Pioglitazone therapy in mouse offspring exposed to maternal obesity

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<ul><li><p>OPeAr thleMa</p><p>OBdinadcepproinsyn</p><p>STmopup ng wsig thylme s. Ttre ecosub g mmo r treinvasive microendoscopic fluorescence confocal imaging and intraperi-</p><p>tesical</p><p>suantriulinnd</p><p>m Pc chlict acs toop m</p><p>mmmaternal obesity, mice, offspring, pioglitazone</p><p>Cite201</p><p>Adrooffol</p><p>anepedingnd</p><p>relative risk of childhood obesity associ-</p><p>Fro ent,By ngiPa nch</p><p>Re</p><p>Thi cieTex nce(UL es,of H rdBu m thInstitute of Allergy and Infectious Diseases, the Eunice Kennedy Shriver National Institute of ChildHe</p><p>TherepKeDir</p><p>The</p><p>Pre6-1</p><p>Re</p><p>000</p><p>Research www.AJOG.org</p><p>30atedwithmaternal obesity in the first tri-mester of pregnancy was found to be in-creased by 2-fold at age 2-3 years and 2.3-fold at 4 years of age.3</p><p>MetS was found to be more commonin adolescents who were born to obesemothers.5 Others have shown that themothers body mass index is one of thestrongest predictors of overweight/obe-</p><p>alth and Human Development, and the Office of the Director, National Institutes of Health.</p><p>views expressed herein are solely the responsibility of the authors and do not necessarilyresent the official views of the National Institute of Allergy and Infectious Diseases, the Eunicennedy Shriver National Institute of Child Health and Human Development, the Office of theector, or the National Institutes of Health.</p><p>authors report no conflict of interest.</p><p>sented at the 32nd annual meeting of the Society for Maternal-Fetal Medicine, Dallas, TX, Feb.1, 2012.</p><p>prints not available from the authors.ccording to the International Dia-betes Federation, metabolic syn-</p><p>me (MetS) is defined by the presencecentral obesity and at least 2 of thelowing components: hypertension,</p><p>glucose intolerance,The MetS has reachedtions in many developsequently, understandpotential of obesity a</p><p>m the Departments of Obstetrics and Gynecology (Drs Kalanderian, Vinctautiene) and Internal Medicine (Dr Abate) and the Center for Biomedical Etrikeev, Wei, Vincent, and Motamedi), The University of Texas Medical Bra</p><p>ceived Sept. 7, 2012; revised Dec. 7, 2012; accepted Jan. 9, 2013.</p><p>s study was conducted with the support of the Institute for Translational Sas Medical Branch, supported in part by a Clinical and Translational Scie1TR000071) from the National Center for Advancing Translational Sciencealth. Dr Bytautiene is supported by a research career development awa</p><p>ilding Interdisciplinary Research Careers in Womens Health Program) fro2-9378/$36.00 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/</p><p>8.e1 American Journal of Obstetrics&amp; Gynecology APRIL 2013d dyslipidemia.idemic propor-countries. Con-the detrimentalits unfavorable</p><p>consequences is vital to the developmentof preventative measures.1</p><p>Traditionally, metabolic syndromeand obesity research has focused on life-style factors in childhood and adult-hood.More recently the role of pregnancyin the obesity epidemic is beginning to berecognized.The rate of obesity inpregnantwomen worldwide is between 18.5% and38.3%, and these rates continue to rise.2</p><p>Epidemiological and experimental studiesdemonstrate that offspring from obesemothers are at an increased risk of obe-sity as well as other features and com-plications of MetS.3-7 For example, the</p><p>Saade, andneering (Drs, Galveston, TX.</p><p>nces, University ofAwardNational Institutes</p><p>(K12HD052023:e Nationalthis article as: Kalanderian A, Abate N, Patrikeev I, et al. Pioglitazone therapy in mouse offspring exposed to maternal obesity. Am J Obstet Gynecol3;208:308.e1-7.BSTETRICS</p><p>ioglitazone therapy ixposed to maternalshag Kalanderian, MD; Nicola Abate, MDssoud Motamedi, PhD; George Robert S</p><p>JECTIVE: Pioglitazone (PIO), an antidiabeticedione family, improves glucose and lipid mipose, and liver tissues via peroxisome proltor gamma activation. We hypothesize thatve the metabolic status of offspring exposea mouse model developmentally programdrome.</p><p>UDY DESIGN: CD-1 female mice were fednths prior to breeding and throughout pregnans were weaned to a standard-fat diet. Offsprined to receive 40 mg/kg of PIO in 0.5% of methyl cellulose by daily oral gavage for 2 weekatment total body weights of the pups were rcutaneous adipose tissue were evaluated usingraphy. Serum analytes were measured. Aftemouse offspringesity</p><p>gor Patrikeev, PhD; Jingna Wei, MD; Kae, MD; Egle Bytautiene, MD, PhD</p><p>g of the thiazoli-bolism in muscle,ator-activated re-therapy will im-maternal obesityed for metabolic</p><p>igh-fat diet for 3and lactation. Theere randomly as-cellulose or 0.5%he pre- and post-rded. Visceral andicrocomputed to-atment, minimally</p><p>toneal glucose toleranceusing appropriate statist</p><p>RESULTS: PIO therapy receral adipose tissue gainPIO significantly loweredmodel assessment of insfemales. There was a tre</p><p>CONCLUSION: Short-terers attenuates metaboliprogramming of metabotential role for drugs thaceptor gamma receptoroffspring at risk to devel</p><p>Key words: fetal prograsitj.ajog.2013.01.013en Listiak Vincent, MD;</p><p>ts were performed. The data were analyzedtests (significance, P .05).</p><p>lted in lower total body weight and lower vis-d increased subcutaneous adipose tissue.glycerides, insulin levels, and homeostasisresistance in males and fasting glucose in</p><p>toward larger adipocyte size.</p><p>IO therapy in the offspring of obese moth-anges associated with the developmentalsyndrome. These novel data suggest a po-tivate peroxisome proliferator-activated re-prevent metabolic syndrome in the adultetabolic alterations.</p><p>ing, in vivo adipose tissue imaging,nob; Iaad</p><p>druetaiferPIO</p><p>d tom</p><p>a hcyy and percentage of body fat in her</p></li><li><p>chersthoovonmaThdecorisMe</p><p>emtotiotiocesancarincthainmofunclucreauhysomgamtivphlev</p><p>ancrianPPingactmopreincspristdin(TabtheveltheofoffthoI</p><p>tha</p><p>ogprosprexpingpe</p><p>MAllAnUnanatuwicycwa</p><p>StuInmofeemonaofsigvatwhmo</p><p>CDRivFewefedD1widuwe(mthe70stesontwanoil</p><p>subsuranMimiagidivgro</p><p>mefemtazMOlosPit(C4)(0.wa2 wT</p><p>thefrowhasinwethetolmeanOntrelecsistheadidian</p><p>InPrforceradrodpromaopmoistke5-1agenetiowe50timdeprowecon</p><p>www.AJOG.org Obstetrics Researchildren.4,6 Adults born to obese moth-were more insulin resistant thanse born to nonobesemothers.8More-er, maternal prenatal fat intake wase of the strongest predictors of fatss in the offspring at 10 years of age.9</p><p>us, there is strong accumulating evi-nce that offspring born of pregnanciesmplicated by obesity are at increasedk of obesity and other features oftS.Visceral adipose tissue dysfunction iserging as the primary defect leadingMetS.10,11 Pathological lipid accumula-n and adipocyte hypertrophy, in addi-n to hypoxia and a variety of other pro-ses, lead to adipose tissue dysfunctiond the development of metabolic and/ordiovascular disorders. Previous studies,luding our own, have demonstratedt adipose tissue dysfunction is presentfetal programmingmodels ofMetS.Thest notable examples of adipose dys-ction in fetal programmingmodels in-de elevated serum leptin levels, in-ased adipose tissue lipid accumulation,gmented expression of angiotensin,poxia-inducible factor-1 and peroxi-e proliferator-activated receptorma (PPAR) receptor, amplified ac-</p><p>itiesof lipoprotein lipaseandglycerol-3-osphate dehydrogenase, and decreasedels of antioxidant enzymes.7,12-15</p><p>PPAR is highly expressed in whited brown adipose tissue and plays atical role in adipocyte differentiationd mature adipocyte maintenance.AR regulates systemic insulin signal-via ligand-dependent transcriptionalivation of target genes. In animaldels, messenger ribonucleic acid ex-ssion of the PPAR is significantlyreased in the adipose tissue of the off-ing of obese dams.7,12 When admin-ered to pregnant animals, thiazoli-ediones (TZDs), PPAR agonistsZDs) improvematernal and fetal met-olic markers.16-20 However, none ofse studies followed up offspring de-opment into adulthood, nor werere studies that investigated the effectsTZDs if administered postnatally tospring at risk to developMetS, such asse born to obese mothers.</p><p>n this study, we tested the hypothesist postnatal administration of the pi- samlitazone, a TZD class drug, will im-ve the metabolic status of adult off-ing at risk to developMetS because ofosure to amaternal high-fat diet dur-the prenatal and the early postnatal</p><p>riods.</p><p>ATERIALS AND METHODSprocedures were approved by theimal Care and Use Committee of theiversity of Texas Medical Branch. Theimalswerehoused separately in temper-re- and humidity-controlled quartersth constant 12 hour light, 12 hour darkles and were provided with food andter ad libitum.</p><p>dy designour laboratory, we have established ause model of developmental MetS byding female mice a high-fat diet for 3nths before pregnancy, during preg-ncy, and until weaning. At 6 monthsage, offspring of these dams developnificant adiposity, hypertension, ele-ed fasting glycemia, and dyslipidemiaen compared with pups born tothers fed standard chow.21,22</p><p>For the current study, female andmale-1 mice were obtained from Charlesers Laboratories (Wilmington, MA).male mice were approximately 4-5eks of age upon arrival. Females werehigh-fat rodent chow (34.9% fat;</p><p>2492; ResearchDiets Inc, NewBruns-ck,NJ) for 3months prior to breeding,ring pregnancy, and until weaning (3eks postpartum), when all animalsothers and offspring) were placed onstandard chow diet (5.6% fat; Teklad</p><p>12: Harlan Teklad LM-485 mouse/ratrilizable diet; Harlan Teklad, Madi-, WI). The fat source differed be-een diets: the high-fat diet used lard,d the standard-fat diet used soybean.At 8 weeks of age, the offspring werejected to fasting blood glucose mea-ements and blood collection for lateralysis of triglyceride and insulin levels.ce also underwent in vivowhole-bodycrocomputed tomography (CT) im-ng. At 10 weeks of age, the pups wereided into control and treatmentups to include animals born to the</p><p>e mother in both groups. The treat- pe</p><p>APRIL 2013 Americannt group (PIO group; males, n 6,ales, n 4) received 40mg/kg piogli-one (PIO; Sigma-Aldrich, St Louis,) dissolved in 0.5% of methyl cellu-</p><p>e by oral gavage (Fisher Scientific,tsburgh, PA).23 The control groupTR group; males, n 6, females, nreceived a similar amount of vehicle5%methyl cellulose) only. Treatments given daily (except on weekends) foreeks, until 12 weeks of age.he selection of dosage and timing oftreatment was based on evidence</p><p>m literature, in which db/db mice,ich are at risk to develop MetS (sameour animals), showed improvementstheir metabolic parameters after 2-3eks of PIO treatment.23,24 Followingtreatment, intraperitoneal glucose</p><p>erance tests (IGTT), fasting glucoseasurements, and in vivo micro-CTd confocal imaging were performed.e male per group died during post-atmentmanipulations. Bloodwas col-ted for insulin and triglyceride analy-from the remaining animals, and thenoffspring were euthanized. Visceral</p><p>ipose tissue, includingmesenteric, ep-dymal, and perirenal fat, was excisedd weighed.</p><p>vivo imagingeviously validated imaging methodsthe quantitative assessment of vis-al (VAT) and subcutaneous (SAT)ipose tissue and liver fat infiltration inents were used.25-27 Experimentaltocols for noninvasive CT and mini-lly invasive confocal imaging weretimized in our laboratory over severalnths prior to the study. After admin-ering anesthesia intraperitoneally withtamine/xylazine (80-100 mg/kg and0mg/kg, respectively), mice were im-d with a small animal micro-CT scan-r (Inveon; Siemens Preclinical Solu-ns,Knoxville, TN). Imagingparametersre set as follows: voltage 70 kV, current0 A, resolution 0.107 mm, exposuree 1000 milliseconds, 520 steps and 360grees of rotations. Scan time was ap-ximately 15 minutes per mouse. Micere given supplemental oxygen via nosee during scanning.</p><p>The transverse views of CT images (1</p><p>r animal) at the level of the fifth lum-</p><p>Journal of Obstetrics&amp; Gynecology 308.e2</p></li><li><p>baVAlevfortioareseacen(%sectiomibyPfor</p><p>athoexpanradthesplinfi</p><p>meoffimcen48Keres(0.themmdomepoInsthetrawianeacuse</p><p>livsiztro</p><p>IntIGweMhog/k</p><p>tiopri60tiodostubloprisulmewasay</p><p>SeBlineS(1fromusstrtritemtal</p><p>DaDafronuasIGus</p><p>meresbydeunnifipatesuteingofAsig</p><p>TB ona</p><p>V No</p><p>B 3. .........</p><p>V 2. .........</p><p>S. .........</p><p>L. .........</p><p>T. .........</p><p>F 17. .........</p><p>F. .........</p><p>H. .........</p><p>DC ssesssa .</p><p>K of m</p><p>Research Obstetrics www.AJOG.org</p><p>30r vertebra were selected for analysis ofT, whereas transverse images at theel of sixth lumbar vertebra were usedanalysis of SAT.25,26 The cross-sec-nal total body area and adipose tissuea were measured using Inveon Re-rch Workplace software. The per-tage of VAT (%VAT) and SATSAT) was calculated from the cross-tional total body area. The correla-ns between the weight of VAT deter-ned after extraction and determinedCTwere significant (Pearson r 0.82,.003 for males and r 0.82, P .01females).</p><p>Liver and spleen radiodensity values intransverse section between the 13thracic and first lumbar vertebrae wereressed as liver to spleen density ratiod compared between the groups. Theiodensity of the liver normalized toradiodensity of the spleen (liver to</p><p>een radiodensity ratio) indicated fatltration in the liver.25,26,28,29</p><p>To quantitatively assess PIO treat-nt-induced changes in adipocyte size,springunderwent high-resolutionmin-ally invasive microendoscopic fluores-ce confocal imagingusing aCell-Vizio-8 confocal microendoscope (Maunaa Technologies, Paris, France). A fluo-cent 1% fluorescein sodium solution2-0.3 mL per animal) was injected viatail vein or intraperitoneally. A 1.8probe was inserted through an ab-</p><p>minal incision. Confocal images ofsenteric adipocytes in VAT were ex-rted into Image J software (Nationaltitutes of Health, Bethesda,MD), andcell area was measured by manuallycing the cell borders.30 Every cellthin 5 imaging fields was measured,d the mean cell area was calculated forh animal. The mean area values wered to compare the 2 groups.Individuals interpreting VAT, SATer/spleen radiodensity, and adipocytee were blinded to the treatment/con-l group allocation.</p><p>raperitoneal glucose tolerance testTTs were performed following 2eks of PIO or vehicle administration.ice were fasted overnight (16-18urs) prior to a 20 % glucose bolus (1</p><p>gpermouse) by intraperitoneal injec-</p><p>Pasen</p><p>8.e3 American Journal of Obstetrics&amp; Gynecologyn. Blood glucose levels were recordedor to glucose injection and at 15, 30,, 90, and 120 minutes after the injec-n. Overnight fasting and the glucosese were selected based on our pilotdies inCD-1mice. Sixtymicroliters ofod were collected via the tail veinor to glucose injection for fasting in-in measurements. The blood was im-diately centrifuged, and the serums stored at80C until the insulin as-was performed.</p><p>rologic analysisod glucose levels were measured us-an OneTouch Ultra glucometer (Lif-an, Milpitas, CA) after an overnight-18 hours) fast. Blood was collectedm the offspring before and after treat-nt. Commercially available kits wered according to themanufacturers in-uctions to determine serum levels ofglycerides, cholesterol (BioAssay Sys-s, Hayward, CA), and insulin (Crys-</p><p>Chem, Downers Grove, IL).</p><p>ta analysista were analyzed as the differencem baseline (posttreatment value mi-s pretreatment value) and expressedthe mean SEM. The area under theTT curve for glucos...</p></li></ul>

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