Post-oral appetite stimulation by sugars and nonmetabolizable sugar analogs

14 Pages·2013·0.27 MB·English

AmJPhysiolRegulIntegrCompPhysiol305:R840–R853,2013.
FirstpublishedAugust7,2013;doi:10.1152/ajpregu.00297.2013.
Post-oral appetite stimulation by sugars and nonmetabolizable sugar analogs
Steven Zukerman, Karen Ackroff, and Anthony Sclafani
DepartmentofPsychology,BrooklynCollege,CityUniversityofNewYork,Brooklyn,NewYork;andCognition,Brain,
andBehaviorDoctoralSubprogram,GraduateSchool,CityUniversityofNewYork,NewYork,NewYork
Submitted17June2013;acceptedinfinalform3August2013
Zukerman S, Ackroff K, Sclafani A. Post-oral appetite stimula- bygastric,duodenal,andjejunalglucoseinfusions,butnotby
tion by sugars and nonmetabolizable sugar analogs. Am J Physiol gastric infusions with a closed pylorus, or by ileal, hepatic-
Regul Integr Comp Physiol 305: R840–R853, 2013. First published portal,orintraperitonealglucoseinfusions(2,15,67).Hepatic-
August 7, 2013; doi:10.1152/ajpregu.00297.2013.—Post-oral sugar
portal glucose infusions produced preferences in other condi-
actions enhance the intake of and preference for sugar-rich foods, a
tioning paradigms, however, which suggest that sugar sensors
process referred to as appetition. Here, we investigated the role of
in both the upper intestinal and hepatic portal regions contrib-
intestinalsodiumglucosecotransporters(SGLTs)insugarappetition
ute to sugar conditioning (39, 58). D
in C57BL/6J mice using sugars and nonmetabolizable sugar analogs
that differ in their affinity for SGLT1 and SGLT3. In experiments 1 Thepresenceoftheoralsweettastereceptor(T1R2(cid:2)T1R3) ow
n
and 2, food-restricted mice were trained (1 h/day) to consume a in the intestinal tract suggested the possibility that the same lo
flavoredsaccharinsolution[conditionedstimulus(CS(cid:1))]pairedwith sensorthatstimulatessugarappetiteinthemouthalsomediates ad
e
intragastric (IG) self-infusions of water and a different flavored sugar appetition in the gut (5). However, this notion is not d
solution (CS(cid:2)) paired with infusions of 8 or 12% sugars (glucose, supported by the findings that sweet ageusic mice missing fro
fructose, and galactose) or sugar analogs ((cid:3)-methyl-D-glucopyrano- critical sweet taste-signaling elements (T1R3, TRPM5, gust- m
sbiodtetl,eMCDS(cid:2)G;v3s-.OC-Sm(cid:1)ethchyol-iDc-egtleusctospwyerarenocsoinddeu,cOteMdGw)i.thSouubtsceoqiunefnutsitownos-. ducin)demonstratenormalflavor-conditioningresponsestoIG http
InfusionsoftheSGLT1ligandsglucose,galactose,MDG,andOMG sugar infusions (50). Furthermore, not all ligands of the sweet ://a
stimulated CS(cid:2) licking above CS(cid:1) levels. However, only glucose, taste receptor are effective in conditioning flavor preferences jp
MDG, and galactose conditioned significant CS(cid:2) preferences, with byIGinfusions.Whereasglucoseandglucose-containingsac- reg
the SGLT3 ligands (glucose, MDG) producing the strongest prefer- charides(sucrose,maltose,andmaltodextrin)conditionrobust u.p
ences.Fructose,whichisnotaligandforSGLTs,failedtostimulate flavor preferences, IG fructose has weaker or no conditioning h
y
CMSD(cid:2)G(cid:2)intpahkleoroidrzpirnef(earneSncGeL.TE1x/p3erainmtaegnotn3isrte)vbelaolcekdetdhMatDIGGianpfpuesitoitnioonf, etifofnecetds,awmhielrdeaasvethrseionnon(5n0u)t.riTtivheeseswfienedteinngesr isnudcircaalotesethcaotngdui-t siolo
whereas phloridzin had minimal effects on glucose-induced appeti- sensors other than T1R2 and T1R3 mediate the appetition gy
tion. However, adding phloretin (a GLUT2 antagonist) to the .o
ggleuthcoers,e(cid:2)thpehsleorfiidnzdiinnginsfussuigognesbtlotchkaetdhgulmucoorasel saipgpneatlistiogne.nTeraakteedn tboy- pcarondduidcaetde sbeynsgoarsstarirce saonddiuimntegsltuicnoaslegcloutcroasnespionrftuerssio(nSsG. LTTw1o) byrg/
intestinalSGLT1andSGLT3,andtoalesserdegree,GLUT2,mediate and SGLT3. The sodium glucose cotransporter SGLT1 is a 1
0
post-oral sugar appetition in mice. The MDG results indicate that transporter of glucose in intestinal enterocytes and is also .2
sugarmetabolismisnotessentialforthepost-oralintake-stimulating thought to serve as a glucose sensor in enteroendocrine cells 20
andpreference-conditioningactionsofsugarsinmice. (56, 63). The related protein SGLT3 has little or no transport .3
3
functionbutmayserveasaglucosesensorinthegut(56,63). .5
post-oralsugarconditioning;glucose;fructose;galactose SGLT1andSGLT3areattractivecandidatesbecausetheybind on
to glucose but not fructose. The two sensors differ in that J
u
FOOD INTAKE AND PREFERENCE are guided by oral sensations glucose and galactose are substrates for SGLT1 but only ne
(taste,odor,andmouthfeel)thatcontributetotheidentification glucosebindstoSGLT3.Inratsandmice,IGgalactosefailed 1
2
and hedonic evaluation of food flavor. Considerable progress toconditionflavorpreferences(49,51),whichwouldappearto , 2
0
hasbeenmadeinidentifyingthetastereceptorsthatrespondto implicate SGLT3 as the sensor responsible for post-oral glu- 1
7
sugar,fat,andaminoacidsthatprovideattractivesweet,fatty, cose appetition. However, a recent study reported that IG
andumamiflavorstofoods(8).Theappetiteforsuchfoodsis intubation of glucose and galactose, but not fructose, condi-
further enhanced by the post-oral actions of ingested nutrients tionedplacepreferencesinmice(30).Inaddition,werecently
(50). This is demonstrated in laboratory rodents by the intake reported that sweet ageusic T1r3 and Trpm5 knockout (KO)
stimulation and learned preferences for arbitrary flavors (con- mice expressed preferences for glucose and galactose, but not
ditioned stimuli, CS) that are paired with gastric or intestinal forfructose,in24-hsugarvs.watertests,whichweattributed
infusionsofsugar,fat,andproteins(orglutamate)(50).How- to the differential post-oral actions of the sugars (68). Taken
ever, relatively little is known about the sites and identities of together,thesefindingssuggestthatgalactose,likeglucose,has
the sensors that mediate the post-oral appetite-stimulating ac- post-oral reward actions in mice in some test situations.
tions of nutrients, a process we refer to as appetition (48). In The present study investigated the role of SGLT1 and
the case of sugars, several findings implicate the upper small SGLT3 in post-oral sugar appetition by using agonists and
intestineasaprimarysiteofactionforglucoseappetition.That antagonistsofthesesugarsensors.Thisstrategyhasbeenused
is,preferencesforflavoredsaccharinsolutionsareconditioned to characterize the sugar sensors involved in gut hormone
release, sugar-induced satiety, and brain neural responses (12,
20, 43, 45, 54, 56). Experiment 1 compared the post-oral
Addressforreprintrequestsandothercorrespondence:A.Sclafani,Dept.of
conditioningeffectsofglucose,galactose,andfructoseusinga
Psychology,BrooklynCollegeofCUNY,2900BedfordAve.,Brooklyn,NY
11210(e-mail:[email protected]). newly developed IG self-infusion paradigm that reveals both
R840 0363-6119/13Copyright©2013theAmericanPhysiologicalSociety http://www.ajpregu.org
POST-ORALSUGARSENSINGANDAPPETITE R841
rapid glucose stimulation of intake and flavor preference con- infusion rate and volume by its licking response. In particular, the
ditioning in C57BL/6J mice (67, 69). Experiment 2 extended computer accumulated licks during 3-s bins and activated the pump
this analysis by comparing the post-oral appetition effects of for 3 s when a criterion number of licks was recorded. The oral-to-
infusion intake ratio was maintained at (cid:5)1:1 by adjusting the lick
glucose with those of two nonmetabolizable glucose analogs:
(cid:3)-methyl-D-glucopyranoside(MDG)and3-O-methyl-D-gluco- criterionforeachmouse.Dailyoralfluidintakesweremeasuredtothe
nearest0.1g,andIGinfusionswererecordedtothenearest0.5ml.
pyranoside (OMG). These analogs were of interest because
Testsolutions.TheCSsolutionscontained0.025%sodiumsaccha-
they are both ligands for the SGLT1 transporter, and MDG is
rin (Sigma Chemical, St. Louis, MO) flavored with 0.02% ethyl
also a ligand for SGLT3 (56, 63). Furthermore, they both acetateorpropylacetate(Sigma).TheCS(cid:1)solutionwaspairedwith
evoke physiological responses similar to glucose, such as IG infusion of water, while the CS(cid:2) solution was paired with IG
inhibition of gastric emptying and release of GLP-1 and GIP infusion of food-grade glucose, fructose (Honeyville Food Products,
hormones, although MDG is more effective than OMG in RanchoCucamonga,CA),orgalactose(Sigma).Inexperiment1A,the
mimickingglucoseactionsinsomeresponses(35,44).Exper- 8%Fructose,8%Galactoseand8%Glucosegroupscontained14,12,
iment 3A determined the effects of the SGLT1/3 antagonist and 10 mice, respectively. In experiment 1B, the 12% Fructose,
Galactose,andGlucosegroupscontained12,9,and12mice,respec-
phloridzin on the flavor-conditioning effects of glucose and
tively.Forabouthalfoftheanimalsineachgroup,theCS(cid:1)solution
MDG. Experiment 3B then determined whether blocking glu-
contained ethyl acetate, and the CS(cid:2) solution contained propyl D
cose actions on SGLT1 and SGLT3, as well as on the sugar o
acetate;theflavorswerereversedfortheremaininganimals. w
transporter GLUT2, using a mixture of phloridzin and phlore- Procedure. The mice were trained (1 h/day) in the test cages for n
tin would more effectively prevent glucose-conditioned flavor two sessions with unflavored 0.025% sodium saccharin solution, loa
d
preferences (see Ref. 29). whilewater-deprivedandthenforfoursessionswhilefood-restricted e
d
MATERIALS AND METHODS tpoai8re5d–9w0i%thomfatthcheierd-avdolluibmiteuminfbuosdioynswoefigwhta;tesra.cTchhaerimnicinetawkeersewtheerne from
Experiment1—Glucose,Galactose,andFructose giventhree1-htestsessionswiththeCS(cid:1)saccharinsolutionpaired h
with IG water infusions followed by three sessions with the CS(cid:2) ttp
This experiment compared the post-oral appetition effects of IG saccharin solution paired with IG infusions of the appropriate sugar ://a
self-infusionsofglucose,galactose,andfructose,whichhavediffer- solution.Themicewerethengiventwoalternating(A)sessions,each jp
entialactionsonSGLT1andSGLT3.Thetestprotocolwasidentical withtheCS(cid:1)/IGwater(days1and3)andtheCS(cid:2)/IGsugar(days2 re
g
tothatusedinourrecentstudyof2–32%glucoseself-infusions(67). and4)toenhancetheirdiscriminationbetweenthetwosolutions(67). u
In experiment 1A, the mice were infused with 8% sugar solutions In the last of each CS(cid:1) and CS(cid:2) session, the mice were given a .ph
basedonourfindingthat8%glucosewasthethresholdconcentration second sipper tube containing water not paired with IG infusions to ys
to condition a CS(cid:2) preference (67). Experiment 1B determined familiarize them with the presence of two sipper tubes for the io
whetherahighersugarconcentration(12%)enhancedthecondition- subsequenttwo-bottletest.Thetwo-bottletestwiththeCS(cid:2)andCS(cid:1) log
y
ingresponsetogalactoseandfructose. solutions,nolongerpairedwithIGinfusions,wasconductedoverfour .o
Animals.AdultmaleC57BL/6J(B6)mice(10wkold)borninthe 1h/daysessions. rg
laboratory from Jackson Laboratories (Bar Harbor, ME) stock were Data analysis. CS(cid:1) licks and total intakes (oral (cid:2) IG infusate) b/
y
singly housed in plastic tub cages kept in a test room maintained at during the last two 1 h/day sessions were averaged. The data from 1
2ch2o°Cww(5i0th01a;1P2M:1I2-Nhultirgithito-ndaIrnktecryncalteio.nTahl,eBmriecnetwwoeored,mMaiOn)tapinreiodrotno dthuersinegtwthoestehsrseioenCs,Sr(cid:2)efesreressdiotnosa(stetsetsst10–,3a)ndwethree alincaklsyzaendduisnitnagkeas 0.22
0
food restriction. During testing, they were maintained at 85–90% of mixed-model ANOVA with a group factor (IG Sugar group) and .3
t(h0e.5iroard1libgi,tuBmiob-oSdeyrvw, eFirgehntcbhytowfene,diNngJ)t,hwemhicfihxeadll-osiwzeedchfoowr ppreelcliestes CreSp(cid:2)eatleidck-msedausruinregsthfaecttworo(atletsetrsna0t–in3g).oSniem-biolatrtlley,sethsesiomnesaanndCSth(cid:1)efaonudr 3.5 o
adjustment of daily food rations. Experimental protocols were ap- two-bottle tests were compared in separate ANOVAs. Additional n J
proved by the Institutional Animal Care and Use Committee at analysesaredescribedintheresults. u
n
Brooklyn College and were performed in accordance with the Na- e
1
triaotonrayl IAnnstiimtuatless.of Health Guidelines for the Care and Use of Labo- RESULTS 2, 2
Surgery.MicewerefittedwithIGcatheters(0.84mmOD(cid:4)0.36 Experiment 1A. Fig. 1A shows the total 1-h lick data for 01
7
mm ID, Micro-Renathane tubing, MRE-033; Braintree Scientific, CS(cid:1)test0andCS(cid:2)tests1–3.Analysisofthesedatarevealed
Braintree,MA)whileanesthetizedwithisoflurane(2%)inhalation,as that the 8% sugar groups did not differ in their CS(cid:1) licks
previouslydescribed(52).About10daysaftersurgerythemicewere
pairedwithIGwaterself-infusion(test0)butdiddifferintheir
briefly(5min)anesthetizedwithisoflurane,andtubingwasattached
CS(cid:2)lickspairedwithIG8%sugarself-infusion[group(cid:4)test
to the gastric catheter and then passed through an infusion harness
withaspringtether(CIH62,InstechLaboratories,PlymouthMeeting, interaction,F(6,99)(cid:6)4.32,P(cid:7)0.001].The8%Glucoseand
PA).Thetubingwasthenattachedtoaninfusionswivelmountedon 8% Galactose groups, but not the 8% Fructose group, signifi-
a counterbalanced lever (Instech Laboratories). The body weight of cantlyincreased1-hlickswhenswitchedfromtheCS(cid:1)tothe
each mouse was measured before and after it was fitted with the CS(cid:2). A comparison of the CS(cid:2) tests 1–3 licks indicated that
infusiontether/swivelsystem;dailybodyweightsweremonitoredby the 8% Glucose group licked more (P (cid:7) 0.01) than the 8%
weighingthemousewiththeattachedinfusiontether/swivelsystem. Fructosegroup,whilethelicksofthe8%Galactosegroupwere
Each animal was then returned to a tub cage, and the swivel- intermediatebetweentheothertwogroups[F(2,32)(cid:6)6.2,P(cid:7)
counterbalancedleverwasattachedabovethecage.
0.01]. Within-group analyses revealed that the 8% Galactose
Apparatus. IG infusion tests were conducted in plastic test cages
group [F(3,33) (cid:6) 10.1, P (cid:7) 0.001] and 8% Glucose group
(52). The sipper spouts were interfaced via electronic lickometers
[F(3,27)(cid:6)14.5,P(cid:7)0.001]lickedmoreineachofCS(cid:2)tests
(Med Electronics, St. Albans, VT) to a computer, which operated a
syringe pump (A-99; Razel Scientific, Stamford, CT) that infused 1–3thaninCS(cid:1)test0andthattheirlicksintests1–3didnot
liquidintothegastriccathetersastheanimalslicked.Thepumprate differ. The 1-h intake data (CS solution (cid:2) IG infusion) re-
was nominally 0.5 ml/min, but the animal controlled the overall vealed a similar pattern of results [group (cid:4) test interaction,
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
R842 POST-ORALSUGARSENSINGANDAPPETITE
A One-Bottle Tests tively), whereas the CS(cid:2) and CS(cid:1) licks did not differ in the
8%Fructosegroup(769.8vs.833.9)[group(cid:4)CSinteraction,
F(2,33) (cid:6) 12.9; P (cid:7) 0.001].
2000 CS+3 Figure 1B presents the lick data for the two-bottle CS(cid:2) vs.
CS+2 CS(cid:1) choice test conducted without IG infusions. Analysis of
11550000 CS+1 ** * * * Pthe(cid:7)sed0a.0ta01re]v.eIanledpaartgicrouulapr,(cid:4)thCeS8i%nterGacluticoonse[Fa(2n,d33G)a(cid:6)lac2t2o.s6e;
CS-0 * * groups licked significantly more (P (cid:7) 0.001 and P (cid:7) 0.05,
h
1 respectively)fortheCS(cid:2)thanCS(cid:1),whiletheCSlicksofthe
s / 1000 8%Fructosegroupdidnotdiffer.Furthermore,theCS(cid:2)licks
ck differed (P (cid:7) 0.001) among the groups: 8% Glucose (cid:8) 8%
Li
Galactose(cid:6)8%Fructosegroups,butCS(cid:1)licksdidnotdiffer.
A similar pattern of results was obtained in the analysis of
500
two-bottle intake data except that the CS(cid:2) intakes of the 8%
Glucose and Galactose groups only marginally differed (P (cid:6) D
0.052). Also, the Glucose group consumed less CS(cid:1) than the o
w
0 Fructose and Galactose groups (data not shown). The groups n
8% FRU 8% GAL 8% GLU lo
alsodifferedintheirpercentCS(cid:2)licks[F(2,33)(cid:6)27.9;P(cid:7) a
B d
Two-Bottle Tests 0.001]. The 8% Glucose group displayed a higher preference ed
2500 (70%, P (cid:7) 0.01) than did 8% Galactose (56%) and Fructose fro
(51%) groups, which did not differ from one another. Note m
2000 CCSS+- 70*% tvthheaartnytChweSe(cid:1)a5k6.,%buCtS1(cid:2)0porfeftehreen1c2emoficteheli8c%kedGamlaocrteosfeorgrthoeupCwS(cid:2)as http://ajp
re
g
h 1500 Experiment 1B u
1 .p
Licks / 1000 56*% tesFtsig1.–23A.sAhnoawlysstihseotfottahle1s-ehdliactkadreavtaeafolerdCtSh(cid:1)atttehset012a%ndsCuSg(cid:2)ar hysiolo
51% groups did not differ in their CS(cid:1) licks paired with IG water gy
self-infusion (test 0) but did differ in their CS(cid:2) licks paired .o
500 with IG 12% sugar self-infusion in tests 1–3 [group (cid:4) test brg/
interaction, F(6,99) (cid:6) 14.80; P (cid:7) 0.001]. The 12% Glucose y
1
andGalactosegroups,butnotthe12%Fructosegroup,signif- 0
0 icantlyincreased1-hlickswhenswitchedfromtheCS(cid:1)tothe .22
8% FRU 8% GAL 8% GLU CS(cid:2). A comparison of the CS(cid:2) tests 1–3 licks indicated that 0.3
Groups the 12% Glucose and Galactose groups did not differ and both 3.5
Fig.1.Experiment1A:Valuesareexpressedasmeans(cid:9)SE.A:1-htotallicks lickedmore(P(cid:7)0.01)thanthe12%Fructosegroup[F(2,33)(cid:6) o
n
areplottedforonebottletests0–3.Themicedrank(1h/day)aconditioned 23.7;P(cid:7)0.001].Within-groupanalysesrevealedthatthe12% J
stimuli (CS)-flavored saccharin solution paired with intragastric (IG) water Glucose group increased (P (cid:7) 0.01) CS licks from test 0 to 1 un
infusionsintest0beforebeingswitchedtoaCS(cid:2)-flavoredsaccharinsolution e
pairedwithIGsugarself-infusionsintests1–3.The8%GLU,8%FRU,and and then in each successive CS(cid:2) test. The 12% Galactose 12
8% GAL groups were infused with 8% glucose, fructose, and galactose, groupincreased(P(cid:7)0.01)theirlicksfromtest0to1andthen , 2
respectively. B: 1-h licks are plotted for CS(cid:2)- and CS(cid:1)-flavored saccharin fromtest2to3.The1-hintakedatarevealedasimilarpattern 0
1
solutionsduringthetwo-bottlepreferencetestforthe8%GLU,8%FRU,and of results [group (cid:4) test interaction, F(6,99) (cid:6) 12.74, P (cid:7) 7
8% GAL groups. CS(cid:2) and CS(cid:1) intakes were not paired with IG infusions
duringtest.NumberatopbarrepresentsmeanpercentpreferencefortheCS(cid:2) 0.001]. Overall, intakes significantly increased from CS(cid:1) test
solution.*Significantdifference(P(cid:7)0.05)betweentest0vs.tests1–3licks 0 to CS(cid:2) test 3 in the 12% Glucose (2.1 to 3.5 g/h) and the
andbetweenCS(cid:2)vs.CS(cid:1)licks. 12% Galactose (2.0 to 3.2 g/h) groups, but not in the 12%
Fructosegroup(2.1to2.2g/h).TheCS(cid:1)intakesofthegroups
F(6,99) (cid:6) 4.77, P (cid:7) 0.001]. Overall, intakes significantly did not differ in test 0, whereas their CS(cid:2) test 1 and test 3
increased from CS(cid:1) test 0 to CS(cid:2) test 3 in the 8% Glucose intakes differed as follows: 12% Glucose (cid:6) 12% Galactose (cid:8)
(2.0 to 3.3 g/h) and the 8% Galactose (1.9 to 2.9 g/h) groups, 12%Fructose.Test2intakesdifferedasfollows:12%Glucose(cid:8)
but not in the 8% Fructose group (1.9 to 2.2 g/h). The CS(cid:1) 12%Galactose(cid:8)12%Fructose.
intakesofthegroupsdidnotdifferintest0,whereastheirCS(cid:2) In the alternating training sessions, the 12% Glucose and
tests 1–3 intakes differed as follows: 8% Glucose (cid:1) 8% Galactose groups licked more than the 12% Fructose group
Galactose (cid:8) 8% Fructose. [F(2,33)(cid:6)13.1;P(cid:7)0.01].Inaddition,the12%Glucoseand
Inthealternatingtrainingsessions,the8%Glucoseand8% Galactose groups licked more (P (cid:7) 0.001) in the CS(cid:2) than
Galactose groups licked more than the 8% Fructose group CS(cid:1)sessions(1,665.0vs.1,252.1,1,375.8vs.1,092.5,respec-
[F(2,33) (cid:6) 7.9, P (cid:7) 0.01]. In addition, the 8% Glucose and tively), whereas the CS(cid:2) and CS- licks did not differ in the
Galactose groups licked more (P (cid:7) 0.001) in the CS(cid:2) than 12%Fructosegroup(931.1vs.939.5)[group(cid:4)CSinteraction;
CS(cid:1) sessions (1,467.0 vs. 1,152.1, 1,129.6 vs. 910.4, respec- F(2,33) (cid:6) 9.7, P (cid:7) 0.001].
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
POST-ORALSUGARSENSINGANDAPPETITE R843
A One-BottleTests alsodifferedintheirpercentCS(cid:2)licks[F(2,33)(cid:6)34.0;P(cid:7)
0.001].The12%Glucosegroupdisplayedahigher(P(cid:7)0.01)
preference(84%)thanthe12%Galactosegroup(64%),which
CS+3
2000 displayed a higher preference than the 12% Fructose (53%)
CS+2 * group.
CS+1 * *
11550000 CS-0 * * DISCUSSION
**
h Glucose. As previously reported, the 8% Glucose mice
ks / 1 1000 lCicSk(cid:2)edtessigtn1ifiacnadndtliyspmlaoyreedfoarstihgeniCficSa(cid:2)nttChaSn(cid:2)CpSr(cid:1)efebreegnicneniinngthine
c
Li two-bottle choice test without IG infusions (67). Experiment
1B revealed an even greater stimulation of CS(cid:2) licking and
500 CS(cid:2)preferenceinthe12%Glucosegroup.Thisconfirmsour
prior concentration-response findings obtained with mice
D
testedwith2,4,8,16,and32%glucoseinfusions.Asdetailed o
w
0 12% FRU 12% GAL 12% GLU irnevoeaulredpritohratretphoertI,Ganglauncaolsyesisgroofupwsithinincr-eseassseidonthleicirk CraSt(cid:2)es nloa
B licking as early as 4–6 min in CS(cid:2) test 1 and showed de
2500 Two-Bottle Tests saunbds3ta.nStiiamliilnacrrcehaasnesgeinsiCnSl(cid:2)icklircakteinsgwienremoinbusteersve1d–3inotfhtees1t2s%2 d fro
84% m
CS+ * Glucose group in the present study (data not shown). h
2000 CS- FruFcrtuocsteosger.oIunpscofnatirleadstttooitnhcerGealsuecothseeigrr1o-uhplsi,ctkhsei8n%CaSn(cid:2)d1te2s%ts ttp://a
1–3 or display a significant preference for the CS(cid:2) over the jp
64% CS(cid:1). This is consistent with prior 1-h lick data obtained with reg
1 h 1500 * B6micedrinking0.8%sucraloseandless“sweet”8%glucose u.ph
s / and8%fructosesolutions.Thatis,themicethatswitchedfrom ys
Lick 1000 53% sthuecrmaliocseethtoatgsluwciotcsheeldicfkreodmmsuuccrhalmosoeretofofrrutchteosseugliacrk,ewdhmeruecahs iolog
y
less sugar than sucralose (66). The present 1-h IG results are .o
also in agreement with our 24-h IG study showing that IG rg
500 infusions of 8% or 16% fructose, unlike glucose infusions, b/
y
failed to stimulate intake or condition CS(cid:2) preferences in B6 1
0
mice(49).The1-and24-hIGconditioningresultsindicatethat .2
2
0 the post-oral actions of fructose have no reinforcing effects in 0
12% FRU 12% GAL 12% GLU B6 mice. This conclusion is compatible with the failure of .33
Groups naïvesweetageusicT1r3KOandTrpm5KOmicetodevelop .5 o
preferences for orally consumed fructose solutions, which n
Fig.2.Experiment1B:Valuesareexpressedasmeans(cid:9)SE.A:1-htotallicks J
areplottedforonebottletests0–3.Themicedrank(1h/day)aCS(cid:1)-flavored contrasts with their robust experience-induced preferences for u
n
saccharin solution paired with IG water infusions in test 0 before being glucose solutions (68). e
switched to a CS(cid:2)-flavored saccharin solution paired with IG sugar self- Galactose.Thepresentresultsprovidethefirstevidencethat 12
infusionsintests1–3.The12%GLU,12%FRU,and12%GALgroupswere IG infusions of galactose can stimulate intake and condition , 2
infusedwith12%glucose,fructose,andgalactose,respectively.B:1-hlicks 0
are plotted for CS(cid:2)- and CS(cid:1)-flavored saccharin solutions during the two- flavor preferences in rodents. The 8% and 12% galactose 17
bottle preference test for the 12% GLU, 12% FRU, and 12% GAL groups. infusionsincreasedlickratesintheveryfirstCS(cid:2)testsession
CS(cid:2) and CS(cid:1) intakes were not paired with IG infusions during the test. and further stimulated licking in subsequent test sessions.
Number atop bar represents mean percent preference for the CS(cid:2) solution. Overall, the 8% and 12% galactose infusions stimulated CS(cid:2)
*Significant differences (P (cid:7) 0.05) between test 0 vs. tests 1–3 licks and
licking as much as did the IG glucose infusions. However,
betweenCS(cid:2)vs.CS(cid:1)licks.
galactose conditioned weaker CS(cid:2) preferences than did glu-
cose. In particular, the preference of the 8% Galactose group,
Fig. 2B presents the lick data for the two-bottle CS(cid:2) vs. while statistically significant, was quite modest (56%) and
CS(cid:1)choicetest.Analysisofthesedatarevealedagroup(cid:4)CS substantially less than the 70% CS(cid:2) preference of the 8%
interaction[F(2,33)(cid:6)15.3,P(cid:7)0.001].Inparticular,the12% Glucose group. The 12% galactose IG infusions increased the
Glucose and Galactose groups licked significantly more (P (cid:7) CS(cid:2)preferenceto64%,butitwassignificantlylowerthanthe
0.01) for the CS(cid:2) than CS(cid:1), while the CS licks of the 12% 84% preference produced by the 12% glucose infusions.
Fructose group did not differ. Furthermore, the CS(cid:2) licks To determine whether a higher galactose concentration
differed(P(cid:7)0.001)amongthegroups:12%Glucose(cid:8)12% wouldconditionastillstrongerCS(cid:2)preference,anothergroup
Galactose (cid:6) 12% Fructose groups. CS(cid:1) licks also differed of mice (n (cid:6) 12) was tested as in experiment 1 but with IG
(P(cid:7)0.01)amongthegroups:12%Fructose(cid:6)12%Galactose(cid:8) infusions of 16% galactose. These mice displayed only a
12% Glucose. A similar pattern of results was obtained in the marginal (P (cid:6) 0.079) increase in CS(cid:2) licks in tests 1–3. The
analysisoftwo-bottleintakedata(datanotshown).Thegroups 16% galactose mice showed a weak (54%), albeit significant
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
R844 POST-ORALSUGARSENSINGANDAPPETITE
(P (cid:7) 0.05), CS(cid:2) preference in the two-bottle tests, which AA One-BottleTests
contrasts with the 80% CS(cid:2) preference displayed by rats
infused with 16% glucose infusions (67). Thus, while IG
CS+3
galactose can stimulate intake and condition a flavor prefer- 2000
ence, it is effective only at a limited concentration range and CS+2
much less so than glucose. Postabsorptive metabolic factors CS+1
*
may limit the appetition effects of galactose (see GENERAL 11550000 CS-0 * * * *
DISCUSSION).
*
h * *
Experiment 2: Glucose Analogs 1
Thepresentexperimentfurtherexploredtheroleofintestinal ks / 1000
c
SGLTproteinsinsugarappetitionbycomparingthecondition- Li
ing effects of two nonmetabolizable glucose analogs: MDG, a
substrate for both SGLT1 and SGLT3, and OMG, a substrate 500
for SGLT1 only. Prior studies have used these two glucose D
analogs to reveal the sugar-sensing mechanisms involved in ow
gastrointestinal hormone release and satiety effects (7, 12, 18, n
0 lo
19, 21, 33, 37), but their abilities to stimulate intake and 8% OMG 8% MDG 8% GLU ad
condition flavor preferences have not been previously investi- B ed
gtiaotnedf.aiOlendetsotucdoyndreitpioonrtead,plhaocweepvreerf,etrheantceOiMnGmigcaest(r3ic0)i.ntuba- 2000 Two-Bottle Tests from
Attheendofbehavioraltesting,theeffectsoftheIGglucose CS+ 70% h
amnedasgulruecdo.sTehaisnawloasgoifnfinutseiorensstboencabuloseodofgtlhuecopsoetenletviaellsrowleeoref CS- 70% * ttp://a
1500 jp
postabsorptive glucose in preference conditioning (39, 58). * re
A prior study indicated that gastric intubation of MDG, gu
unlikeglucose,didnotincreaseplasmaglucoselevelsinB6 h .p
mice (37). s / 1 1000 56% hysio
ck lo
MATERIALS AND METHODS Li gy
.o
Adult male B6 mice were housed and tested as in experiment 1. rg
Experiment2Aincluded8%MDG(n(cid:6)14)and8%OMG(n(cid:6)15) 500 b/
groups and experiment 2B included 12% MDG (n (cid:6) 14) and 12% y
OMG (n (cid:6) 9) groups. Their licking responses were compared with 10
thoseofthe8%and12%Glucosegroupsofexperiment1. .2
2
Following the two-bottle tests, blood glucose levels were mea- 0
0 .3
sured. Each mouse was infused with 0.6 ml of its respective sugar 8% OMG 8% MDG 8% GLU 3
solution(8%or12%glucose,MDG,orOMG)overa10-minperiod Groups .5 o
(see Ref. 67). Tail blood samples were measured using a FreeStyle n
Freedom Lite (Abbott Diabetes Care, Alameda, CA) blood glucose Fig.3.Experiment2A:Valuesareexpressedasmeans(cid:9)SE.A:1-htotallicks J
meterjustbefore(0min),and15,30,and60minfollowingthestart areplottedforonebottletests0–3.Themicedrank(1h/day)aCS(cid:1)-flavored un
saccharin solution paired with IG water infusions in test 0 before being e
omficthee(nIG(cid:6)in1f2u)sitohna.tDwaetraewinefruesecdolIlGectwedithfro0m.6amnlaodfdwitiaotenralingsrtoeuapd ooff sinwfiutscihoendsitnotaesCtsS1(cid:2)–-3fl.aTvhoere8d%saGccLhUar,in8%soMluDtioGn,apnadire8d%wOitMhGIGgrsouugpasrwseelrfe- 12, 2
glucose(67).ThebloodglucosedatawereanalyzedusinganANOVA infused with 8% glucose, MDG and OMG, respectively. B: 1-h licks are 0
1
with a group factor and a repeated-measure factor (sample time plottedforCS(cid:2)-andCS(cid:1)-flavoredsaccharinsolutionsduringthetwo-bottle 7
points). In addition, incremental areas under the curve (IAUC) were preference test for the 8% GLU, MDG, and OMG groups. CS(cid:2) and CS(cid:1)
calculatedandcomparedacrossgroupswithaone-wayANOVA. intakes were not paired with IG infusions during the test. Number atop bar
representsmeanpercentpreferencefortheCS(cid:2)solution.*Significantdiffer-
RESULTS ences(P(cid:7)0.05)betweentest0vs.tests1–3licksandbetweenCS(cid:2)vs.CS(cid:1)
licks.
Experiment 2A
Fig.3Ashowsthetotal1-hlickdataforCS(cid:1)test0andCS(cid:2)
tests 1–3. Analysis of these data revealed that the 8% sugar than in CS(cid:1) test 0, whereas the 8% MDG group licked more
groups did not differ in their CS- licks paired with IG water only in CS(cid:2) tests 2 and 3 than in CS(cid:1) test 0. The 1-h intake
self-infusion (test 0), and all three groups increased their data revealed a similar pattern of results [group (cid:4) test inter-
lickingresponseintheCS(cid:2)tests,althoughtodifferentdegrees action,F(6,108)(cid:6)4.46;P(cid:7)0.001].Overall,allthreegroups
[group (cid:4) test interaction, F(6,108) (cid:6) 4.4; P (cid:7) 0.001]. A significantly increased their solution intakes from CS(cid:1) test 0
comparison of the CS(cid:2) tests 1–3 revealed that the Glucose toCS(cid:2)test3(2.0to3.3g/hfor8%Glucose,2.4to3.2g/hfor
group licked more than the 8% MDG group in test 1; there 8%MDG,and1.9to2.7g/hfor8%OMG).TheCS(cid:1)intakes
werenoothergroupdifferences[F(4,72)(cid:6)4.9;P(cid:7)0.01].In ofthegroupsdidnotdifferintest0,whereastheirCS(cid:2)test1
addition, within-group analyses revealed that the 8% Glucose intakes differed as follows: 8% Glucose (cid:8) 8% OMG (cid:6) 8%
and8%OMGgroupslicked(P(cid:7)0.01)moreinCS(cid:2)tests1–3 MDG; CS(cid:2) intakes did not differ in tests 2 and 3.
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
POST-ORALSUGARSENSINGANDAPPETITE R845
In the alternating training sessions, the groups differed in A One-BottleTests
theirCS(cid:2)andCS(cid:1)licks[group(cid:4)CSinteraction,F(2,36)(cid:6)
4.4; P (cid:7) 0.05]. Only the 8% Glucose and 8% MDG groups
CS+3
licked more (P (cid:7) 0.001) in the CS(cid:2) than CS(cid:1) sessions
2000
(1,467.0 vs. 1,152.1, 1,391.4 vs. 1,102.2, respectively); CS(cid:2) CS+2
*
and CS(cid:1) licks did not significantly differ in the 8% OMG CS+1
*
group (1,102.9 vs. 1,006.4).
Fig. 3B presents the lick data for the two-bottle CS(cid:2) vs. h 11550000 CS-0 ** *
CS(cid:1)choicetest.Analysisofthesedatarevealedagroup(cid:4)CS 1 * * * *

interaction[F(2,36)(cid:6)9.75;P(cid:7)0.001].Inparticular,the8% /
s
Glucose and MDG groups licked significantly more (P (cid:7) k 1000
c
0.001) for the CS(cid:2) than CS(cid:1), while the CS licks of the 8% Li
OMGgroupdidnotdiffer.Furthermore,theCS(cid:2)licksdiffered
(P(cid:7)0.001)amongthegroups:8%Glucose(cid:8)8%MDG(cid:8)8%
500
OMGgroups,butCS(cid:1)licksdidnotdiffer.Asimilarpatternof
D
results was obtained in the analysis of the two-bottle intake o
data, except that the CS(cid:2) intakes of the 8% Glucose and 8% wn
MDGgroupsdidnotsignificantlydiffer(datanotshown).The 0 loa
g1pr0lao.y2ue2pd;ssPaimls(cid:7)oilad0ri.p0ffr0ee1rfe]e.dreTnihnceetsh8(e%7ir0%Gpe)lurtccheoanstteexCacnSed(cid:2)edMeldiDc(kGPs(cid:7)g[Fr0o(2.u0,p31s6))dthi(cid:6)sa-t B 2500 12% OMG T w o -1B2%ot MtleDG T e s t s 12% GLU ded from
of the 8% OMG group (56%). CS+ 84% h
* ttp
Experiment 2B 2000 CS- 71% ://a
* jp
Fig. 4A shows the total 1-h licks for CS(cid:1) test 0 and CS(cid:2) reg
h u
tests 1–3. Analysis of these data revealed that the 12% sugar 1 1500 .p
groups did not differ in their CS(cid:1) licks paired with IG water / hy
sl[igeclrkfo-iuinnpgfur(cid:4)esisoptnoesnt(steeinsinttetr0ha)ec,tCiaoSnnd(cid:2), Fate(ll6st,s9th6ar)lethe(cid:6)ougg7roh.6ut5po;sdPiifnfc(cid:7)erreean0st.e0dd0e1gt]rh.eeeAisr Licks 1000 55% siology.o
comparisonoftheCS(cid:2)tests1–3revealedthatoveralltheCS(cid:2) rg
licks of the 12%GlucosegroupexceededthatoftheMDGand 500 by/
OMGgroups,whichdidnotdifferfromoneanother[F(2,32)(cid:6) 1
6.74; P (cid:7) 0.001]. Within-group analyses indicated that the 0.2
12%GlucoseandMDGgroupslickedmoreinCS(cid:2)tests1–3 0 20
thaninCS(cid:1)test0,whereasthe12%OMGgrouplickedmore 12% OMG 12% MDG 12% GLU .33
onlyinCS(cid:2)test3comparedwithCS(cid:1)test0.The1-hintake Groups .5
data revealed a similar pattern of results [group (cid:4) test inter- on
aincctiroenas,eFd(6th,9e6ir)s(cid:6)ol4u.t8io;nPi(cid:7)nta0k.e0s1]f.roAmllCthSr(cid:1)eetgersotu0pstosiCgSni(cid:2)fictaensttly3 Fasariecgc.ph4lao.rtEitnexdpseforolirumtoeinonent2bpBoat:itrlVeedatleuswetssith0ar–eI3Ge.xTpwhreeastmesreidcienafsdurmsaineokanns(s1i(cid:9)nh/dSteaEsy.t)Aa0:C1b-Seh(cid:1)foto-rfletaalvbloeicirnekdgs June
(12% glucose: 2.1 to 3.5 g/h; 12% MDG: 2.1 to 2.8 g/h; 12% switched to a CS(cid:2)-flavored saccharin solution paired with IG sugar self- 12
OMG: 1.9 to 2.4 g/h). infusionsintests1–3.The12%GLU,12%MDG,and12%OMGgroupswere , 2
infused with 12% glucose, MDG, and OMG, respectively. B: 1-h licks are 0
In the alternating training sessions, the groups differed in 1
plottedforCS(cid:2)-andCS(cid:1)-flavoredsaccharinsolutionsduringthetwo-bottle 7
theirCS(cid:2)andCS(cid:1)licks[group(cid:4)CSinteraction,F(2,32)(cid:6) preferencetestforthe12%GLU,MDG,andOMGgroups.CS(cid:2)andCS(cid:1)
7.9;P(cid:7)0.01].Onlythe12%GlucoseandMDGgroupslicked intakes were not paired with IG infusions during the test. Number atop bar
more (P (cid:7) 0.05) in the CS(cid:2) than CS(cid:1) sessions (1,665.0 vs. representsmeanpercentpreferencefortheCS(cid:2)solution.*Significantdiffer-
1,252.1, 1,368.8 vs. 1,230.3, respectively); CS(cid:2) and CS(cid:1) ences(P(cid:7)0.05)betweentest0vs.tests1–3licksandbetweenCS(cid:2)vs.CS(cid:1)
licks.
licksweresimilarforthe12%OMGgroup(1,037.6vs.992.4).
Fig. 4B presents the lick data for the two-bottle CS(cid:2) vs.
CS(cid:1)choicetest.Analysisofthesedatarevealedagroup(cid:4)CS was greater in the 12% glucose group (84%) than the MDG
interaction[F(2,32)(cid:6)8.6;P(cid:7)0.001].Inparticular,the12% group(71%),which,inturn,wasgreaterthanthatoftheOMG
Glucose and MDG groups licked significantly more (P (cid:7) group (56%).
0.001)fortheCS(cid:2)thanCS(cid:1),whiletheCS(cid:2)andCS(cid:1)licks Blood glucose measures. Fig. 5 presents the blood glucose
of the 12% OMG group did not differ. Furthermore, the (BG) data following IG infusions of water, glucose, MDG, or
number of CS(cid:2) licks was greater in the 12% Glucose and OMGinthedifferentgroups.AnalysisoftheabsoluteBGdata
MDG groups than in the 12% OMG group (P (cid:7) 0.01), while revealed that overall, the three 8% sugar groups differed in
the CS(cid:1) licks did not differ. Similar results were obtained in theirBGresponse[F(3,47)(cid:6)30.3,P(cid:7)0.001],andtheeffect
the analysis of CS(cid:2) and CS(cid:1) intakes during the two-bottle varied as a function of time [group (cid:4) time interaction,
tests(datanotshown).Thegroupsalsodifferedintheirpercent F(9,141) (cid:6) 66.4; P (cid:7) 0.001]. The groups did not differ at 0
CS(cid:2)licks[F(2,32)(cid:6)9.6;P(cid:7)0.001]andtheCS(cid:2)preference min. At 15 min, they differed (P (cid:7) 0.05) as follows: 8%
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
R846 POST-ORALSUGARSENSINGANDAPPETITE
300 0.001)asfollows:12%OMG(cid:8)12%Glucose(cid:6)H2O(cid:8)12%
12% GLU MDG.AnalysisoftheIAUCalsorevealedthefollowinggroup
8% GLU differences:12%Glucose(cid:8)12%OMG(cid:8)H O(cid:6)12%MDG
dl) 250 182%% M MDDGG [F(3,43) (cid:6) 27.89; P (cid:7) 0.001]. 2
mg / 200 182%% O OMMGG DISCUSSION
( HH22OO
e MDG. This experiment reports for the first time that a
s
co 150 nonmetabolizable glucose analog can stimulate CS(cid:2) licking
u
Gl and condition a flavor preference; these responses occurred in
d theabsenceofchangesinbloodglucoselevels.The8%MDG
oo 100 infusionswerenearlyaseffectiveinstimulatingCS(cid:2)intakeas
Bl
8% glucose and conditioned an identical preference (70%).
50 The 8% infusions differed only in that MDG, unlike glucose,
didnotstimulateCS(cid:2)lickingintest1(butseeexperiment3).
In contrast, the 12% MDG infusions were less effective than D
0 0 15 30 60 12% glucose in stimulating intake in CS(cid:2) tests 1–3 and ow
Time(min) conditioned a weaker CS(cid:2) preference (71% vs. 84%). To nlo
a
determine whether a stronger preference could be obtained d
3000 with a more concentrated MDG infusion, another group of ed
mice (n (cid:6) 12) was tested as in this experiment but with IG fro
-1g * min * dl) 22050000 iit0wnnw.a0fcousr5-es)baiwoosthneettaeslek(oPCefcrSh(cid:6)1(cid:2)ot6hi%ca0teno.0Mt7ttehh5sDeo)t,GsCietn.hSToe(cid:1)1fh-1,het6sah%eCletShm8Mo(cid:2)i%ucDgelhGiaschnkthgodserwoii1rneu2dpp%treoepsnftrMesleyrfeD1eanr–Grc3meeda.g(rr(6IgoPn2iun%p(cid:7)tahs)le http://ajprem
m g
se ( 1500 (I7t0m–a7y1b%e)t,haastwatehlligahsetrhcaotnocfean1tr6a%tioGnsl,utchoeseacgcruomupul(a8t0io%n)o(f6t7h)e. u.ph
o y
c nonmetabolizable MDG in the body generates satiating or s
u io
Gl 1000 other inhibitory signals that counteract the appetition signals lo
d generated by this sugar analog. gy
Bloo 500 OMG. The 8% and 12% OMG infusions stimulated CS(cid:2) .org
licking about as much as did the MDG infusions but, unlike b/
MDG (and glucose), did not condition significant CS(cid:2) pref- y 1
0 erences.ThedifferentialeffectivenessofOMGandMDGmay 0
H2O OMG MDG GLU OMG MDG GLU .2
beduetothefactthatOMG,unlikeMDG,isnotasubstratefor 2
0
-------- 8% -------- -------- 12% -------- SGLT3 (3). OMG results are consistent with the findings that .3
Groups galactose, which also only binds to SGLT1, conditioned 3.5
Fig.5.Experiment2:Valuesareexpressedasmeans(cid:9)SE.A:bloodglucose weakerCS(cid:2)preferencesthandidglucose.However,itisalso o
n
at0,15,30,and60minaftera0.6-mlinfusionofglucose,MDG,orOMGin possible that, like galactose, OMG may have postabsorptive J
8%and12%groupsorwaterintheH2Ogroup.B:incrementalareaunderthe actionsthatinterferewithCS(cid:2)conditioning.Inparticular,the un
curve(IAUC)forbloodglucoseaftera0.6-mlinfusionofglucose,MDG,or e
IGOMGinfusionproducedasmallbutsustainedincreasedBG 1
OMGin8%and12%groupsorwaterintheH2Ogroup. 2
level.Himsworth(23)previouslyreportedasustainedincrease , 2
in BG following an intravenous infusion of OMG, which he 0
1
Glucose(cid:8)8%OMG(cid:8)H O(cid:6)8%MDG.At30and60min, attributedtoaglucoprivationresponseduetoOMGcompeting 7
they differed (P (cid:7) 0.05) a2s follows: 8% OMG (cid:8) H O (cid:6) 8% with glucose for cellular uptake. Thus, OMG-induced gluco-
Glucose (cid:6) 8% MDG. Analysis of the IAUC data rev2ealed the privation in the B6 mice may have prevented CS(cid:2) flavor
followinggroupdifferences:8%OMG(cid:6)8%Glucose(cid:8)H O(cid:6) preference conditioning and perhaps place preference condi-
8%MDG[F(3,47)(cid:6)26.49;P(cid:7)0.001](Fig.5). 2 tioning, as previously reported (30).
AnalysisoftheabsoluteBGdatarevealedthatoverall,thethree
Experiment 3: Phlorizdin and Phloretin Effects on Glucose
12% sugar groups differed in their BG response [F(3,43) (cid:6)
and MDG Appetition
53.76; P (cid:7) 0.001], and the effect varied as a function of time
point [group (cid:4) time interaction, F(9,129) (cid:6) 50.23; P (cid:7) The findings of the first two experiments that ligands of
0.001]. The groups differed (P (cid:7) 0.001) at the 0-min time SGLT1and/orSGLT3(glucose,galactose,MDG,OMG)stim-
point in that the starting value in the 12% MDG group was ulate CS(cid:2) intake and, except for OMG, condition CS(cid:2) pref-
lower than that of the other groups. At the 15-min point, the erencessuggestthattheSGLTsensorsmediatepost-oralsugar
groupsdiffered(P(cid:7)0.001)inthatthe12%Glucosedisplayed appetition.Ifthisisthecase,thenIGsugarconditioningshould
higher BG levels than the other three groups, which did not be blocked by the SGLT1/SGLT3 antagonist, phloridzin (17,
differfromoneanother.Atthe30-mintimepoint,theydiffered 33). Experiment 3A tested this possibility by determining the
(P (cid:7) 0.05) as follows: 12% OMG (cid:6) 12% Glucose (cid:8) H O (cid:8) effects of adding phloridzin to 8% glucose and 8% MDG
2
12% MDG. At the 60-min time point, they differed (P (cid:7) infusions on stimulation of CS(cid:2) licking and preference con-
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
POST-ORALSUGARSENSINGANDAPPETITE R847
ditioning. Galactose and OMG were not included in this A One-Bottle Tests
experiment because of their weaker conditioning effects.
Experiment 3B determined whether combining the GLUT2
CS+3
2000
inhibitor phloretin with phloridzin would more completely
CS+2
block the flavor-conditioning actions of IG glucose self-infu-
*
sions. This manipulation was based on a recent report that CS+1
*
phloridzin alone only partially blocked glucose stimulation of 1500 * * *
CS-0
ginutteshtionramloinnfeussi[oGnLpPr-e1p,arGatIiPo,n,pwephteidreeaYsaYph(PloYreYti)n](cid:2)inphalnoraidcuzitne h * * * * *
1
cocktail was fully effective (29). s / 1000
k
MATERIALS AND METHODS Lic
Experiment3A
500
ThreegroupsofadultmaleB6micewerehousedandtestedasin
the prior experiments: an 8% Glucose(cid:2)0.4% phloridzin (GLU(cid:2)Pz, D
o
n(cid:6)12),an8%MDG(cid:2)0.4%phloridzingroup(MDG(cid:2)Pz;n(cid:6)12), w
and an 8% MDG group (8% MDG, n (cid:6) 9). The 0.4% (8.4 mM) 0 nlo
phloridzin (Sigma) concentration was based on the report of Savas- MDG+Pz MDG GLU+Pz GLU a
d
tano et al. (46), which investigated the drug’s effect on the feeding B ed
icnohnicbeintitornatipornodwuacsednebayrtdhueodliemniatlomfasoltloudbeixlittryinofinpfhulsoiorindsziinnirnattsh.eT8h%is 2500 Two-Bottle Tests from
sugar solutions. The data from the 8% GLU(cid:2)Pz group were com- CS+ h
paredwiththe8%Glucosegroupdatafromexperiment1A.Anew8% ttp
MDG group was included in this experiment to replicate the novel 2000 CS- 68% 70% ://a
fistnimdiunlgateodf eCxSp(cid:2)eriminetnatke2 athnadt cthoendnitoinomneedtabaolpizraebfelerengclue.coFseollaonwailnogg 72% * * jpre
* g
behavioral testing, the effect of phloridzin on the blood glucose u
h 1500 .p
rabEenexsacpplaeoyurnssiismesee8wtn%oatsI3MGBpeD8r%fGorgdmliudecdnooosetnaitnlhtfeeurs8ibo%lnooMwdaDgsGlmu(cid:2)ceoaPsszeuraienndde.xN8p%oerbiMmloDeondGtg2gl.urocuopses Licks / 1 1000 57% hysiology.o
testTewdoagsroinuptshe(np(cid:6)rio1r0eexapcehr)imofenatdsu.ltOmnealegrBo6upm(icGeLwUe(cid:2)rePhzo(cid:2)uPset)dwanads 500 byrg/
tested with 8% glucose self-infusions containing 0.4% (8.4 mM) 10
phloridzinand0.23%(8.4mM)phloretin.Isomolardrugdoseswere .2
2
usedbasedonapriorstudy(29).[Thisstudyusedamuchlowerdose 0
(0.5 mM), but the inhibitors were added to a more dilute glucose 0 MDG+Pz MDG GLU+Pz GLU .33
solution(1.8%)thanthatusedhere.]Becauseofthelimitedsolubility .5
of phloretin, the glucose(cid:2)phloridzin(cid:2)phloretin mixture contained Groups on
3.25% 1 N NaOH to dissolve the drugs (38). A second group Fig.6.Experiment3A:Valuesareexpressedasmeans(cid:9)SE.A:1-htotallicks Ju
(GLU(cid:2)Pz) was tested with 8% glucose self-infusions containing n
areplottedforonebottletests0–3.Themicedrank(1h/day)aCS-flavored e
0.4% phloridzin and 3.25% NaOH. This group was included to saccharin solution paired with IG water infusions in test 0 before being 1
dreestperomnsienetow8h%ethgelructohsee(cid:2)adpdhitlioornidzoifnNinafOusHionasl.teTrehdetphHe ocofngdliutcioonsien(cid:2)g sTwhietcMheDdGto(cid:2)aphClSo(cid:2)rid-zflianvo(Prezd),sMacDchGa,riGnLsoUl(cid:2)utiPozn,paanidreGdLwUithgrIoGupsusgwarerienfiunsfiuosnesd. 2, 20
phloretin(cid:2)phloridzin and glucose(cid:2)phloridzin solutions were 10.1 with8%MDG,8%MDG(cid:2)0.4%Pz,8%glucose,and8%glucose(cid:2)0.4%Pz, 17
and 10.8, respectively. Following behavioral testing, the effects of respectively. B: 1-h licks are plotted for CS(cid:2) and CS(cid:1)-flavored saccharin
phloridzin and phloretin on the blood glucose response to IG 8% solutionsduringthetwo-bottlepreferencetestfortheMDG,MDG(cid:2)Pz,GLU,
glucose infusion were measured as in experiment 2 in the GLU(cid:2)Pz and GLU(cid:2)Pz groups. CS(cid:2) and CS(cid:1) intakes were not paired with IG
and GLU(cid:2)Pt(cid:2)Pz groups. Two days after the BG test, a second BG ifnofrutshioenCsSd(cid:2)urisnogluthtieonte.s*t.SNigunmifibcearnattdoipffbearernrceepsre(sPen(cid:7)tsm0.e0a5n)pbeertwceenetnprteesfetr0envcse.
test was conducted in some mice (5 GLU(cid:2)Pz and 4 GLU(cid:2)Pz(cid:2)Pt) tests1–3licksandbetweenCS(cid:2)vs.CS(cid:1)licks.
duringwhichtheywereinfusedintragastricallywithwatercontaining
3.25%of1NNaOH.
test1butthatthe8%MDGgrouplickedmoreinCS(cid:2)tests2
RESULTS and 3 than did the MDG(cid:2)Pz group. A within-group analysis
revealed that the 8% MDG group licked more in CS(cid:2) tests 1
Experiment 3A
and2thaninCS(cid:1)test0,andmoreinCS(cid:2)test3thanintests
Analysis of 8% MDG and MDG(cid:2)Pz groups revealed that 0–2.The1-hsolutionintakedatarevealedasimilarpatternof
thetwogroupsdidnotdifferintheirCS(cid:1)lickspairedwithIG results [group (cid:4) test interaction, F(3,57) (cid:6) 8.4, P (cid:7) 0.001].
water self-infusion (test 0) but only the 8% MDG group The 8% MDG Group increased (P (cid:7) 0.01) its intakes from
increased licks in CS(cid:2) tests 1–3 [group (cid:4) test interaction, CS(cid:1) test 0 to CS(cid:2) test 3 (2.1 to 3.6 g/h), whereas the 8%
F(3,57)(cid:6)7.0,P(cid:7)0.001](Fig.6A).AcomparisonoftheCS(cid:2) MDG(cid:2)Pzgroupintakesdidnotsignificantlychangefromtest
tests 1–3 licks indicated that the groups did not differ in CS(cid:2) 0 to test 3 (2.0 vs. 2.2 g/h).
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
R848 POST-ORALSUGARSENSINGANDAPPETITE
In the alternating training sessions, the 8% MDG group 300
lickedmoreintheCS(cid:2)thanintheCS(cid:1)sessions(1,586.8vs.
8% GLU
1,107.4),whereastheCS(cid:2)andCS(cid:1)licksdidnotdifferinthe
8%MDG(cid:2)Pzgroup[975.2vs.870.7;group(cid:4)CSinteraction, 250 8H%2O GLU+Pz
F(1,19)(cid:6)22.5,P(cid:7)0.01].Inaddition,theMDGgrouplicked dl)
more overall than did the MDG(cid:2)Pz group [F(1,19) (cid:6) 15.6; g / 200
P (cid:7) 0.001]. (m
e
In the two-bottle choice test, the 8% MDG group licked s
significantlymore(P(cid:7)0.001)fortheCS(cid:2)thanCS(cid:1),whilethe co 150
u
CS licks of the 8% MDG(cid:2)Pz group did not differ [F(1,19) (cid:6) Gl
11.1, P (cid:7) 0.01, Fig. 6B]. Furthermore, the number of CS(cid:2) d 100
o
o
licks was greater in the 8% MDG group than in the 8% Bl
MDG(cid:2)Pz group (P (cid:7) 0.001), while CS(cid:1) licks did not
50
differ. Similar results were obtained in the analysis of CS(cid:2)
and CS(cid:1) intakes during the two-bottle tests (data not D
shown). The CS(cid:2) preference was also greater in the 8% 0 0 15 30 60 ow
MDG than in the 8% MDG(cid:2)Pz group [72% vs. 57%, t(19) (cid:6) nlo
3.1, P (cid:7) 0.01]. Time (min) ad
Analysis of the 1-h lick data for the GLU(cid:2)Pz and GLU 2500 ed
groupsindicatedthatoverall,thegroupsdidnotdifferintheir fro
C0StoliCckSs(cid:2), atnedstbso1th–3gr[oFu(p3s,6i3n)cr(cid:6)eas2e9d.2t1h;eiPr l(cid:7)ick0s.0fr0o1m] (CFSig(cid:1). 6tAes)t. -1* dl) 2000 httpm
pdTw(CPeuhSirrte(cid:1)ih(cid:7)ionidnggl0-riitsoc.nhe0kutes1esps)lrsiaaiondscndtutiti2roefCi0fsnneSt,gmr(cid:2)Fe0itd.(nh2,leTic,c4hhofik2aomrs)twspt(cid:6)iieans2vr,e90ettde5rh,ms.ew6tsii;iGnntP1hLo–tt(cid:7)Uhf3he,e(cid:2)t0haG.Pdel0tiLz0hs1Ut1o-grh]uir.bgogtuTruheothpsiuotensplnoi1c[tb-gkouhirenftoidnumtttphhlaoeeek(cid:4)rsiieesrr ucose (mg * min 11050000 ://ajpregu.physio
data revealed that both groups significantly increased their Gl log
GtihnLetaUk8e%asnfdrGo2mL.0UCtoSg2(cid:1)r.o9utgeps/hte0fxoctreo8eCd%eSdG(cid:2)L(PtUes(cid:2)t(cid:7)P3z0()2,.0.a05lt)thoothu3ag.3thgoin/fhtatfhkoeers88f%o%r Blood 500 by.org/
y
GLU(cid:2)PzgroupinCS(cid:2)tests1and2[group(cid:4)testinteraction, 1
0 0
F(3,63)(cid:6)2.8,P(cid:6)0.047]. H2O GLU+Pz GLU .2
2
In the alternating training sessions, the groups differed in Groups 0.3
theirCS(cid:2)andCS(cid:1)licks[group(cid:4)CSinteraction,F(1,21)(cid:6) 3
13.2;P(cid:6)0.01].Whereasthe8%GLUgrouplickedmore(P(cid:7) aFtig0.,7.1E5,xp3e0r,imaenndt36A0:mVianluaefstearreaex0p.6r-emssledinafsusmioenanosf(cid:9)8%SE.gAlu:cbolsoeo,d8g%lucgoluse- .5 o
0.001)inCS(cid:2)thanCS(cid:1)sessions(1,467.0vs.1,152.1),theCS cose(cid:2)0.4% phloridzin, or water in the GLU, GLU(cid:2)Pz, and H2O groups. n J
licks of the 8% GLU(cid:2)Pz group did not differ (1,349.9 vs. B:bloodglucoseIAUCintheGLU,GLU(cid:2)Pz,andH2Ogroups. un
e
1,318.5). 1
moTrhee(PG(cid:7)LU0(cid:2).0P01z)mfoicret,hleikCeSt(cid:2)hethGaLnUCSmiincet,hleictwkeod-bsoigttnlieficchaonitclye Experiment 3B 2, 20
1
tests [F(1,21) (cid:6) 22.85; P (cid:7) 0.001], and there were no group 7
Analysisofthe1-hlickdataindicatedthattheGLU(cid:2)Pzand
differences (Fig. 6B). The GLU(cid:2)Pz and GLU groups also
GLU(cid:2)Pz(cid:2)PtgroupsdidnotdifferintheirCS(cid:1)licksintest0,
displayed comparable CS(cid:2) preferences (68% and 70%, re-
but only the GLU(cid:2)Pz group increased its licking response in
spectively). Similar results were obtained in the analysis of the CS(cid:2) tests [group (cid:4) test interaction, F(3,54) (cid:6) 10.6, P (cid:7)
CS(cid:2) and CS(cid:1) intakes during the two-bottle tests (data not
0.001] (Fig. 8A). Within-group analyses indicated that the
shown). GLU(cid:2)Pz group licked more in test 1 than in test 0 and more
Analysis of the BG data for the GLU, GLU(cid:2)Pz, and water intests2and3thanintest1[F(3,27)(cid:6)22.5;P(cid:7)0.001].The
groupsindicatedagroup(cid:4)concentrationinteraction[F(6,96)(cid:6) 1-h intake data revealed a similar pattern of results [group (cid:4)
58.40, P (cid:7) 0.001] (Fig. 7). Individual comparisons revealed test interaction, F(3,54) (cid:6) 7.47; P (cid:7) 0.001]: the GLU(cid:2)Pz
higher(P(cid:7)0.05)BGlevelsintheGLUgroupcomparedwith miceincreasedtheirintakefromCS(cid:1)test0toCS(cid:2)test3(2.1
the GLU(cid:2)Pz and water groups at the 15-min time point, to 2.6 g/h), while intake did not significantly change in the
whereas the GLU(cid:2)Pz had a higher blood glucose than the GLU(cid:2)Pz(cid:2)Pt (2.1 to 1.9 g/h).
GLU group at the 30-min time point. The IAUC analysis In the alternating training sessions, the GLU(cid:2)Pz group
indicatedthatthe8%GLUgrouphadagreateroverallincrease licked more overall than the GLU(cid:2)Pz(cid:2)Pt group [F(1,18) (cid:6)
in BG than the 8% GLU(cid:2)Pz group, which, in turn, had a 26.8;P(cid:7)0.001].TheGLU(cid:2)Pzgrouplickedsomewhatmore
greater increase than the Water group [F(2,32) (cid:6) 13.90; P (cid:7) in the CS(cid:2) than CS(cid:1) sessions (1,226.4 vs. 1,172.0), whereas
0.001] (Fig. 7). theGLU(cid:2)Pz(cid:2)Ptgroupshowedtheoppositepattern(863.9vs.
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org
POST-ORALSUGARSENSINGANDAPPETITE R849
A One-Bottle Tests [group (cid:4) concentration interaction, F(6,75) (cid:6) 8.32; P (cid:7)
0.001] (Fig. 9). Individual comparisons revealed that groups
didnotdifferatthe15-mintimepoint,buttheydiffered(P(cid:7)
2000 CS+3 0.05) at the other time points as follows: time 0: Water (cid:8)
CS+2 GLU(cid:2)Pz(cid:8)GLU(cid:2)Pz(cid:2)PT;time30:GLU(cid:2)Pt(cid:6)GLU(cid:2)Pz(cid:2)
CS+1 Pt(cid:8)Water;time60:Water(cid:8)GLU(cid:2)Pz(cid:6)GLU(cid:2)Pz(cid:2)Pt.The
11550000 CS-0 ** IAUC analysis indicated that the GLU(cid:2)Pz(cid:2)Pt group had a
h * * greater overall increase in BG than the GLU(cid:2)Pz and Water
1 groups [F(2,25) (cid:6) 14.46, P (cid:7) 0.001].
s / 1000
k
c DISCUSSION
Li
The phloridzin findings of experiment 3A provide partial
500 support for the involvement of SGLT1/SGLT3 in preference
conditioning. In particular, phloridzin completely blocked
MDG-induced stimulation of CS(cid:2) licking and preference D
o
0 conditioning. Yet, the same phloridzin dose had a relatively w
GLU+Pz+Pt GLU+Pz minor effect on glucose conditioning. Glucose(cid:2)phloridzin nlo
B Two-Bottle Tests stimulated licking nearly as much as glucose alone and pro- ad
2500 duced a comparable CS(cid:2) preference. Phloridzin, however, ed
CS+ fro
m
CS- 300 h
2000 ttp
s / 1 h 1500 6633*%% mg / dl) 222050000 88H%%2O GGLLUU ++ PPzz +Pt ://ajpregu.ph
Lick 1000 50% ose ( 150 ysiolo
c g
u y
500 Gl .o
d 100 rg
oo b/
0 Bl y 1
GLU+Pz+Pt GLU+Pz 50 0.2
Groups 20
Fig.8.Experiment3B:Valuesareexpressedasmeans(cid:9)SE.A:1-htotallicks 0 .33
areplottedforonebottletests0–3.Themicedrank(1h/day)aCS(cid:1)-flavored 0 15 30 60 .5
saccharin solution paired with IG water infusions in test 0 before being Time (min) on
switchedtoaCS(cid:2)-flavoredsaccharinsolutionpairedwithIGsugarinfusions. J
The GLU(cid:2)Pz(cid:2)phloretin (Pt) and GLU(cid:2)Pz groups were infused with 8% 2500 un
gNsalaucOcchoHas,eri(cid:2)nre0ss.op4le%ucttiiPovnze(cid:2)slyd0.u.2rBi3n:%g1t-Phhtealtiwncdkos-8b%aortetglleuppcloroetstfeee(cid:2)dre0nf.oc4er%tCePsStz(cid:2)wc-oitnhatnathdineiCnGgSL(cid:1)3U.-2(cid:2)fl5aP%vzo(cid:2)1rePNdt -1dl) 2000 e 12, 2
ainnfdusiGoLnsUd(cid:2)uPriznggtrhoeuptess.t.CNSu(cid:2)mbaenrdatoCpSb(cid:1)arirnetparkeessenwtsemreeannopterpcaeinretdprwefietrhenIcGe min * 017
fteosrtsth1e–C3Sli(cid:2)ckssoalnudtiobne.tw*SeeignnCifiSc(cid:2)antvds.ifCfeSre(cid:1)ncleicsk(sP. (cid:7)0.05)betweentest0vs. g * 1500
m
e (
s
909.2)butthegroup(cid:4)CSinteractionwasnotsignificant[P(cid:6) o 1000
c
u
0.095]. Gl
The two-bottle test data revealed significant group differ- d
ences(Fig.8B):theGLU(cid:2)Pzmicelickedmore(P(cid:7)0.01)for oo 550000
the CS(cid:2) than CS(cid:1), whereas the GLU(cid:2)Pz(cid:2)Pt mice did not Bl
differ in their CS licks [group (cid:4) CS interaction, F(1,18) (cid:6)
26.4,P(cid:7)0.001].Similarresultswereobtainedintheanalysis 0
H2O GLU+Pz GLU+Pz+Pt
ofthetwo-bottleCS(cid:2)andCS(cid:1)intakedata(datanotshown).
Groups
The CS(cid:2) preference was also greater in the GLU(cid:2)Pz group
than in the GLU(cid:2)Pz(cid:2)Pt [63% vs. 50%, t(18) (cid:6) 4.9; P (cid:7) Fig.9.Experiment3B:Valuesareexpressedasmeans(cid:9)SE.A:bloodglucose
at0,15,30,and60minaftera0.6-mlinfusionof8%glucose(cid:2)0.4%Pz,8%
0.001].
glucose(cid:2)0.4%Pz(cid:2)0.23%Pt,andwaterintheGLU(cid:2)Pz,GLU(cid:2)Pz(cid:2)Pt,and
Analysis of the BG data for the GLU(cid:2)Pz, GLU(cid:2)Pz(cid:2)Pt,
HOgroups,respectively.Allinfusionscontained3.25%of1NNaOH.B:blood
2
and Water groups indicated small but significant differences glucoseIAUCintheGLU(cid:2)Pz,GLU(cid:2)Pz(cid:2)Pt,andHOgroups.
2
AJP-RegulIntegrCompPhysiol•doi:10.1152/ajpregu.00297.2013•www.ajpregu.org