Role of adipose and hepatic atypical protein kinase C lambda (PKC?) in the development of obesity and glucose intolerance.

2012·1.9 MB·English

by  Habegger, Kirk M.

RESEARCHPAPER
Adipocyte1:4,203–214;October/November/December2012;G2012LandesBioscience
Role of adipose and hepatic atypical protein
l
kinase C lambda (PKC ) in the development
of obesity and glucose intolerance
Kirk M. Habegger,1 Daniela Matzke,2 Nickki Ottaway,1 Jazzminn Hembree,1 Jenna Holland,1 Christine Raver,1
JohannesMansfeld,3TimoD.Müller,1,4DiegoPerez-Tilve,1PaulT.Pfluger,1,4SangJunLee,5MariaDiaz-Meco,5JorgeMoscat,5
Michael Leitges,6 Matthias H. Tschöp1,4 and Susanna M. Hofmann7,*
1DivisionofEndocrinology;DepartmentofInternalMedicine;ObesityResearchCenter;MetabolicDiseasesInstitute;UniversityofCincinnati;Cincinnati,OHUSA;2Departmentof
ExperimentalDiabetology;GermanInstituteofHumanNutrition;Potsdam-Rehbrücke,Germany;3DepartmentofHumanNutrition;Friedrich-Schiller-UniversityofJena;Jena,
Germany;4InstituteforDiabetesandObesity;HelmholtzZentrumMünchen;GermanResearchCenterforEnvironmentalHealth;München/Neuherberg,Germany;5Sanford-
BurnhamMedicalResearchInstitute;LaJolla,CAUSA;6TheBiotechnologyCentreofOslo;UniversityofOslo;Oslo,Norway;7InstituteforExperimentalGenetics;Helmholtz
ZentrumMünchen;GermanResearchCenterforEnvironmentalHealth;München/Neuherberg,Germany
Keywords: atypical protein kinase C-lambda, diet induced obesity, energy expenditure, insulin resistance, glucose tolerance
Abbreviations: PKCl, protein kinase C-lambda; DIO, diet induced obesity; HFD, high fat diet; ip, intraperitoneal; GTT, glucose
tolerance test; ITT, insulin tolerance test; PTT, pyruvate tolerance test, SREBP-1c, sterol regulatory element-binding protein-1c;
FAS, fatty acid synthase; NFkB, nuclear factor kappa B
PKCl,anatypicalmemberofthemultifunctionalproteinkinaseCfamily,hasbeenimplicatedintheregulationofinsulin-
stimulated glucose transport and of the intracellular immune response. To further elucidate the role of this cellular
regulatorindiet-inducedobesityandinsulinresistance,wegeneratedbothliver(PKC-Alb)andadiposetissue(PKC-Ap2)
specificknockoutmice.Bodyweight,fatmass,foodintake,glucosehomeostasisandenergyexpenditurewereevaluated
inmicemaintainedoneitherchoworhighfatdiet(HFD).AblationofPKClfromtheadiposetissueresultedinmicethat
were indistinguishable from their wild-type littermates. However, PKC-Alb mice were resistant to diet-induced obesity
(DIO).SurprisinglythisDIOresistancewasnotassociatedwitheitherareductionincaloricintakeoranincreaseinenergy
expenditureascomparedwiththeirwild-typelittermates.Furthermore,thesemicedisplayedanimprovementinglucose
tolerance.Whenmaintainedonchowdiet,thesemiceweresimilartowildtypesinrespecttobodyweightandfatmass,
yetinsulinsensitivitywasimpairedcomparedwithwtlittermates.TakentogetherthesedatasuggestthathepaticPKClis
modulating insulin-mediated glucose turnover and response to high fat diet feeding, thus offering a deeper
understanding ofan important target foranti-obesity therapeutics.
Introduction insulin effects on glucose transport in muscle and adipose tissue
and lipid synthesis in liver.3-5
Dissecting molecular signaling pathways involved in the patho- PKCl, together with PKCf, are the two highly homologous
genesis of the metabolic syndrome through the regulation of members of the aPKC subfamily. Both are serine/threonine
nutrient and energy homeostasis is of pivotal importance for the kinases activated by phosphorylation and phosphatidylserine, but
generation of novel strategies to prevent and treat obesity, and are insensitive to either calcium (Ca2+) or diacylglycerol (DAG)
associated diseases such as glucose intolerance, type 2 diabetes, (reviewedinref.6).Inmetabolicsignaling,insulinactivatesaPKC
cancerandpulmonarydiseases.Severalstudiesoverthelastdecade throughphosphatidylinositol-3 kinase(PI3K)tofacilitate glucose
haveestablishedacloseinteractionbetweenimpairmentofinsulin transporter4(GLUT4)translocationtotheplasmamembranein
signaling and dysfunction of cellular pathways in inflammation skeletal muscle and fat.7 Consistent with this regulatory role,
and innate immunity.1 This convergence of metabolic and ablation of PKCl in muscle impairs glucose uptake and this
inflammatory signals is exemplified by the role of the signaling tissue-specificlimitationissufficienttoinduceabdominalobesity,
molecule protein kinase l (PKCl), member of the atypical hyperlipidemiaandhepatosteatosis,allhallmarksofthemetabolic
subfamily of PKC isoforms (aPKC), as a critical regulator of the syndrome.8 Hepatic deletion of PKCl paradoxically enhanced
immune response2 and at the same time as a crucial mediator of insulin-mediated glucose uptake in muscle and fat, but also
*Correspondenceto:SusannaM.Hofmann;Email:[email protected]
Submitted:03/26/12;Revised:05/24/12;Accepted:05/25/12
http://dx.doi.org/10.4161/adip.20891
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Figure1.Bodyweight(AandB),fatmass(CandD)andleanmass(EandF)growthcurvesofchow-orHFD-fedPKClfl/flCreAlb(closedcircles)and
PKClwt/wtmice(opencircles).Cumulativefoodintakewasmonitoredthroughoutthestudy(GandH).Alldataarerepresentedasmean±SEM
in6–7age-matched,malemice.*p,0.05,**p,0.01,***p,0.001,asdeterminedby2-wayANOVA.
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accomplished by breeding mice expressing the Cre recombinase
transgene controlledby the albumin (Alb) or the adipocyte lipid-
bindingprotein(aP2)promotor/enhancerwithmicecarryingtwo
targeted PKC-l alleles with loxP sites (PKClfl/fl) flanking an
essential exon of the PKCl gene as previously described.2
Hepatic-specific PKCl deletion. Body weight regulation and
energy expenditure. Mice deficient for hepatic PKCl (PKClfl/fl
CreAlb) were born in normal Mendelian ratios and had similar
bodyweightatweaningascomparedwiththeirwild-typesiblings
(PKClwt/wt; Fig.1A and B). When maintained on chow diet, we
did not detect differences in body weight and composition
throughout the initial 23 weeks of study (Fig.1A, C and E).
However, when fed a high fat diet (HFD), PKClfl/fl CreAlb mice
displayedresistancetodietinducedobesity(DIO; Fig.1BandD)
as compared with their PKClwt/wt littermates. Lean mass was not
different between the groups (Fig.1E and F). Regardless of diet,
mice from both genotypes displayed similar food intake
throughout the initial 23 weeks of study (Fig.1G and H).
Prolonged exposure (37 weeks) of these mice to either chow or
HFD resulted in a reciprocal effect on body weight. Specifically,
chow-fed PKClfl/fl CreAlb mice displayed increased body weight
(Fig.2A) as compared with their PKClwt/wt littermates.
Consistent with their earlier DIO resistance (see Fig.1B and
Figure2.Bodyweightof40-week-old,chow-(A)orHFD-fed(B)PKClfl/fl D), HFD-fed PKClfl/fl CreAlb mice displayed decreased body
CreAlb(closedbars)andPKClwt/wtmice(openbars).Alldataare weight as compared with their PKClwt/wt littermates (Fig.2B).
representedasmean±SEMin6–7age-matched,malemice.*p,0.05 The resistance to DIO in PKClfl/fl CreAlb mice, accompanied
asdeterminedbyStudent’st-test.
bynormalfoodintake(Fig.1H),suggestedthatthesemicemight
exhibit increased energy expenditure as compared with their
prevented insulin-stimulated lipogenesis in liver,9,10 resulting in PKClwt/wtlittermates.Totestthishypothesis,energyexpenditure
decreased triglyceride content and expression of the sterol wasmonitoredin23-week-oldmiceonchoworHFDviaindirect
regulatory element-binding protein-1c (SREBP-1c) gene. In calorimetery over the course of one week. However, energy
inflammatory signaling, aPKCs activate the NFkB pathway expenditure was not different from PKClwt/wt littermates in
through phosphorylation of inhibitory-kBa (IkBa) and the p65 PKClfl/fl CreAlb mice fed either chow (Fig.3A and B) or HFD
subunit of NFkB.11,12 In addition, aPKCs regulate receptor- (Fig. 4A and B). Likewise, locomotor activity and respiratory
interacting protein and TNF receptor-associated factor 6 by quotient were unaffected by genotype, regardless of diet (Figs.3
bindingtothescaffoldingproteinp62,thusmodulatingToll-like and 4C–F).
receptor 4 (TLR4) and TNF-a receptor signaling cascades.13,14 Glucose metabolism. PKClfl/fl CreAlb mice on chow exhibited
aPKCactivationisfurtherrequiredfortheLPS-inducedsignaling lower fasting blood glucose levels compared with their wt
through TLR4 in macrophages15,16 and for T-cell helper (Th2) littermates (Fig.5A) with a similar trend observed in HFD-fed
activation, a pathway relevant in inflammatory disorders.2 mice(Fig.5B).HFDfeedingincreasedfastinginsulinlevelsabout
Since aPKCs are uniquely positioned to mediate convergent 4-fold (from an average of 0.47 to 1.93 ng/ml) in wt mice
intracellular immune and metabolic responses we sought to compared with 1.9-fold (from an average of 0.49 to 0.95 ng/ml)
investigate whether PKCl may also regulate cellular signaling in PKClfl/fl CreAlb mice (Fig.5C and D), suggesting that that
cascadesrelevantforenergyhomeostasisininsulin-sensitivetissues hepatic ablation of PKCl results in enhanced insulin sensitivity.
suchasliverandfatunderhighfatdiet(HFD)feedingconditions. This hypothesis was supported by calculating the HOMA-Index,
HereinwedemonstratethatmicedeficientforhepaticPKCl,are a widely used clinical surrogate marker for insulin sensitivity: We
resistant to diet-induced obesity and diet-induced glucose detectedatrendtowardimprovedHOMA-Indexvaluesinchow-
intolerance without increases in energy intake or expenditure. fed PKClfl/fl CreAlb mice (p = 0.0556; Fig.5E) and significantly
In contrast, we show that adipose-specific PKCl does not play a improvedHOMA-IndexvaluesinHFD-fedPKClfl/flCreAlbmice
role in diet-induced obesity and associated insulin resistance. (Fig.5F), as compared with their respective wild-type controls.
The regulation of glucose homeostasis by hepatic PKCl was
Results further examined by performing intraperitoneal glucose tolerance
test (ipGTT) and insulin tolerance test (ipITT). Although blood
To test the hypothesis that hepatic or adipose PKCl modulates glucose levels were similar during GTT (Fig.6A, inset), the
signalingpathwaysofenergy homeostasis,wecreatedhepaticand glucose lowering effect of exogenously administered insulin was
adipose tissue-specific knockouts for the PKCl gene. This was reducedinPKClfl/flCreAlbmiceonchowcomparedwithwtmice
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Figure3.Dynamicandaverageenergyexpenditure(AandB),respiratoryquotient(CandD)andlocomotoractivity(EandF)inchow-fedPKClfl/flCreAlb
(closedcircles/bars)andPKClwt/wt(opencircles/bars)miceasmeasuredbyindirectcalorimetry.Alldataarerepresentedasmean±SEMin6–7age-
matched,malemice.Nodifferencesweredeterminedby2-wayANOVA.
(Fig.6C and E), indicating that contrary to our hypothesis, We next conducted pyruvate tolerance tests (ipPTT) to
insulin-mediated glucose transport was impaired. However, elucidate the contribution of hepatic glucose output to the
PKClfl/fl CreAlb mice on HFD exhibited an improved response divergent ipGTT and fasting glucose levels observed in these
toipGTTcomparedwiththeirPKClwt/wtlittermates.Specifically, mice. Chow-fed PKClfl/fl CreAlb mice displayed similar glucose
theglucoseexcursion60minafterglucosechallenge(Fig.6B)and excursions (Fig.7A) and increase of glucose levels of ipPTT
the area under the curve for the duration of the test (Fig.6B, (Fig.7C) as compared with their PKClwt/wt littermates. Under
inset)werereducedinPKClfl/flCreAlbmice.AnalysisofipITTin HFD feeding conditions, relative glucose appearance revealed
HFD-fed PKClfl/fl CreAlb mice and their PKClwt/wt littermates similar glucose excursions (Fig.7B) but, an enhanced hepatic
revealed no difference in glucose clearance (Fig.6D and F), glucose output in PKClfl/fl CreAlb mice (Fig.7D) during the first
suggesting that hepatic glucose output may be different in the 30 min, suggesting a higher gluconeogenic potential in the
absenceofPKCl.Sincewealsoobservedsimilarglucoselowering absenceofPKCl. We concluded that theobserveddifferences in
effectsofexogenouslyadministeredinsulininchowandHFDfed fasting glucose levels can neither be explained by increased
PKClfl/flCreAlbmice(Fig.6CandD),weconcludedthatablation insulin-mediated glucose uptake nor by reduced hepatic gluco-
of hepatic PKCl makes mice resistant to the effect of HFD on neogenicpotential.Takentogetherourresultsdrawacomplicated
insulin-mediated glucose uptake. pictureofPKCl’svariousrolesinglucosehomeostasis,indicating
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Figure4.Dynamicandaverageenergyexpenditure(AandB),respiratoryquotient(CandD)andlocomotoractivity(EandF)inHFD-fedPKClfl/flCreAlb
(closedcircles/bars)andPKClwt/wt(opencircles/bars)miceasmeasuredbyindirectcalorimetry.Alldataarerepresentedasmean±SEMin6–7age-
matched,malemice.Nodifferencesweredeterminedby2-wayANOVA.
that ablation of hepatic PKCl protects against DIO-induced HFD-fedPKClfl/flCreAlbmice(Fig.8B).Theseeffectsonplasma
hyperglycemia, but at the same time it impairs insulin-mediated triglycerides were not associated with liver injury/dysfunction as
glucose uptake under lean, chow feeding conditions. determined by Alanine transaminase (Fig.8C). Together these
Lipidmetabolism.PriorreportsonthehepaticactionofPKCl findings suggest that in our model, ablation of hepatic PKCl
suggestthatthispathwayregulateshepaticlipogenesis.9,10Togain modulates triglyceride metabolism in chow fed animals, an effect
insight into lipid metabolism in our model, we measured that is not evident under high fat feeding conditions.
cholesterolandtriglyceridesattheterminationofthestudy(week Adipose-specific PKCl deletion. As in the hepatic specific
40). Plasma cholesterol levels were similar in PKClfl/fl CreAlb and ablation, mice deficient for adipose PKCl (PKClfl/fl CreaP2) were
their PKClwt/wt littermates, regardless of diet (Fig.8A). borninnormalMendelianratiosandhadsimilarbodyandfatmass
Furthermore,wefoundthatplasmatriglyceridesweresignificantly at weaning as compared with their wild-type siblings (PKClwt/wt;
elevatedinchow-fedPKClfl/flCreAlbmiceascomparedwiththeir Fig.9A and B). When maintained on chow diet, these mice
PKClwt/wt littermates (Fig.8B). However, while HFD exposure showed similar body composition compared with wt littermates
inPKClwt/wtmiceelevatedplasmatriglyceridesascomparedwith throughouttheinitial20weeksofstudy(Fig.9BandC).Atweek
chow-fed PKClwt/wt mice, we observed no further increase in 20,allmiceweremonitoredforenergyexpendituredifferencesand
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Figure5.Bloodglucoselevels(AandB)ofchow-andHFD-fedPKClfl/flCreAlb(closedbars)andPKClwt/wt(openbars)micefollowing6hfast.Plasma
insulin(CandD)inHFD-fedPKClfl/flCreAlb(closedbars)andPKClwt/wt(openbars)micefollowing6hfast.HOMA-INDEX(eandf)inHFD-fedPKClfl/flCreAlb
(closedbars)andPKClwt/wt(openbars)mice.Alldataarerepresentedasmean±SEMin6–7age-matched,malemiceatweek25.*p,0.05as
determinedbyone-wayANOVA.
then exposed to HFD. We found that PKClfl/fl CreaP2 mice indicating that adipose tissue specific deficiency of PKCl has no
developeddiet-inducedobesityatsimilarratesthanwtlittermates, effectonwholebodyglucosemetabolism.
suggesting that ablation of PKCl in adipose tissue does not
modulate body composition (40 weeks; Fig.9A). Despite the Discussion
similarbodyandfatmass,foodintakeoverthecourseofthisstudy
showed a trend to be decreased in PKClfl/fl CreaP2 mice as We herein report for the first time to our knowledge that mice
comparedwiththeirPKClwt/wtlittermates(Fig.9D).Surprisingly, deficient in hepatic PKCl are protected against diet-induced
but consistent with our observations in PKClfl/fl CreAlb mice, obesityandhyperglycemia.Sincethisresistancewasnotaresultof
energyexpenditureinPKClfl/flCreaP2micewasnotdifferentfrom reduced energy intake or increased expenditure, we propose that
PKClwt/wt littermates (Fig.10A and B). Likewise, respiratory the observed difference in body weight may be due to a small
quotient(Fig.10CandD)andlocomotoractivity(Fig.10EandF) difference in energy balance that is below the detection limit of
wereunaffectedbygenotype.Glucosemetabolismanalysisatweek the generally insensitive 24 h indirect calorimetry, but neverthe-
20 revealed no differences in fasting glucose levels (Fig.9F) and less relevant for chronic body composition development, as
similar response to glucose tolerance test in chow-fed PKClfl/fl previouslydescribed.17Consistentwithreducedadiposity,glucose
CreaP2 mice and their PKClwt/wt littermates (Fig.9E, inset), tolerance was improved in HFD-fed PKClfl/fl CreAlb mice
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Figure6.Glucosetoleranceasassessedbyglucoseexcursion(AandB)andareaunderthecurveanalyses(insetofAandB)ofchow-andHFD-fed
PKClfl/flCreAlb(closedcircles)andPKClwt/wt(opencircles)micefollowinga2g/kgdoseofipglucoseatweek20.Insulintoleranceasassessedbyglucose
excursion(CandD)andglucoseclearance[as%ofinitialbloodglucose(EandF)]ofchow-andHFD-fedPKClfl/flCreAlb(closedcircles)andPKClwt/wt
(opencircles)micefollowinga0.75U/kgdoseofipinsulinatweek25.Allstudieswereconductedaftera6hfastandalldataarerepresentedas
mean±SEMin6–7age-matched,malemice.*p,0.05asdeterminedby2-wayANOVAorStudent’st-test.
compared with theirwtlittermates,pointingtoabeneficialeffect enhanced in wt mice compared with PKClfl/fl CreAlb mice
on diet induced hyperglycemia and glucose intolerance by indicatingapossibleimpairedearly-phaseinsulinsecretionduring
silencingPKClinliver.Ourfindingsareconfirmedbyemerging glucose challenge in wt mice.20 To our surprise, results obtained
data utilizing small-molecule inhibitors of hepatic PKCl in with ipGTT and ipPTT showed that the observed improvement
rodents.18 in glucose metabolism is not due to enhanced insulin-mediated
Lower fasting glucose and insulin levels reflected significantly glucose transport or hepatic gluconeogenesis. We therefore
improved HOMA-IR index values, a well-established surrogate speculate that hepatic deficiency of PKCl may reduce glycogen-
measure of insulin action,32,33 suggesting that HFD-induced olysis and consequently lowering fasting glucose levels.21
insulin resistance is reduced in the absence of hepatic PKCl. Furthermore, we observed that lean and obese PKClfl/fl CreAlb
Furthermore, HFD-induced fasting hyperinsulinemia was mice exhibited similar glucose disappearance rates during ipITT
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Figure7.Pyruvatetoleranceasassessedbyglucoseexcursion(AandB)andglucoseappearanceduringthefirst30minfollowingippyruvatechallenge
(2g/kg)(CandD)ofchow-andHFD-fedPKClfl/flCreAlb(closedcircles)andPKClwt/wt(opencircles)micefollowinga2g/kgdoseofippyruvateatweek26.
Alldataarerepresentedasmean±SEMin6–7age-matched,malemice.*p,0.05asdeterminedbyStudent’st-test.
in contrast to wt mice in which insulin-mediated glucose uptake ablation in liver may disrupt insulin and/or inflammatory
wasmarkedlybluntedbyHFDfeeding.Thislackofdiet-induced signalingcascadessimilartothehepaticinsulinresistanceobserved
worsening of insulin action indicates that alterations in hepatic in patients with hyperglycemia and dyslipidemia.19 The resulting
nutrient metabolism due to the absence of PKCl translates into increaseindenovolipogenesisandVLDLsecretion19maythusbe
impaired insulin-mediated glucose transport that is no longer responsibleforthedeterioration oftheobservedinsulin-mediated
responsivetotheeffectofHFD.Inlightoftheseobservationswe glucose uptake in PKClfl/fl CreAlb mice.
hypothesize that while insulin-mediated glucose transport is While our investigation confirms certain findings reported
principally impaired, glucose-stimulated insulin secretion may be previouslyontheablationofhepaticPKClinmice,wealsomade
preservedinPKClfl/flCreAlbmiceunderHFDfeedingconditions. observations that contradict prior investigations on some fronts:
Ourfindingthatfastingtriglyceridelevelsareincreasedinlean First,similartoMatsumotoetal.,wefoundthatchowfedPKClfl/fl
PKClfl/fl CreAlb mice to similar levels detected in obese wt and CreAlb mice exhibited a body composition and response to GTT
PKClfl/fl CreAlb mice suggests that at least lipid metabolism may similartotheirwild-typecontrols.9However,inthesamereportthe
bemodulatedbythedeficiencyofPKClinliver.Althoughatthis authors describe a paradoxically beneficial effect of hepatic PKCl
time we do not fully understand the underlying mechanisms, ablation on insulin sensitivity in chow-fed mice. In contrast, our
thereisgrowingevidencesuggestingthatalterationsoffattyacids/ studiessuggestthatinsulin-mediatedglucosetransportisimpaired
triglyceride metabolism are an important determinant of insulin intheabsenceofhepaticPKCl. Ourdataareconsistentwiththe
sensitivity.22 In addition, as outlined in our introduction, PKCl hypothesisthatPKClisimportantforproperinsulinsignalingand
modulates inflammatory responses by activating the NFkB are in line with the results obtained in muscle specific deletion of
pathway11,12 and modulating Toll-like receptor 4 (TLR4) and PKCl.8 We thus speculate that the contradicting findings by
TNF-areceptorsignalingcascades.13,14Sinceitiswellestablished Matsumoto may be a consequence of changes in the genetic
that these pathways are implicated in the development of diet- background, based on emerging evidence that even slight
induced insulin resistance and dyslipidemia,23 it seems plausible differenceshaveprofoundeffectsonanobservedphenotype.24
that ablation of PKCl in the liver may diminish HFD- Inourmodel,fastingplasmacholesterollevelsareunaffectedby
induced inflammation and protect against diet-induced obesity, PKCl ablation, regardless of diet. However, fasting plasma
dyslipidemia and glucose intolerance through inhibition of its triglyceride levels are significantly elevated in chow-fed PKClfl/fl
inflammatory signaling function. We thus propose that PKCl CreAlbmice.Thoughwedidnotdirectlymeasuregenesimportant
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negative inhibition of atypical PKC signaling.29,30 However,
PKClablationfromalltissuesisembryoniclethal,sotheseearlier
studies were limited to cell-based assays. Our current strategy of
targeted PKCl deletion in adipose tissue allowed for the
assessment of the effects of this tissue specific kinase on energy
and glucose homeostasis in the whole organism. In light of the
prior evidence surrounding PKCl in adipocytes and its role in
insulin-stimulated glucose transport, we expected these mice to
exhibit impaired glucose tolerance. As such, our observation that
mice lacking adipose PKCl were of similar glucose tolerance to
theirWTlittermateswasunexpected,butnotunique.Inarecent
reviewFareseetal.pointedtounpublishedresultswheretheirlean
adipocyte-specific PKCl knockout mice have mild glucose
intolerance but, unlike lean muscle-specific PKCl-knockout
mice,donotdevelopobesityorhyperlipidemia,perhapsreflecting
a failure to develop increases in either serum insulin levels or
hepatic SREBP-1c expression.5 Based on our and their findings
that adipocyte PKCl deficient mice have a relatively normal
response to glucose challenge suggests that other tissues (skeletal
muscleandliver)areabletocompensateforthepresumedlackof
uptake into adipose tissue. This hypothesis is supported by the
observation that adipose tissue is responsible for less than 5% of
the postprandial glucose load.31 In view of our findings in mice
lacking hepatic PKCl, and prior studies in mice deficient for
skeletal muscle PKCl8, these data suggest that adipose PKCl
plays a subtle role, if any, in whole body glucose homeostasis.
Furthermore, for the first time to our knowledge we have
determined that adipose PKCl does not impact energy
homeostasis and the development of diet-induced obesity
In summary, this report suggests that hepatic PKCl is an
importantregulatorofglucoseandenergyhomeostasis.Specifically
these data implicate hepatic PKCl in the development of diet
induced obesity and impaired insulin action. Furthermore, this
reportdetailstherelativecontributionsofhepaticPKClincontrast
toPKClinadiposetissue.Importantly,ourdataconfirmandbuild
Figure8.Fastingcholesterol(A),triglyceride(B)andalaninetransami- upon previous observations and suggest that inhibition of hepatic
naseconcentrationsofchow-andHFD-fedPKClfl/flCreAlb(closedbars) PKClmaybeanimportanttargetinthetreatmentofobesity.
andPKClwt/wt(openbars)miceatsacrifice.Alldataarerepresentedas
mean±SEMin6–7age-matched,malemice.**p,0.01asdetermined
by1-wayANOVA.
Materials and Methods
for lipogenic programming, this observation was unexpected Animals. Mice carrying the atypical protein kinase C PKClfl/fl
basedonpreviousreports.9,10Onereasonforthesenon-consistent allele2 have been described previously. Alb- and Ap2-Cre mice
results is the fact that Matsumoto et al. determined lipid wereobtainedfromtheJacksonLaboratory(BarHarbor,Maine).
parameters during a randomly fed state whereas we fasted the Micefromalllineswerebredinourfacilitiesandmaintainedona
mice for 6 h to shed light into hepatic triglyceride metabolism. C57/B6 background. The PKClfl/fl mice were crossed with Alb-
Our results seem to confirm a growing body of evidence that Cre or Ap2-Cre mice to get PKClfl/fl CreAlb or PKClfl/fl CreaP2
PKCl may have divergent metabolic functions depending upon genotypes.DNAwasextractedfromtailsnipsandPCRwasused
nutritionalstatusaspostulatedbyFareseetal.inarecentreview.5 to establish genotypes. All animals were housed on a 12:12 h
In addition to its hepatic role, PKCl is also known to light-darkcycleat22°C.Onlymalemicewereusedforthestudies
participateininsulin-stimulatedglucoseuptakeinadipocytes.25-27 andhadfreeaccesstowaterandwerefedadlibitumwitheithera
Activation of the atypical PKCs by insulin appears to occur regular chow diet (LM-485; Teklad, 5.6% fat) or a high fat diet
downstream of PI3K, as the relative activity of this kinase is (D12331; Research Diets Inc., 58% kcal fat) after weaning. All
subject to wortmanin inhibition.28 The regulatory importance of studies were approved by, and performed according to, the
this interaction is evidenced by the impaired glucose transport guidelinesoftheInstitutionalAnimalCareandUseCommitteeof
observed in studies utilizing pharmacological or dominant- the University of Cincinnati.
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Figure9.Bodyweight(A),fatmass(B),leanmass(C)andcumulativefoodintake(D)inPKClfl/flCreaP2(closedcircles)andPKClwt/wtmice(opencircles).
Glucosetoleranceasassessedbyglucoseexcursion(E)andareaunderthecurveanalyses(insetofE)of20-week-old,chow-fedPKClfl/flCreAlb(closed
circles)andPKClwt/wt(opencircles)micefollowinga2g/kgdoseofipglucoseatweek20.Alldataarerepresentedasmean±SEMin6–7age-matched,
malemice.Nodifferencesweredeterminedby2-wayANOVAorStudent’st-test.
Body composition and plasma analysis. Whole body com- kg body weight in all animals. Insulin tolerance tests were
position(fatandleanmass)wasmeasuredusingNMRtechnology conducted with 100 U/ml human insulin (Lilly Humolog) in
(EchoMRI). Individual samples for plasma triglyceride and 0.9% saline, such that the final dose was 0.75 U/kg. Pyruvate
cholesterol levels were measured by enzymatic assay kits tolerance tests were conducted with sodium pyruvate (P2256;
(TR22421 and TR13421; Thermo Electron) in mice that have Sigma) in 0.9% saline, such that the final dose was 2 g/kg.
been fasted for 6 h. Glucose levels (mg/dl) were measured from tail blood using a
Insulin, pyruvate and glucose tolerance test. For measure- handheldglucometer(Freestylelite)before(0min)andat15,30,
ments of insulin, pyruvate, and glucose tolerance, mice were 60and120minafterinjection.Glucosetolerancewasassessedby
fasted for 6 h before the intraperitoneal challenge. Glucose area under the curve analysis, insulin tolerance by glucose
tolerance tests were conducted with 25% D-glucose (G8270; clearance over the initial 60 min of the insulin challenge, and
Sigma)in0.9%saline,suchthatthefinaldosewas2.0gglucose/ pyruvate tolerance by glucose appearance over the initial 30 min
212 Adipocyte Volume1Issue4