owing to technical reasons, resultsfrom only seven volunteers were available for butyrate levels.There was no change in the serum propionate (baseline:1.637 0.24 mmol/l; study end: 1.59 7 0.25 mmol/l, n 10,P 4 0.05) during the course of the bre supplementationperiod (Figure 1b).

Butyrate decreases basal TGF- b1 generation. Addition of butyrate led to a dose-dependent reduction in the generationof TGF-b1 (Figure 2). A signicant reduction in thisunstimulated constitutive generation was seen at concentra-tions of butyrate of 5 m M and above. In contrast, addition of propionate over the concentration range 010 m M did notinuence TGF- b1 synthesis at any of the time points studied(Figure 3).

The doses of the SCFA used were non-toxic. Over theconcentration range used, there were no alterations inuorescence readings following addition of butyrate orpropionate, as assessed by alamarBlue assay (data notshown).

Our previous studies have demonstrated independentmechanism regulating transcription and translation of TGF-b1.6,7 Furthermore, signalling via the p38 mitogen-activated

protein (MAP) kinase pathway is involved in the stimulationof promoter activity, whereas the extracellular signal-regulated kinase (ERK)/MAP kinase pathway may regulatepost-transcriptional events. 8 The potential involvement of these two signalling pathways in TGF- b1 generation underbasal conditions was assessed by determining the impact of

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Figure 1 | Dietary supplementation with SUPERGUM TM increasesserum butyrate. At the end of a 10-week study period in whichnormal healthy volunteers were fed 25 g/day of SUPERGUMTM , serumsamples were collected and ( a) butyrate or (b) propionate werequantified as detailed in Materials and Methods. Data representmedian and inter-quartile range; for butyrate n 7 and for

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Figure 2 | Butyrate suppresses basalTGF- b1 production bytubular epithelial cells. Increasing doses of butyrate were added toconfluent monolayers of serum-deprived HK2 cells under serum-freeconditions. Cell culture supernatant samples were collected after(a) 24, (b) 48, and (c) 72h and TGF-b1 was quantified by ELISA. Datarepresent mean 7 s.d.; n 6. *P o 0.05 compared to serum-freemedium in the absence of butyrate.

258 Kidney International (2006) 69 , 257265

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inhibition of these pathways on TGF- b1 concentration.SB203580 and PD98059 (Calbiochem, Nottingham, UK)are cell-permeable inhibitors of p38 kinase and MAP kinasekinase (MEK), respectively. Addition of the MEK inhibitorPD98059 (Figure 4a), but not the p38 MAP kinase inhibitor(Figure 4b), mimicked the effect of butyrate and led to asignicant reduction in TGF- b1 concentration in the culturesupernatant (Figure 5). Neither compound over the con-centration ranges used led to any toxic effect on cells as

assessed by the alamarBlue assay (data not shown). Thehypothesis that butyrate may mediate its effect by inhibitionof ERK/MAP kinase was also supported by immunoblotanalysis demonstrating both dose-(Figure 4c) and time-dependent (Figure 4d) suppression of ERK phosphorylationin unstimulated monolayers of HK-2 cells.

Glucose-stimulated TGF- b1 synthesis is inhibited by butyrate.We have previously demonstrated, in our in vitro model of the diabetic state, that exposure of HK-2 cells to elevatedconcentrations of D-glucose for 7 days stimulates de novoTGF-b1 synthesis.8 Having determined the effect of SCFA onbasal/constitutive TGF- b1 synthesis, we next sought toexamine their effect on glucose-stimulated TGF- b1 synthesis.

Addition of 25 m M D-glucose led to a signicant increasein the concentration of TGF- b1 in the culture supernatant, aspreviously demonstrated. 8 Glucose-stimulated increase inTGF-b1 was signicantly attenuated when cells were culturedin the presence of 25 m M D-glucose together with butyrate(Figure 5a). Propionate in contrast did not inuence glucose-stimulated TGF- b1. To conrm that reduced TGF- b1concentration following addition of butyrate was the resultof decreased synthesis, we assessed de novo synthesis of TGF-b1 by detection of radioactive amino-acid incorporation intoTGF-b1.8 Consistent with the enzyme-linked immuno-sorbent assay (ELISA) data, 25m M D-glucose-stimulatedsynthesis of radiolabelled TGF- b1 was also reduced in the

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Figure 3 | Propionate has no effect on basal TGF- b1productionby tubular epithelial cells. Increasing doses of propionate wereadded to confluent monolayers of serum-deprived HK2 cells underserum-free conditions. Cell culture supernatant samples werecollected after ( a) 24, (b) 48, and (c) 72 h and TGF-b1 was quantifiedby ELISA. Data represent mean7 s.d.; n 6. P 4 0.05 for all samplescompared to serum-free medium in the absence of propionate.

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Figure 4 | Butyrate-mediated suppression of TGF- b1 is associated with suppression of ERK activation. Increasing doses of either ( a) theMEK inhibitor PD98059 or (b) p38 MAP kinase inhibitor SB203580 were added to confluent monolayers of HK-2 cells under serum-freeconditions for 24 h. Subsequently, cell culture supernatant samples were collected for analysis of TGF- b1 concentration by ELISA. Datarepresent mean 7 s.d.; n 3. *P o 0.05 compared to serum-free medium alone in the absence of inhibitor. In parallel experiments, the effect of butyrate on ERK activation was examined by immunoblot analysis using antibodies to either dually phosphorylated active form of MAPK (p44/ERK1 and p42/ERK2) or total MAPK. (c) To determine the dose-dependent effect of SCFA, butyrate or propionate at the concentrations shownwas added to confluent HK2 cell monolayers in the absence of serum for 30 min. ( d) Time-dependent effects of butyrate were examined byaddition of 1m M butyrate to confluent monolayers of HK2 cells for up to 30 min. Cell lysis and immunoblot analysis were carried out as detailedin Materials and Methods.


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presence of butyrate, but was unaffected by the addition of propionate (Figure 5b).

The involvement of the p38 and MAP kinase pathways in25 mM D-glucose-stimulated TGF- b1 synthesis was deter-mined by addition of glucose in the presence of eitherSB203580 or PD98059. Stimulation of cells with 25m MD-glucose in the presence of PD98059 led to a dose-dependentdecrease in glucose-stimulated TGF- b1 concentration (Figure6a). In contrast, inhibition of the p38 MAP kinase pathway did not affect glucose-stimulated TGF- b1 generation (Figure6b). D-glucose (25m M)-stimulated activation of the ERK/MAP kinase pathway was examined by immunoblot analysisof ERK phosphorylation. A time-dependent increase inp-ERK was demonstrated 10 min after addition of 25 m MD-glucose, with maximal stimulation seen at 15 min (Figure 6c).Addition of 25m M D-glucose had no effect on the expression of total ERK. The effect of butyrate on glucose-stimulated ERKactivation was examined by stimulation of HK-2 cells with25 mM D-glucose in the presence of 1 m M butyrate for 15 min.These experiments demonstrated abrogation of 25m MD-glucose activation of ERK such that there was no differencein ERK phosphorylation following addition of 25m M

D-glucose in the presence of butyrate compared with thatseen in the control cell (Figure 6d).

Butyrate suppresses TGF- b1-dependent signalling. Activa-tion of the Smad signalling pathway was examined using the(SBE)4-Lux reporter, which contains four repeats of theCAGACA sequence identied as a SMAD-binding element.Addition of TGF- b1 led to a signicant increase in luciferaseactivity (Figure 7). Stimulation of HK-2 cells transfected withthe reporter construct with TGF- b1 in the presence of butyrate blunted TGF- b1-dependent activation of Smadsignalling, as luciferase activity was signicantly lower inthe presence of either 1 or 10m M butyrate (Figure 7a).Stimulation with TGF- b1 in the presence of propionate incontrast did not affect TGF- b1-dependent activation of thereporter construct (Figure 7b).

Activation of the Smad signalling pathway was alsoexamined by immnuoblot analysis of phosphorylated Smad2/3 (Figure 8). Addition of TGF- b1 increased Smad phospho-rylation. As with reporter construct activity, TGF- b1-dependent phosphorylation of Smad was blunted in thepresence of butyrate in a dose-dependent manner, whilepropionate did not inuence Smad phosphorylation.

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Figure 5 | Butyrate suppresses 25 mM D-glucose-stimulatedTGF-b1 synthesis. (a) Confluent monolayers of HK2 cells werestimulated with 25 m M D-glucose either alone or in the presence of butyrate ( ) or propionate ( ) at the concentrations indicated for atotal of 7 days under serum-free conditions. In control experiments,serum-free medium containing 5m M D-glucose ( ) was added tothe cells. TGF-b1 concentration in the cell culture supernatant wasexamined by ELISA. (b) Data represent mean 7 s.d.; n 6, *P o 0.05compared to 25m M D-glucose alone. In parallel experiments, de novoTGF-b1 protein synthesis was examined by stimulation of cellswith 25 mM D-glucose either alone or in the presence of butyrateor priopionate in tissue culture medium to which 40 mCi of 3 H-radiolabelled amino-acid mixture was added. Following 7 days

of stimulation, TGF-b1 in the cell culture supernatant wasimmunoprecipitated and visualized by autoradiography.

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Figure 6 | Butyrate-mediated suppression of 25 mMD-glucose-stimulated TGF- b1 is associated with suppression of

ERK activations. (a) Increasing doses of either the MEK inhibitorPD98059 or (b) p38 MAP kinase inhibitor SB203580 were added toconfluent monolayers of HK-2 cells stimulated with 25m M D-glucoseunder serum-free conditions for 24 h. Subsequently, cell culturesupernatant samples were collected for analysis of TGF- b1concentration by ELISA. Data represent mean 7 s.d.; n 3. *P o 0.05compared to serum-free medium alone in the absence of inhibitor.In parallel experiments, the effect of butyrate on 25 m M D-glucose-stimulated ERK activation was examined by immunoblot analysisusing antibodies to either dually phosphorylated active form of MAPK (p44/ERK1 and p42/ERK2) or total MAPK. (c) To demonstrateactivation of ERK, HK2 cells were stimulated with 25 mM D-glucoseunder serum-free conditions for up to 180 min. At the time pointsindicated, cells were lysed and ERK activation was assessed. (d) Todetermine the effect of butyrate, HK-2 cells were stimulated with25 mM D-glucose either alone or in the presence of 1 m M butyrate for15 min before cell lysis and immunoblot analysis.




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As previously demonstrated, 9,10 addition of TGF- b1 andstimulation of TGF- b1-dependent signalling increased traf-cking of receptors to non-lipid raft membrane-associatedfractions, as assessed by sucrose gradient density separationof membrane fractions and detection of TGF- b RI by immunoblot analysis (Figure 8b). Following TGF- b1, 69%of TGFb RI was in the non-raft membrane fraction ascompared to only 39% in the control cell (Figure 8c).Addition of TGF- b1 in the presence of butyrate decreasedTGF-b-mediated trafcking of TGF- b1 receptors into non-lipid raft-associated membrane fractions, which are associatedwith endosomal internalization and enhanced signalling. 11

Under these conditions, only 38% of total TGF- b RI detectedwas in the non-raft-associated fractions.

DISCUSSIONRecent changes in industrialized countries have led to dietary patterns based on animal foodstuffs and rened cereals, with

a reduced supply of dietary bre. Until the last 30 years or so,dietary bre had long been considered a non-useful foodcomponent. Nutrition scientists, however, have long agreedthat a quality diet consists of eating plenty of high-brefoods. Currently, this view is reinforced by numerousepidemiological, clinical and cell biological observations.The bre hypothesis proposed by Burkitt and Trowel 12,13

suggested, some three decades ago, that there was a link between the consumption of a diet rich in bre and the levelof protection against many of the rst world diseases such asconstipation, diverticulosis, cancer of the colon diabetes,obesity, and cardiovascular disease.

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Figure 7 | Antagonism of TGF- b1-mediated increase of SMAD-dependent signalling. HK-2 cells were transfected with a

SMAD-responsive promoter (SBE)4 -Lux, before stimulation withTGF-b1 (1 ng/ml) either alone or in the presence of either ( a) butyrateor (b) propionate. All stimuli were applied for 6 h, subsequentlyluciferase content was quantified as described in Materials andMethods and the results were normalized for transfection efficiency(using b-galactosidase) expressed as the fold increase above thenon-stimulated control. Data represent mean 7 s.d.; n 6.

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Figure 8 | Suppression of Smad phosphorylation and distributionof TGF-b receptors. (a) Confluent monolayers of HK2 cells werestimulated with TGF-b1 (1 ng/ml) either alone or in the presenceof increasing doses of either butyrate or propionate as indicated.Subsequently, 6 h after stimulation, cells were lysed and Smadphosphorylation was assessed by immuoblot analysis using anantibody. ( b) TGF-b receptor distribution in lipid raft and non-raft

fractions was analysed in HK-2 cells following sucrose gradientsubcellular fractionation to separate lipid rafts from other cellularcomponents. Confluent monolayers were stimulated with TGF- b1(1 ng/ml) either alone or in combination with 1 m M butyrate for 24hbefore cell lysis and fractionation as described in Materials andMethods. In control experiments, cells were exposed to serum-freemedium alone. Membrane fractions were subsequently analysed byTGF-b RI immunoblot analysis. (c) Following scanning densitometryof autoradiographs, the distribution of TGF- b receptor into the raft(fractions 36) and non-raft (fractions 712) was quantified and thedata from three separate experiments were expressed graphicallyand expressed as the percentage of the receptor in the raft-andnon-raft-associated membrane fractions.




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Studies dating back over 20 years have shown that locustbean gum, a non-digestible polymer of mannose andgalactose derived from the seeds of the Ceratonia siliquatree, was an efcient sorbent, which bound to many of thepotential toxic substances found in patients with chronicrenal failure. 14 Subsequently, in a study of only two patients,it was suggested that dietary supplementation with locustbean gum improved blood pressure control and reducedserum creatinine. 15 These observations, which have not beeninvestigated further, if conrmed would suggest a mechanismother than that related to its sorbent properties by whichlocust bean gum may inuence progressive renal disease. Inaddition, a single study using dietary supplementation withthe hemicellulosic ispaghula husk also suggested a benecialeffect on renal function beyond increased faecal bacterialloss.16

Our interest in the potential health benet of gum arabicstems from two studies demonstrating that supplementationof the diet with gum arabic increases faecal nitrogen excretionand lower serum urea nitrogen concentration in patients withchronic renal failure, 3 and work in rat models of acute renalfailure, which suggests that gum arabic may improve renalfunction independently of its action on faecal bacterialammonia metabolism. 4

The aim of this study was to examine the hypothesis thatdietary supplementation with gum arabic in humans willincrease systemic levels of the SCFA, which in turn may haveactions at the cellular level and a benecial effect on thedevelopment and progression of renal brosis.

The rst signicant observation is the demonstration of elevated serum butyrate levels in human subjects who

received 25 g/day of specic gum arabic (Acacia(sen) SUPER-GUMTM

). Completion of the full 10-week protocol by all of the subjects conrms the tolerability of such an approach.Butyrate is considered a preferred fuel for colonocytes and isbelieved to be selectively extracted by them, 17 resulting inrelatively low butyrate concentrations in the portal blood. 18

Furthermore, as the vast majority of butyrate is likely to beextracted by the liver, 19 alterations in systemic butyrate arelikely to be at low levels.18 Our data, however, are consistentwith other studies, which suggest that increased colonicbutyrate production in human subjects can be detected by increased serum butyrate. 20 It is interesting to note that incontrast to the increase in butyrate levels, we were unable todemonstrate any alteration in serum propionate in thesubjects given the test material. We postulate that this isrelated to the fact that the majority of colonic propionate istaken up by the liver 19 where it inhibits the incorporation of acetate into lipids 21 and can be utilized as a gluconeogenicsubstrate. 22

Many studies have demonstrated that the SCFA butyratedisplays a host of biologically important effects in vitro,including modulation of cell proliferation, apoptosis, regu-lation of angiogenesis and inammation. 2

In order to provide a mechanistic basis to the hypothesisthat gum arabic supplementation of the diet may have

benecial effects in renal disease, we have examined the invitro effects of butyrate on the generation of the pro-broticcytokine TGF-b1 and also the responsiveness of cells to theeffects of TGF-b1 in vitro. There is clear evidence that thiscytokine is involved in the nal common pathway of renalbrosis in all renal diseases, and antagonism of its action hasbeen proposed as a potential therapeutic goal. 2327 There is aclose correlation between rate of progression of renal diseaseand the degree of interstitial brosis. Furthermore, it is wellestablished that the epithelial cells of the proximal tubulecontribute to this brotic process, both by generation of cytokines such as TGF-b1 6,7,28 and by a process of epithelialmesenchymal transdifferentiation under the in-uence of TGF-b1, through which contribute to thepopulation of renal interstitial broblasts, which mediatedeposition of interstitial extracellular matrix in disease. 29 Wehave therefore focused on the modulation effect of butyrateon TGF-b1 in this renal epithelial cell. The results clearly demonstrate that at non-toxic doses, exposure to elevatedconcentrations of butyrate prevents TGF- b1 generation.More specically, the data demonstrate that both basal/constitutive TGF- b1 synthesis and glucose-stimulated gen-eration of TGF- b1 are suppressed. Previously, we havedemonstrated the involvement of both the p38 MAP kinaseand ERK/MAP kinase pathways in the regulation of transcriptional and translational regulation of TGF- b1,respectively. 8 In this study, the data suggest that butyrate-mediated inhibition of TGF- b1 synthesis is related to thespecic inhibition of the ERK/MAP kinase pathway. Thiscoupled to our previous work would suggest that butyratemay therefore specically modulate the synthesis of TGF- b1

translation. It is of note that the effects of butyrate in the invitro situation were seen at concentrations far in excess of thecirculating serum levels seen in the in vivo feeding study. How circulating levels relate to tissue levels, and in particular tolevels in the kidney and more specically in the environmentof the proximal tubular epithelial cell, remains a matter forspeculation. The concentrations used, however, are in linewith previous studies in which the effects of butyrate on renalcell function have been studied. 30,31 These in vitro observa-tions therefore merit further clinical studies to validate theirpotential importance.

TGF-b1 receptors internalize into both caveolin- andEEA-1-positive vesicles and reside in both lipid raft and non-raft membrane domains. 11 Clathrin-dependent internaliza-tion into the EEA-1-positive endosome promotes TGF- b1signalling. In contrast, the lipid raft-caveolar internalizationpathway contains Smad7-bound receptor and is required forreceptor turnover. Altered receptor trafcking between thesetwo membrane receptor pools may inuence TGF- b1signalling in a stimulus-specic manner resulting in eitherenhancement 10 or attenuation 9 of TGF-b1 signalling. In thisstudy, we have demonstrated attenuation of TGF- b1 signal-ling in the presence of butyrate, using a TGF- b1-responsivepromoter construct, and also phosphorylation of Smadproteins as markers of TGF- b1 response. Furthermore,




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attenuation of TGF- b1 signalling was associated withinhibition of trafcking of TGF- b RI in the cell membrane.

In conclusion, the data support the hypothesis that dietary supplementation with Acacia(sen) SUPERGUM

TM

(gum ara-bic), by increasing systemic levels of butyrate, may have apotential benecial effect in renal disease by suppression of TGF-b1 activity. Conrmation that the alterations insystemic butyrate levels documented in this study may havecomparable in vivo effects to those demonstrated in this cellculture system therefore warrants further in vivo investiga-tion. TGF- b1 is a multifunctional cytokine, which controls aplethora of biological responses, and alterations in itssignalling pathway are associated with a range of humandiseases. It is important in development and normal woundhealing through its effects on cell proliferation, migration,differentiation and apoptosis. 32 In addition, it is a criticalregulator of the normal inammatory response, as illustratedby the death of TGF-b1 / mice of a multifocal inamma-tory syndrome soon after weaning. 33 It has growth inhibitory effects, which explains its role as a tumor suppressor early inthe course of disease. Its expression by tumor cells, however,also contributes to cancer progression and metastasis at laterstages of disease.34,35 TGF-b1 also plays a central role in thepathological process of brosis, which may lead to organfailure.36,37 Although the data presented in this paper have

been generated in a renal epithelial cell line, given this widerrole of TGF-b in health and disease, the general applicability of our ndings to other organ systems may have importanthealth implications.

MATERIALS AND METHODSEffect of gum arabic on SCFA in vivo

Test material. The test gum Acacia(sen) SUPERGUM TM EM2(SUPERGUM TM ) has precisely structured molecular dimensionsand chemical composition as shown in Tables 16.

Protocol for dietary supplementation. Healthy volunteers(n 10) each had their normal diet supplemented with 25gSUPERGUMTM daily. The daily amount of SUPERGUM TM wasprovided in a dried form in individual sealed foil pouches. TheSUPERGUMTM was reconstituted in 250ml of water and shaken toensure adequate mixing. The volunteers then added avouring totaste and consumed the SUPERGUM TM mixture.

Compliance was assessed by counting the foil pouches returnedto the department. Volunteers were also questioned about ill effectsfrom the bre supplementation.

Quantitation of butyrate and propionate. Serum for mea-surement of SCFA was obtained before and 10 weeks aftercommencing dietary supplementation with SUPERGUM TM . Volun-teers attended after an overnight fast, and a 12 ml blood sample wasobtained and placed into two 6 ml tubes containing serum clotactivator (Vacuette, Greiner labortechnik, Kremsmunter, Austria).Once clotted, the serum was immediately separated by centrifuga-tion for 7 min at 2000r.p.m. and 4 1 C. Aliquots of serum were storedat 701 C and batched for measurement of SCFA.

Serum butyrate and propionate were measured in quadruplicateby gas chromatography as previously described. 38 Statisticalsignicance was assessed by standard non-parametric tests (Wilcox-on) using SPSS 11 statistical computer package.

Table 1 | Percentage loss on drying and specific rotationof the matured gum arabic

Sample name% loss on

drying (%wt)Specific rotation(deg/dm cm 3 /g)

Matured gum arabic (SUPERGUMTM EM2) 6.25 31

Table 2 | Molecular weight of matured gum arabic byGPC-MALLS analysis

Sample name Processing

Molecularweight

(Mw, g/mol)%

MassRg

(nm)

Matured gum arabic One peak 1.77 106 68SUPERGUMTM EM2 Two peaks (1, AGP)a 7.84 106 18.2 75

Two peaks (2, AG+GP) 4.16 105 81.8 a The method allows fractionation of the gum into its components as described byAl-Assaf et al. 5

Table 3 | Sugar composition (mol%) of the matured gumarabic

Sample name Gal Ara Rha Uronic acid

SUPERGUMTM EM2 32.8 31.4 13.1 24.3

Table 4 | Amino-acid composition (mol%) of the matured gum arabic

Sample name % N Ala Arg Asp Cys Glu Gly His Hyp Ile Leu Lys Met Phe Pro Ser Thr Tyr Val

SUPERGUM EM2 0.325 2.8 1.0 5.4 3.6 5.7 5.4 26.2 1.4 8.3 2.6 0.1 3.5 7.4 13.8 8.1 0.7 4.0

Table 5 | Trace metal analysis (ICP) of the matured gum arabic

(l g/g) Supergum EM2

K 7810Na 100Ca 6580Mg 2250P 4Fe 6Zn 1Cu 1Mn 2Si 39S 97

Table 6 | Intrinsic viscosity of the matured gum arabic

Sample name (code no.) Intrinsic viscosity ( g) (ml/g)

Matured gum arabic (SUPERGUM EM2) 19.4




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In vitro effects of SCFACell culture. HK-2 cells (human renal proximal tubular epithelial

cells immortalized by transduction with human papilloma virus (HPV16 E6/E7 genes39 ) were cultured as previously described. 4043

Toxicity/cell viability. Cell viability was assessed by the ability of monolayers to reduce the dye alamarBlue. At the end of eachexperimental period/manipulation, alamarBlue reagent was added

to make up a nal volume of 10% of the culture medium. The cellswere returned to the incubator for 1 h, after which 100 ml aliquots of medium were quantied for alamarBlue uorescence using aFluostar Optima Fluorescence Meter of excitation wavelength544nm and detection wavelength 590 nm.

Quantitation of TGF- b1. Total TGF-b1 in the cell culturesupernatant was measured by specic ELISA (R&D Systems,Abingdon, UK) of cell culture supernatant samples collected fromgrowth-arrested HK-2 cells, stimulated under serum-free condi-tions. Active TGF-b1 was measured directly and latent TGF- b1 canbe measured indirectly following acid activation of samples.

Assessment of de novo TGF-b1 synthesis. De novo TGF-b1synthesis was examined by detection of radioactive amino-acid

incorporation into newly synthesized protein by metabolic labellingand detection by TGF- b1 immunoprecipitation and autoradiogra-phy as previously described. 8

Western blot analysis. Equal numbers of cells were lysed by addition of sodium dodecyl sulphate (SDS) sample buffer, and100 ml of the resultant lysates was boiled for 5 min at 951 C beforeloading onto SDS-polyacrylamide electrophoresis (SDS-PAGE) gel(515%). Electrophoresis was carried out under reducing condi-tions. 44 Separated proteins were transferred to a nitrocellulosemembrane (Amersham, Little Chalfont, UK). Following a blockingstep, membranes were incubated with the appropriate primary antibody in Tris-buffered saline containing 0.1% Tween 20(phosphate-buffered saline-Tween) overnight at 4 1 C. Primary antibodies were as follows:

K rabbit polyclonal anti-TGF b type I receptor antibody (dilution1:500; Santa Cruz Biotechnology, Wiltshire, UK);

K antibody to dually phosphorylated active form of (mitogen-activated protein kinase) MAPK (p44/ERK1 and p42/ERK2;final dilution 1:5000; New England Biolabs (UK) Ltd, HitchinHerts, UK;

K antibody to total MAPK (dilution 1:5000; New England Biolabs(UK) Ltd);

K antibody to phosphorylated SMAD3 final dilution 1:1000;Cambridge Bioscience, Cambridge, UK).

The blots were subsequently incubated with an appropriatehorseradish peroxidase-conjugated secondary antibody (Sigma,

Poole, UK) in Tris-buffered saline-Tween. Proteins were visualizedusing enhanced chemiluminescence (Amersham, UK).

Assessment of TGF-b1 responsiveness. The SMAD-responsivepromoter (SBE) 4 -Lux 45 was a gift from Aristidis Moustakas. Fortransfection of the reporter construct, 30 103 cells/well wereseeded onto a 24-well plate (cells at this density produced a 70%conuence monolayer the following day). The next day, cells weretransfected with 0.2 mg of the SMAD-responsive promoter-luciferaseconstruct using the mixed lipofection reagent FuGene 6 (Roche, IN,USA) at a ratio of 0.6ml Fugene to 0.2 mg DNA in serum-free andinsulin-free medium. Transfection efciency was monitored by co-transfection with a b-galactosidase reporter plasmid. At 24 h aftertransfection, cells were stimulated with TGF- b1. Following lysis of

the cells in Reporter Lysis Buffer (Promega, WI, USA), the luciferasecontent was quantied by glow-type luminescence assay (Bright-Glo, Promega), and b-galactosidase activity determined by acommercial assay (Promega). Luciferase activity was normalized tob-galactosidase activity.

Detergent-free purification of the lipid raft-rich membranefraction. HK-2 cells were grown to near conuence in 100mm

dishes. After two washes with ice-cold phosphate-buffered saline,two conuent dishes were scraped into 2 ml of 500m M sodiumcarbonate, pH 11.0. Homogenization was carried out by three 20 sbursts of ultrasonic disintegrator (Soniprep 150, Fisher Scientic) todisrupt cellular membranes as previously described. 46 The homo-genates were adjusted to 45% sucrose by addition of 2 ml of 90%sucrose prepared in MBS (2-( N -morpholino) ethanesulphonic acid-buffered saline;, 25 mM 2-( N -morpholino) ethanesulfonic acid, pH6.5, 0.15M NaCl) and placed at the bottom of an ultracentrifugetube. A discontinuous sucrose gradient (4ml of 35% sucrose, 4ml of 5% sucrose, both prepared in MBS) was formed above andcentrifuged at 39000r.p.m. for 1620h, in an SW40 TI rotor(Beckman Instruments, Palo Alto, CA, USA). A light-scatteringband was observed at the 335% sucrose interface. A total of 12 1-mlfractions were collected from the top of the tubes, and a portion of each fraction was analysed by SDS-PAGE (10% gel) and immuno-blot analysis for TGF-b RI, which was quantied by densitometry (Chemi Doc, Bio-Rad, Hemel Hempstead Herts, UK). Data areexpressed as a percentage of the total TGF- b:receptor complex forthat experiment in each fraction.

Statistical analysisStatistical analysis was performed using the unpaired Studentst -test, with a value of P o 0.05 considered to represent a signicantdifference. The data are presented as means 7 s.d. of n experimentsas indicated in the gure legends. For each individual experiment,the mean of duplicate determinations was calculated.

ACKNOWLEDGMENTSThis work was supported by a research grant from San-Ei Gen Inc.,Japan. AOP is supported by a GlaxoSmithKline Advanced Fellowship.The Institute of Nephrology is supported by Kidney WalesFoundation.

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