maternal high-fat intake predisposes nonalcoholic fatty liver disease in c57bl/6 offspring

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ASIC SCIENCE: OBSTETRICS

aternal high-fat intake predisposes nonalcoholicatty liver disease in C57BL/6 offspringianca M. Gregorio, RD, PhD; Vanessa Souza-Mello, RD, PhD; Jorge J. Carvalho, PhD;arlos A. Mandarim-de-Lacerda, MD, PhD; Marcia B. Aguila, RD, PhD

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BJECTIVE: This work aimed to verify the hypothesis that maternal in-ake of high-fat diet in critical periods of pregnancy and/or suckling pe-iod predisposes nonalcoholic fatty liver disease in adult C57BL/6 miceffspring.

TUDY DESIGN: Male pups were divided into 5 groups: (1) SC, fromtandard chow–fed dams; (2) G, from high-fat chow (HF)–fed damsuring the gestation (G) period; (3) L, from HF-fed dams during the lac-ation (L) period; (4) GL, from HF-fed dams during the gestation and lac-ation (GL) periods; and (5) GL/HF, from HF-fed dams during GL, main-aining an HF diet from postweaning to adulthood. We analyzed body

m J Obstet Gynecol 2010;203:495.e1-8.

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oi: 10.1016/j.ajog.2010.06.042

ESULTS: The G offspring showed insulin resistance and lower glucoseransporter-2 expression. Hepatic steatosis was present in the G, L, GL,nd mainly in GL/HF offspring. Sterol regulatory element-binding pro-ein-1c expression was higher in G, GL, and GL/HF offspring.

ONCLUSION: Programming by HF chow predisposes hepatic adverseemodeling in the liver of adult offspring.

ey words: fatty liver, fetal programming, liver steatosis, liver
ass, plasma blood, and liver structure. ultrastructure, maternal high-fat diet
ite this article as: Gregorio BM, Souza-Mello V, Carvalho JJ, et al. Maternal high-fat intake predisposes nonalcoholic fatty liver disease in C57BL/6 offspring.

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he term nonalcoholic fatty liver dis-ease (NAFLD) is used to describe a

pectrum of structural findings rangingrom simple steatosis to nonalcoholicteatohepatitis (NASH) with progressivebrosis and liver failure.1 The conditionine qua non of NAFLD patients in-ludes macro- and microvesicular ste-tosis and in NASH patients includeacrovesicular or a mix between micro-

tate University of Rio de Janeiro,iomedical Center, Institute of Biology,aboratory of Morphometry andardiovascular Morphology, Rio de Janeiro,razil.

eceived March 11, 2010; revised May 25,010; accepted June 17, 2010.

eprints: Marcia B. Aguila, RD, PhD, Stateniversity of Rio de Janeiro, Laboratory oforphometry and Cardiovascular Morphology,iomedical Center, Rio de Janeiro, Brazil0551-030. [email protected].

he Laboratory of Morphometry andardiovascular Morphology is supported byrazilian agencies CNPq (National Council forcience and Technology, www.cnpq.br) andaperj (Rio de Janeiro Foundation foresearch, www.faperj.br).

002-9378/$36.002010 Mosby, Inc. All rights reserved.

nd macrovesicular steatosis with mildobular inflammation.2 As fibrosis devel-ps, changes occur within the subendo-helial space and within the hepaticinusoid.

These changes include alterations inoth cellular responses and extracellularatrix composition. Activation of the

epatic stellate cells leads to accumula-ion of a scar (fibril-forming) matrix,hich results in widening of the space ofisse and loss of endothelial fenestrae.ransport across the sinusoidal wall isence reduced, leading to deteriorationf hepatic function.3

In NAFLD, triglycerides are ultimatelyynthesized from fatty acids (FAs). Therere numerous potential sources of FAssed to generate triglycerides, but di-tary FAs are a main source. The FAs de-ived from lipolysis of adipose tissue tri-lyceride depots are also delivered to theiver, taken up by hepatocytes, and con-erted into triglycerides.4

De novo lipogenesis is another factorhat leads to steatosis in NAFLD, a pro-ess regulated by transcription factorshat are activated by insulin, particularlyterol regulatory element binding pro-ein (SREBP)-1c.5 The high insulin levels

on of SREBP-1c, n

NOVEMBER 2010 Americ

hich increases the expression of all li-ogenic enzymes, thus increasing he-atic free fatty acid (FFA) synthesis,hich also increases the hepatic expres-

ion of all hepatic lipogenic genes,6,7 thusurther increasing hepatic FFA synthesis.

FAs may also accumulate within hepa-ocytes because their metabolism is im-aired. In healthy hepatocytes, FAs arexidized by enzymes in peroxisomes,itochondria, and the endoplasmic re-

iculum (microsomes).8 Regardless ofhe source of FAs that hepatocytes use toroduce triglycerides, this triglyceride isormally packaged into lipoproteins in

he hepatocyte endoplasmatic reticulumnd then exported to adipose depots fortorage.9 Therefore, accumulation of fatn the liver leads to an excessive deliveryf FFAs from visceral adipose tissue intohe liver and from a misbalance in deovo synthesis and catabolism inepatocytes.4

Some models, such as nonhuman pri-ate and murine, are used to correlate

he intrauterine environment and devel-pment of NAFLD.10,11 Different mech-nisms are involved in the offspringnsult because of adverse maternal nutri-ion. Up-regulation of specific placental
utrient transporter isoforms consti-
an Journal of Obstetrics & Gynecology 495.e1

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4

utes a mechanism linking maternaligh-fat (HF) diet and obesity to fetalvergrowth12 and modulates the off-pring glucose homeostasis.13 Therefore,his study aimed to investigate how the

aternal intake of HF diet in differentritical periods of pregnancy and/oruckling period influences carbohy-

FIGURE 1Formation of the groups

, gestation; GL, maternal HF diet during gestation and lactation;

regorio. Hepatic adverse remodeling by HF programming. A

FIGURE 2Body mass evolution (mean and SE

ne-way ANOVA and post-hoc test of Tukey: inNOVA, analysis of variance; G, gestation; GL, maternal HF diettandard chow.


95.e2 American Journal of Obstetrics & Gynecolo

rates metabolism alterations andAFLD in adulthood offspring.

ATERIALS AND METHODSnimals and dietxperimental protocols were approvedy the local committee according to con-

igh-fat chow; L, lactation; SC, standard chow.

Obstet Gynecol 2010.

aled cases with the same symbols, P � .05.ng gestation and lactation; HF, high-fat chow; L, lactation; SC,


l

gy NOVEMBER 2010

entional guidelines for experimenta-ion with animals.14 Animals were main-ained under controlled conditions ofemperature and humidity, and 12 hourark, 12 hour light cycle, with free accesso water and food. C57BL/6 virgin ma-ure females were caged with males over-ight, and mating was confirmed theext morning (vaginal plug). Then fe-ales were allocated to be fed either a

tandard chow (SC; 17% fat, 19% pro-ein, and 64% carbohydrate, equivalento 16.5 kJ/g) or a HF chow (49% fat, 19%rotein, and 32% carbohydrate, equiva-

ent to 20.7 kJ/g, 25.5% more energyhan the SC diet). Both diets, includinghe micronutrient mineral mix, followedhe American Institute of Nutrition rec-mmendation (AIN-93G).15

HF feeding was targeted to specific pe-iods in gestation (G) and/or (L) lacta-ion (Figure 1): G (HF diet was takenuring the gestation); L (HF diet wasaken during lactation); GL (HF diet wasaken in both gestation and lactation),nd GL/HF (HF diet was taken in bothestation and lactation and continuedrom postweaning until 3 months old).mmediately after delivery, litters weredjusted to 6 animals per mother (to as-ure adequate and standardized nutri-ion until weaning), and at weaning, 1

ale pup per litter was randomly as-igned to form the groups of study7 animals/group).

Offspring body mass evolution wasonitored until the euthanasia. Food in-

ake was estimated by subtracting themount of food left on the grid and themount of spilled food from the initialeight of food supplied. Energy intakesere calculated based on energetic valuef diets. Feed efficiency was calculated asrams of the body mass gain per kilo-oules of food consumed per animal.

ral glucose tolerance testOGTT) and plasma insulinGTT was made at 3 months old with

5% glucose in sterile saline (0.9%aCl) (1 g/kg body mass [BM]) ad-inistered by orogastric gavage after 6

ours of a fasting period. Blood wasollected from the tail vein at 0, 15, 30,0, and 120 minutes after the glucose

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www.AJOG.org Basic Science: Obstetrics Research

sing a glucometer (Accu-Chek;oche, Sao Paulo, Brazil. The analysisonsidered the area under the curve tossess glucose intolerance (Prism ver-ion 5.03 for Windows; GraphPadoftware, San Diego, CA).Plasma insulin concentrations wereeasured by radioimmunoassay usingmouse insulin radioimmunoassay kit

catalog #RI-13K; Linco Research, Stharles, MO). All samples were ana-

yzed in a double assay, for which thentraassay coefficient of variation was.4%. Insulin resistance was estimatedy homeostasis model assessment for

nsulin resistance index (HOMA-IR)s [(fasting glucose � fasting insulin)/2.5].16

uthanasiaffspring (3 months old) were deeply

nesthetized and blood was collected byardiac puncture. Then the liver was rap-dly excised, weighed, and prepared foroth light and electron microscopy.enital fat deposit (adipose tissue sur-

ounding the ureters, bladder, and epi-

TABLEFeed efficiency, biometry, and bloo

Data Group SC

FE, g/kJ (� 10�2) 5.6 � 0.3...................................................................................................................

LM, mg 0.84 � 0.03...................................................................................................................

GFP, mg 0.45 � 0.02...................................................................................................................

ALT, mg/dL 10.4 � 0.7...................................................................................................................

AST, mg/dL 164.0 � 13.1...................................................................................................................

ASP, mg/dL 45.2 � 5.8...................................................................................................................

OGTT, AUC 17.27 � 1.08...................................................................................................................

HOMA-IR 1.45 � 0.3...................................................................................................................

TC, mg/dL 88.2 � 2.5...................................................................................................................

TG, mg/dL 36.0 � 1.1...................................................................................................................

TNF-alpha, pg/mL 61.3 � 3.5...................................................................................................................

Adiponectin, ng/mL 5.3 � 0.4...................................................................................................................

ALT, alanine aminotransferase; ANOVA, analysis of variance; Afat pad mass; GL, maternal HF diet during gestation and lactatiotolerance test; SC, standard chow; TC, total cholesterol; TG, ta When compared with SC, 1-way ANOVA and post-hoc test o

cases, when compared, P � .05; c When compared with L,post-hoc test of Tukey: in signaled cases, when compared,

Gregorio. Hepatic adverse remodeling by HF programmin

idymis) was dissected and weighed. t

iver enzymes and TNF-alphaotal cholesterol (TC), triglyceride

TG), and hepatic enzymes concentra-ions were measured by a colorimetricssay (Bioclin; Belo Horizonte, Minaserais, Brazil): alanine aminotransferase

ALT), aspartate aminotransferase (AST),nd alkaline phosphatase (ASP). Mice se-um analysis for tumor necrosis factorTNF)-alpha was performed using a com-ercially available enzyme-linked immu-

osorbent assay (ELISA) kit (human/ouse TNF-alpha ELISA Ready-SET-go;

ioscience, San Diego, CA).

estern blottingotal hepatic proteins were extracted inomogenizing buffer and protease in-ibitors. Thereafter the homogenatesere centrifuged for 20 minutes at 4°C,

nd the supernatants were collected.qual quantities of total protein were re-uspended in sodium dodecyl sulfateSDS)– containing sample buffer, heatedor 5 minutes at 100°C, and separated byDS-polyacrylamide gel electrophoresis.fter electrophoresis, proteins were elec-

iochemistry (mean and SEM)Groups HF

G L

6.7 � 0.3 6.4 � 0.2.........................................................................................................................

0.92 � 0.03 0.82 � 0.07.........................................................................................................................

0.48 � 0.02 0.48 � 0.02.........................................................................................................................

24.2 � 1.5a 10.7 � 1.8b

.........................................................................................................................

217.6 � 9.6a 163.4 � 5.6b

.........................................................................................................................

50.2 � 4.2 50.6 � 5.5.........................................................................................................................

23.48 � 1.38a 18.43 � 1.14b

.........................................................................................................................

5.1 � 1.3a 1.3 � 0.2b

.........................................................................................................................

102.0 � 2.3a 89.2 � 2.1b

.........................................................................................................................

32.4 � 2.0 60.5 � 2.2a,b

.........................................................................................................................

70.7 � 1.8 54.5 � 2.1b

.........................................................................................................................

5.3 � 0.2 5.8 � 0.2.........................................................................................................................

lkaline phosphatase; AST, aspartate aminotransferase; AUC, area, high-fat; HOMA-IR, homeostasis model assessment for insulin reeride; TNF, tumor necrosis factor.

ey: in signaled cases, when compared, P � .05; b When compay ANOVA and post-hoc test of Tukey: in signaled cases, when co

.05.

m J Obstet Gynecol 2010.

roblotted on a polyvinyl difluoride t


ransfer membrane (Amersham Bio-ciences, Piscataway, NJ).

The efficiency of the transfer was visu-lized by Ponceau solution staining. Theembrane was blocked by incubationith nonfat dry milk (6% in Tween 20 –ris-buffered saline), incubated witholyclonal antibody against anti-rabbitREBP-1c (68 kDa, SC-367; Santa Cruziotechnology, Santa Cruz, CA) and glu-ose transporter (GLUT)-2 (53-61 kDa,B1342; Chemicon, Temecula, CA),ashed, and incubated with antirabbit

econdary antibody. SREBP-1c andLUT-2 protein expressions were de-

ected using an enhanced chemilumines-ence detection system (Amersham).

Signals were visualized by autoradiog-aphy and determined by quantitativenalysis of digital images of gels usingmagePro plus software, version 7.0Media Cybernetics, Silver Spring, MD).he integral optical density values wereeasured.

iver stereology and ultrastructureiver fragments were immediately ob-

GL GL/HF

6.2 � 0.3 7.2 � 0.4a

..................................................................................................................

0.85 � 0.02 1.08 � 0.02a-d

..................................................................................................................

0.50 � 0.01 1.41 � 0.23a-d

..................................................................................................................

24.0 � 1.8a,c 16.8 � 1.5a,b,d

..................................................................................................................

248.5 � 14.8a,c 186.0 � 14.9d

..................................................................................................................

80.8 � 9.4a-c 41.6 � 4.1d

..................................................................................................................

20.86 � 0.68 20.03 � 0.95..................................................................................................................

3.8 � 0.5 2.1 � 0.1b

..................................................................................................................

110.6 � 2.3a,c 89.8 � 2.7b,d

..................................................................................................................

39.2 � 1.4c 44.6 � 2.5a-c

..................................................................................................................

53.2 � 1.2b 53.2 � 2.1b

..................................................................................................................

3.7 � 0.3a-c 5.1 � 0.4d

..................................................................................................................

er the curve; FE, feed efficiency; G, gestation; GFP, genitalnce index; L, lactation; LM, liver mass; OGTT, oral glucose

ith G, 1-way ANOVA and post-hoc test of Tukey: in signaledred, P � .05; d When compared with GL, 1-way ANOVA and

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tives either for light microscopy (1.27ol/L formaldehyde in 0.1 M phosphate

uffer, pH 7.2) for 48 hours at roomemperature or for transmission electron

icroscopy (0.1 M cacodylate buffer.5% glutaraldehyde, pH 7.2) for 2ours, followed by postfixation in 1%smium tetroxide for 45 minutes. For

ight microscopy material was embed-ed in Paraplast plus, sectioned at 3 �mf thickness, and stained by hematoxy-

in-eosin. For electron microscopy ma-erial was embedded in Epon, ultrathinections were obtained using a Leica Ul-racut ultramicrotome, counterstainedith uranyl acetate and lead citrate, ob-

erved in a Zeiss EM 906 microscope (at0 KV; Zeiss, Oberköchen, Germany).The liver steatosis was assessed with

tereology using a test system made up of6 test points (PT) superimposed to theicroscopic fields as described else-here.17 Briefly, 6 microscopic fields per

nimal were analyzed at random withideo-microscopy (Leica DMRBE mi-roscope; Leica, Wetzlar, Germany;ideo camera; Kappa Gleichen, Ger-any; and Sony Trinitron monitor;

FIGURE 3Volume density of liversteatosis (mean and SEM)

ne-way ANOVA and post-hoc test of Tukey: inignaled cases, when compared, P � .05; if: ahen compared with SC, b with G, c with L, dith GL.

NOVA, analysis of variance; G, gestation; GL, maternal HF dieturing gestation and lactation;HF, high-fat chow; L, lactation; SC,tandard chow.

regorio. Hepatic adverse remodeling by HF programming.m J Obstet Gynecol 2010.

ony, Pencoed, UK). The steatosis vol- 1


me density (Vv) was estimated by pointounting: Vv [steatosis, liver] � PP [ste-tosis, liver]/PT, where PP is the numberf points that hit the structure.18

ata analysisata were tested for normal distribu-

ion and homogeneity of the variancesnd then reported as mean and SEM.ifferences among the groups were ana-

yzed using 1-way analysis of varianceANOVA) followed by a post-hoc test ofukey. A P value � .05 was considered

tatistically significant (Prism version.03 for Windows; GraphPad Software).

ESULTSaternal datall HF mothers tolerated the diet and didot have other complications. No signif-

cant differences were found in the BMain and food intake during the gestationnd lactation periods in both the SCams (10.39 � 0.58 g and 5.57 � 0.42 g)nd HF dams (10.32 � 0.46 and 4.73 �.29 g; P � .05). Food intake was esti-ated by subtracting the total amount of

eed and the amount remaining in theox. It is important to mention thatams were used to obtain the studyroups. Therefore, all metabolic param-ters were evaluated in their offspring.

ffspring dataiometry and food efficiency: the analysisf the offspring biometry considered theM evolution (Figure 2) and also the

eed efficiency (FE), liver mass (LM), andenital fat pad mass (GFP), detailed inhe Table.

The offspring showed differences inM starting at the 5th week. The off-

pring from HF mothers was signifi-antly heavier than those from SC moth-rs. In the period from the seventh to the3th week, 3 different patterns of BMvolution were observed: GL/HF off-pring had the greatest BM during all theeriod, whereas the offspring from theroups G, L, and GL showed a parallelM evolution until the 11th week, beinglaced between those of the GL/HF andC offspring. The BM of the GL offspringecreased at that moment, and from the
1th week, and mainly at the 13th week, 1
gy NOVEMBER 2010

L offspring joined the SC offspringith respect to their BM.During all periods, the SC offspringere the lightest animals. At the 13theek, comparing the SC offspring with

he GL/HF offspring, the BM was almost5% greater in GL/HF (from 22.2 � 0.5o 32.2 � 1.9 g; 1-way ANOVA and post-oc test of Tukey; P � .001).The 3 other parameters, FE, LM, andFP, were equally greatest in GL/HF off-

pring. The FE was different only be-ween the offspring SC and GL/HF (5.6

0.3 and 7.2 � 0.4 g/Kj; P � .007), tohich FE was nearly 30% greater than SCffspring. The others groups, fromothers taking the HF diet in different

eriods of development, had FE betweenC and GL/HF offspring but with no sig-ificant difference. In the GL/HF off-pring, both the measurements LM (1.08

0.02 g) and GFP (1.41 g � 0.23 g) werereater than the SC offspring (0.84 �.03 and 0.45 � 0.02 g; P � .005), the Gffspring (0.92 � 0.03 g and 0.48 �.02 g; P � .0004), the L offspring (0.82

0.07 and 0.48 � 0.02 g; P � .001), andhe GL offspring (0.85 � 0.02 and 0.50 �.01 g; P � .01).Liver enzymes: ALT and AST were signif-

cantly higher in both the G (24.2 � 1.5nd 217.6 � 9.6 mg/dL) and GL (24.0 �.8 and 248.5 � 14.8 mg/dL groups than inoth the SC (10.4 � 0.7 and 164.0 � 13.1g/dL; P � .05) and L (10.7 � 1.8 and

63.4 � 5.6 mg/dL; P � .05) groups, indi-ating that the maternal HF diet intake inhe gestational period was worse than theF intake in the lactation period. Interest-

ngly, there was ALT and AST reduction inL/HF (16.8 � 1.5 and 186.0 � 14.9 mg/L) offspring than in G (24.2 � 1.5 and17.6 � 9.6 mg/dL; P � .05) and GL (24.0

1.8 and 248.5 � 14.8 mg/dL; P � .05)ffspring, but ALT continued to be greater

n GL/HF mice (16.8 � 1.5 mg/dL) than inC mice (10.4 � 0.7 mg/dL; P � .05). ASPas significantly higher in the GL group

han all the other groups (80.8 � 9.4 mg/L; P � .05) (Table).Glucose and HOMA-IR: no difference

n plasma insulin was detected amonghe groups (data not shown). The Tablehows greater OGTT value in G (23.48 �.38 arbitrary units) than in SC (17.27 �
.08 arbitrary units; P � .05) offspring,

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hich, in consequence, had higherOMA-IR than the other groups.Plasma lipids and TNF-alpha: the

alues of TC and TG had a differentattern among the groups. The TC pat-ern among the groups followed theame pattern of both ALT and AST,ith significantly higher values in G

102.0 � 2.3 mg/dL) and GL (110.6 �.3 mg/dL) offspring than in SC (88.2

2.5 mg/dL; P � .005) and L (89.2 �.1 mg/dL; P � .005) offspring. Thesendings suggest that the maternal HFiet intake in the gestational periodas worse than the maternal HF diet

FIGURE 4Liver ultrastructure

roups and main findings: A, SC, preserved hacuoles; B, G, hepatocyte with no significant aicrovesicular steatosis compromising the inne

umerous macro- and microvesicular steatosis; Fepatocyte nucleus.NOVA, analysis of variance; G, gestation; GL, maternal HF diet d


ntake in the lactation period. T

The groups GL/HF (89.8 � 2.7 mg/dL),C (88.2 � 2.5 mg/dL), and L (89.2 � 2.1g/dL) showed close TC values. On the

ontrary, the groups L (60.5 � 2.2 mg/dL)nd GL/HF (44.6 � 2.5 mg/dL) had higherG values than SC (36.0 � 1.1 mg/dL; P �

0001) and G (32.4 � 2.0 mg/dL; P � .05)roups. The groups L (54.5 � 2.1 pg/mL),L (53.2 � 1.2 pg/mL), and GL/HF (53.22.1 pg/mL) had lower TNF-alpha values

han the G group (70.7 � 1.8 pg/mL; P �001) (Table).

iver structure and ultrastructurehese data are shown in Figures 3 and 4.

tocyte architecture showing numerous mitochoation showing few and sparse lipid vacuoles; Cpatocyte architecture; D and E, GL, the hepatoL, showing an activated stellate cell (open arrow

gestation and lactation; HF, high-fat diet; L, lactation; M, macro


he group SC showed a small quantity of a


iver steatosis (7.34 � 0.71% that coulde considered normal). The steatosiseached around 20% in the group G19.52 � 2.39%) and surpassed 30% inhe groups L (33.64 � 1.58%) and GL36.41 � 1.49%). The group GL/HF had

steatosis rate nearing 25% (24.85 �.01%) (Figure 3).The ultrastructural analysis showed

ipid accumulation in altered hepato-ytes. In the SC mice, as expected, theepatocytes were mostly preserved hav-

ng numerous mitochondria and abun-ant endoplasmic reticulum, a commonnding in a normally active cell. This was

ia and endoplasmatic reticulum with rare lipidnumerous vacuoles characterizing macro- ande inner ultrastructure is similar to group L withnd G, GL/HF, some lipid vacuoles surrounds the

ular steatosis; m, microvesicular steatosis; SC, standard chow.

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roup, showing no significant structurallteration and only few and sparse lipidacuoles in the cytoplasm. However, inhe groups L and GL, hepatocytes usuallyave numerous cytoplasmatic vacuolesharacterizing macro- and microvesicu-ar steatosis compromising the inner cel-ular architecture. In addition, the pres-nce of the hepatic stellate cells was morerequent in the GL group than the otherroups. Lipid vacuoles surrounding theepatocyte nuclei like the descriptions ofacrovesicular steatosis were frequently

bserved in the group GL/HF (Figure 4).

REBP-1c and GLUT-2 expressionhese data are shown in Figures 5 and. The SREBP-1c, a positive transcrip-ion factor that stimulates key lipo-enic genes, promoting de novo fattycid synthesis, was expressed more inhe liver of the groups G, GL, and GL/F, whereas it was expressed less in the

roups SC and L (Figure 5). On theontrary, the liver expression of theLUT-2 was smaller in the group G

FIGURE 5Liver SREBP-1 expressionand Western blot bands

iver SREBP-1 expression (mean and SEM) andepresentative Western blot bands. One-wayNOVA and post-hoc test of Tukey: in signaledases, when compared, P � .05; if: a, whenompared with SC, b, with G, and c, with L.NOVA, analysis of variance; a.u., arbitrary units; G, gestation; GL,aternal HF diet during gestation and lactation;HF, high-fat diet; L,

actation; SC, standard chow.


han the group L (Figure 6). c


OMMENT

utritional status during critical periodsf early life has important influences onevelopment, and modification of theuality of maternal nutrition duringregnancy has been shown to have con-equences on the later health of the off-pring, changing their responses to envi-onmental challenges and thus theirredisposition to disease.19,20 Thus, weeproduced a mouse model of humanAFLD, which shows that exposure to aF diet (rich in saturated fatty acid) intero and during lactation exacerbates

he NAFLD phenotype exhibited in off-pring that was also fed a HF diet posteaning.21

Although our study had not shown anlevation in the maternal BM and theirood intake, probably because the shortime of administration, offspring mani-ested significant differences in birth-eight, adult body biometry, carbohydrateetabolism, lipid profile, TNF-alpha lev-

ls, and liver structure. HF offspring wereeavier than the SC offspring at birth. Inddition, the GL/HF offspring were pre-isposed to become fatter in adulthoodnd had higher food efficiency than the SCffspring.Interestingly, the G offspring were hy-

erglycemic and had high HOMA-IRevels, agreeing with previous reports re-arding the effects of HF diets during de-elopment.22,23 Because the G offspringere exposed to earlier HF diet, the re-

ults suggest that this period is central tohe offspring’s glycemic control. A pos-ible explanation relies on the hypothesisf the predicitive adaptative responseshat the contact with altered nutritionalactors during early development causes

etabolic adaptations based on the pre-iction that the same environment willxist in later life. If a mismatch occursetween the predicted and the existentonditions, rather than representing andvantage, this may lead to disease.24

In the present study, the intrauterinexposure to HF diet (G offspring) wasccompanied by reduced expression ofhe GLUT-2, showing an impaired me-abolism of carbohydrate, and an ele-ated production of TNF-alpha, that
ould bring in the genesis of liver dam- i
gy NOVEMBER 2010

ge. Recent experimental evidence inice demonstrated insulin resistance in

ssociation with fatty infiltration and in-reased TNF-alpha expression in theiver in 22 week old KK/Ta mice exposedo an HF diet that mimic, in the mice,ome signals of the human metabolicyndrome.25

Hepatic injuries, including steatosisnd mitochondrial abnormalities suchs ultrastructural lesions, were seen in allF offspring. Notably, once hepatic ste-

tosis is present, other alterations are ex-ected, such as microvascular dysfunc-ion (changes in hepatic sinusoids) andctivated hepatic stellate cells, contribut-ng to the progression of hepatic dis-ase.26 In general, mitochondria are cru-ial to hepatocyte metabolism, being therimary site for the oxidation of FAs andxidative phosphorylation.27,28 The acti-ation of the stellate cells, seen mainly inhe GL offspring of the present study, cane considered as the first step to NASH

nstallation.29

These data are broadly in support ofiver enzymes and lipid profile. Circulat-

FIGURE 6Liver GLUT-2 expressionand Western blot bands

iver GLUT-2 expression (mean and SEM) andepresentative Western blot bands. One-wayNOVA and post-hoc test of Tukey: in signaledases, when compared, P � .05; if: a, whenompared with SC, b, with G.NOVA, analysis of variance; a.u., arbitrary units; G, gestation; GL,aternal HF diet during gestation and lactation; HF, high-fat diet;

, lactation; SC, standard chow.


ng concentrations of mainly ALT, and

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C, were altered in HF offspring, exceptn L offspring, suggesting that maternal

F has a stronger effect on offspringhen administrated during gestation oruring both gestation and lactation. Theajor changes in AST were seen in bothL and G groups, indicating some de-

ree of liver injury in these offspring.Thus, with our findings showing an

nfluence of intrauterine nutritional ex-osure to affect the risk of the NAFLDhenotype, we suggest that intrauterineutrition may have the potential to mod-

fy hepatic lipogenesis through an influ-nce on SREBP-1c function. The off-pring of the groups G, GL, and GL/HFad increased hepatic expression ofREBP-1c, allowing the speculation thatncreased dietary exposure to a HF dietn the pregnant and suckling mother re-ults in an augmented placental transferf FAs to the fetus. This consequentlyan increase the hepatic lipogenesis andxidative stress in the vulnerable fetal

iver, a situation that could contribute tohe subsequent development of NAFLDn adulthood.30

In fact, mice do not imitate entirely therue human situation, and this is the ma-or limitation of the present study. Be-ause main developing differences be-ween rodents and human, rodents noteing born until postpartum day 12, theeriod of the first 12 days of life is prob-bly most comparable with the late ges-ational period of humans.31

Another point to remark is that vari-us diets with very different FA compo-itions are summarized under the termF diet. This has inevitably led to con-

iderable variability in the results re-orted. Furthermore, it has already beenescribed in the literature that malesave a more accelerated developmenthan females during critical periods ofevelopment. Thus, males’ organs areost affected in these models of meta-

olic programming.32 Moreover, studiesre still controversial when it comes toetal programming in females. Gallou-abani et al33 reported resistance to theevelopment of metabolic changes stem-ing from programming by HF diet in

emale offspring.With this mice model, we conclude

hat factors affecting early fetal liver de- R

elopment, such as increased maternalietary fat consumption, may act to in-rease the vulnerability of developing fe-al liver to fat accumulation in adult-ood. We found that the permanency orhe change of the postweaning diet andF diet during gestation are worst to off-

pring because they had hepatic steato-is, liver ultrastructural changes, andltered carbohydrate metabolism. Al-hough GL/HF offspring did not mani-est alterations in lipid profile and carbo-ydrate metabolism, they had crucialodifications in hepatic tissue, intensi-

ying the risk of NAFLD. Therefore, fur-her investigations are needed to deter-

ine whether NAFLD programmed byhe maternal HF in adult offspring willeduce or increase their susceptibility toevelop NAFLD with different lipidource in HF diets. Understanding the

echanisms of fetal HF programmingAFLD may be an approach to new die-

etic recommendations in perinatalutrition. f

EFERENCES. Charlton M. Nonalcoholic fatty liver disease:review of current understanding and future

mpact. Clin Gastroenterol Hepatol 2004;2:048-58.. Varela-Rey M, Embade N, Ariz U, Lu SC,ato JM, Martinez-Chantar ML. Non-alcoholic

teatohepatitis and animal models: under-tanding the human disease. Int J Biochem Celliol 2009;41:969-76.. Hui AY, Friedman SL. Molecular basis of he-atic fibrosis. Expert Rev Mol Med 2003;5:-23.. Jou J, Choi SS, Diehl AM. Mechanisms ofisease progression in nonalcoholic fatty liverisease. Semin Liver Dis 2008;28:370-9.. Chen G, Liang G, Ou J, Goldstein JL, BrownS. Central role for liver X receptor in insulin-ediated activation of Srebp-1c transcription

nd stimulation of fatty acid synthesis in liver.roc Natl Acad Sci USA 2004;101:11245-50.. Yamashita H, Takenoshita M, Sakurai M, etl. A glucose-responsive transcription factorhat regulates carbohydrate metabolism in theiver. Proc Natl Acad Sci USA 2001;98:116-21.. Iizuka K Bruick RK, Liang G, Horton JD,yeda K. Deficiency of carbohydrate responselement-binding protein (ChREBP) reduces li-ogenesis as well as glycolysis. Proc Natl Acadci USA 2004;101:7281-6.. Musso G, Gambino R, Cassader M. Recent

nsights into hepatic lipid metabolism in non-lcoholic fatty liver disease (NAFLD). Prog Lipid
es 2009;48:1-26. L

. Syn WK, Teaberry V, Choi SS, Diehl AM.imilarities and differences in the pathogenesisf alcoholic and nonalcoholic steatohepatitis.emin Liver Dis 2009;29:200-10.0. McCurdy CE, Bishop JM, Williams SM, etl. Maternal high-fat diet triggers lipotoxicity inhe fetal livers of nonhuman primates. J Clinnvest 2009;119:323-35.1. Hartil K, Vuguin PM, Kruse M, et al. Mater-al substrate utilization programs the develop-ent of the metabolic syndrome in male mice

xposed to high fat in utero. Pediatr Res009;66:368-73.2. Jones HN, Woollett LA, Barbour N, PrasadD, Powell TL, Jansson T. High-fat diet beforend during pregnancy causes marked up-reg-lation of placental nutrient transport and fetalvergrowth in C57/BL6 mice. FASEB J 2009;3:271-8.3. Dunn GA, Bale TL. Maternal high-fat dietromotes body length increases and insulin in-ensitivity in second-generation mice. Endocri-ology 2009;150:4999-5009.4. National Institutes of Health. Guide for theare and use of laboratory animals. Publication5-23, revised 1996. Washington, DC: Institutef Laboratory Animal Resources, Commissionn Life Sciences, National Research Council;996:128.5. Reeves PG, Nielsen FH, Fahey GC Jr.IN-93 purified diets for laboratory rodents: final

eport of the American Institute of Nutrition Adoc Writing Committee on the Reformulation of

he AIN-76A Rodent Diet. J Nutr 1993;123:939-51.6. Matthews DR, Hosker JP, Rudenski AS,aylor BA, Treacher DF, Turner RC. Homeosta-is model assessment: insulin resistance andeta-cell function from fasting plasma glucosend insulin concentrations in man. Diabetologia985;28:412-9.7. Aguila MB, Pinheiro AR, Parente LB, Man-arim-de-Lacerda CA. Dietary effect of differentigh-fat diet on rat liver stereology. Liver Int003;23:363-70.8. Souza-Mello V, Mandarim-de-Lacerda CA,guila MB. Hepatic structural alteration in adultrogrammed offspring (severe maternal proteinestriction) is aggravated by post-weaning high-at diet. Br J Nutr 2007;98:1159-69.9. Srinivasan M, Katewa SD, Palaniyappan A,andya JD, Patel MS. Maternal high-fat dietonsumption results in fetal malprogrammingredisposing to the onset of metabolic syn-rome-like phenotype in adulthood. Am Jhysiol Endocrinol Metab 2006;291:E792-9.0. Brion MJ, Ness AR, Rogers I, et al. Maternalacronutrient and energy intakes in pregnancy

nd offspring intake at 10 y: exploring parentalomparisons and prenatal effects. Am J Clinutr 2010;91:748-56.1. Gallou-Kabani C, Vige A, Gross MS, et al.57BL/6J and A/J mice fed a high-fat diet de-

ineate components of metabolic syndrome.besity (Silver Spring) 2007;15:1996-2005.2. Parente LB, Aguila MB, Mandarim-de-
acerda CA. Deleterious effects of high-fat diet

or2ftL2pd2miohc2C

vs32Nd12c2Nhst3M

Cr3h23g23Repo2


4

n perinatal and postweaning periods in adultat offspring. Clin Nutr 2008;27:623-34.3. Gniuli D, Calcagno A, Caristo ME, et al. Ef-

ects of high-fat diet exposure during fetal life onype 2 diabetes development in the progeny. Jipid Res 2008;49:1936-45.4. Gluckman PD, Hanson MA. Living with theast: evolution, development, and patterns ofisease. Science 2004;305:1733-6.5. Akagiri S, Naito Y, Ichikawa H, et al. Aouse model of metabolic syndrome; increase

n visceral adipose tissue precedes the devel-pment of fatty liver and insulin resistance inigh-fat diet-fed male KK/Ta mice. J Clin Bio-hem Nutr 2008;42:150-7.6. McCuskey RS, Ito Y, Robertson GR, Mc-
uskey MK, Perry M, Farrell GC. Hepatic micro- d

ascular dysfunction during evolution of dietaryteatohepatitis in mice. Hepatology 2004;40:86-93.7. Wei Y, Rector RS, Thyfault JP, Ibdah JA.onalcoholic fatty liver disease and mitochon-rial dysfunction. World J Gastroenterol 2008;4:193-9.8. Pessayre D, Fromenty B. NASH: a mito-hondrial disease. J Hepatol 2005;42:928-40.9. Brunt EM, Janney CG, Di Bisceglie AM,euschwander-Tetri BA, Bacon BR. Nonalco-olic steatohepatitis: a proposal for grading andtaging the histological lesions. Am J Gastroen-erol 1999;94:2467-74.0. Marseille-Tremblay C, Gravel A, Lafond J,ounier C. Effect of an enriched cholesterol diet
uring gestation on fatty acid synthase, HMG-
gy NOVEMBER 2010

oA reductase and SREBP-1/2 expressions inabbits. Life Sci 2007;81:772-8.1. Quinn R. Comparing rat’s to human’s age:ow old is my rat in people years? Nutrition005;21:775-7.2. Ozanne SE, Hales CN. Lifespan: catch-uprowth and obesity in male mice. Nature004;427:411-2.3. Gallou-Kabani C, Vige A, Gross MS, et al.esistance to high-fat diet in the female prog-ny of obese mice fed a control diet during theericonceptual, gestation, and lactation peri-ds. Am J Physiol Endocrinol Metab 2007;92:E1095-100.

maternal high-fat intake predisposes nonalcoholic fatty liver disease in c57bl/6 offspring

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