Insulin Therapy for Gestational Diabetes Mellitus DoesNot Fully Protect Offspring From Diet-InducedMetabolic DisordersHong Zhu,1 Bin Chen,1 Yi Cheng,1,2 Yin Zhou,1 Yi-Shang Yan,1 Qiong Luo,3 Ying Jiang,3 Jian-Zhong Sheng,1,2

Guo-Lian Ding,4,5 and He-Feng Huang1,4,5

Diabetes 2019;68:696–708 | https://doi.org/10.2337/db18-1151

Gestational diabetes mellitus (GDM) is associated withan increased risk of metabolic disorders in offspring inlater life. Although mounting evidence suggests thattherapy for GDM could improve neonatal health, whetherthe therapy confers long-term metabolic benefits to off-spring in their later adult lives is not known. Here, usinga mouse model of diabetes in the latter half of pregnancyto mimic human GDM, we find that the efficient insulintherapy for GDM confers significant protection againstglucose intolerance and obesity in offspring fed a normalchow diet. However, the therapy fails to protect offspringwhen challenged with a high-fat diet, especially for maleoffspring. Genome-wide DNA methylation profiling ofpancreatic islets from male offspring identified hyper-methylated regions in several genes that regulate insulinsecretion, including Abcc8, Cav1.2, and Cav2.3 that en-code KATP or Ca2+ channels, which are associated withreduced gene expression and impaired insulin secretion.This finding suggests amethylation-mediated epigeneticmechanism for GDM-induced intergenerational glucoseintolerance. It highlights that even efficient insulin ther-apy for GDM is insufficient to fully protect adult offspringfrom diet-induced metabolic disorders.

Gestational diabetes mellitus (GDM), defined as glucoseintolerance first diagnosed during pregnancy, affects up to15% of pregnancies in the world (1). GDM is associated

with adverse consequences not only during fetal develop-ment, such as stillbirth, visceromegaly, and macrosomia,but also later in life (2,3). Accumulating evidence suggeststhat GDM, independent of maternal obesity and geneticbackground, predisposes offspring to metabolic disordersin later life, such as obesity, impaired glucose tolerance,and diabetes (4–6). Longitudinal studies of GDM offspringindicate that the maternal glucose level is a strong pre-dictor of altered carbohydrate metabolism during child-hood, which can be extended into adulthood (7,8).

The therapeutic management of GDM is critical tominimize these complications. Glycemic control is thecornerstone of GDM management (9). Randomized trialshave confirmed that the therapy for GDM confers imme-diate benefits, such as reduction of perinatal complicationsand prevalence of macrosomia (10,11). However, whetherthe therapy for GDM confers long-term metabolic benefitsto offspring is still unclear (12,13). Importantly, theappropriate offspring follow-up period for assessing theeffects of GDM therapy is still debatable. Offspring en-rolled in most follow-up studies were prepubertal (age 5–10 years), yet the long-term effect of GDM on offspringmetabolic disorders or its reduction through therapymight not be evident until adolescence or adulthood(12,14).

In addition, offspring sex also has a profound effect.Sexual dimorphism in the response to insult in utero

1The Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry ofEducation, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China2Department of Pathology and Pathophysiology, Zhejiang University School ofMedicine, Hangzhou, Zhejiang, China3Department of Obstetrics, Women’s Hospital, Zhejiang University School ofMedicine, Hangzhou, China4The International Peace Maternity and Child Health Hospital, Institute of Embryo-Fetal Original Adult Disease, School of Medicine, Shanghai Jiao Tong University,Shanghai, China5Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China

Corresponding author: He-Feng Huang, hhf57@zju.edu.cn, or Guo-Lian Ding,dingguolian@hotmail.com

Received 25 October 2018 and accepted 18 January 2019

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1151/-/DC1.

H.Z. and B.C. contributed equally to this study.

© 2019 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

696 Diabetes Volume 68, April 2019

METABOLISM

presents with an uneven disease susceptibility: althoughboth sexes can be affected, one is more susceptible (15).Metabolic differences exist between male and femalefetuses (15), and differing sensitivities to maternal hyper-glycemia may result in sex-specific disease risks later inlife (6,16). Evidence from epidemiological studies demon-strates that the effect of therapy for GDM also differs withfetal sex in infancy and childhood (11,16). Therefore, fetalsex may influence the effect that therapy for GDM has onoffspring long-term health. Moreover, social and environ-mental factors could also confound the follow-up results.Thus, whether therapy for GDM is a modifiable risk factorfor offspring metabolic disorders remains unknown.

Mechanistically, epigenetic modifications, such as DNAmethylation, histone modification, and noncoding RNAs,provide a plausible link between environmental exposuresearly in development and the susceptibility to diseaseslater in life (17). DNA methylation, the most studiedepigenetic modification, can alter the status of gene ex-pression and be inherited mitotically in somatic cells(17,18), which provides a potential mechanism by whichenvironmental effects on the epigenome could have long-term effects on gene expression. Results from animal andhuman studies support that intrauterine hyperglycemiacould result in altered fetal DNA methylation patterns andsubsequent changes in the risk of developing disease(6,19). In epidemiological and experimental studies, gly-cemic control improved GDM neonatal outcomes. How-ever, no studies investigated whether these positive effectswere accompanied by a favorable restoration of DNAmethylation (20), which may be critically important forthe susceptibility to disease later in life. Thus, the possi-bility that fetal metabolic programming in GDM can havelong-term effect on health that may in turn be modified bytherapy for GDM remains open to question.

Because excluding the confounders and analyzing theunderlying mechanisms in humans is difficult, we estab-lished a mouse model of diabetes in the latter half ofpregnancy to mimic human GDM with a high incidenceduring the third trimester of gestation (21). We treatedmaternal hyperglycemia with insulin and evaluated thepancreatic islet b-cell function in offspring. We alsoaddressed whether lifestyle factors in adulthood, such asa high-fat diet (HFD), may increase the risk of developingmetabolic disorders in offspring. Finally, we performedgenome-wide DNA methylation sequencing in offspringpancreatic islets and assessed the alterations of candidategenes that may contribute to metabolic phenotypes inoffspring.

RESEARCH DESIGN AND METHODS

Animal CareThe Zhejiang University Animal Care and Use Committeeapproved all animal protocols. All experiments were per-formed with Institute of Cancer Research (ICR) mice (22),which were purchased from Shanghai SLAC LaboratoryAnimal Co. (Shanghai, China). Virgin ICR females (age,

6–8 weeks; weight, 26–28 g) were mated with normal ICRmales. Pregnancy was dated by the presence of a vaginalplug (day 0.5). Pregnant females were randomly assignedto control (Ctrl), GDM, or GDM + insulin therapy (INS)groups. On day 6 and day 12 of pregnancy, GDM and INSdams were fasted 8 h and received a streptozotocin (STZ)injection (100 mg/kg i.p.) (Sigma-Aldrich, St. Louis, MO)(23,24) (Fig. 1A). Control pregnant mice received an equalvolume of citrate buffer. Blood glucose level was measuredvia the tail vein 48–72 h after the second STZ injection,and diabetes was defined as a blood glucose level between14 and 19 mmol/L (6).

INS dams were treated with recombinant insulin (Novo-lin R; Novo Nordisk, Bagsvaerd, Denmark) by miniosmoticpumps (Alzet model 1007D; Durect, Cupertino, CA) ata dose of 0.35 IU/day. Pumps were implanted in INS damson day 14.5 or 15 of pregnancy posterior to the scapulaeunder Avertin (Sigma-Aldrich) anesthesia (0.1 mL/20 gbody wt). Control and GDM dams were implanted withpumps containing normal saline. To maintain stable gly-cemic levels, INS dams received another injection of 0.1units of long-acting insulin (Levemir; Novo Nordisk) ;1 hbefore the fed state (darkness) during late gestation. Onlymice with nearly normal blood glucose levels in INS groupwere included in the further study.

At birth, litter size was equalized to eight. Pups from theGDM and INS groups were fostered by normal femalesuntil the age of 3 weeks. Offspring were designated as Ctrl-F1, GDM-F1, and INS-F1. At 8 weeks of age, offspring weredivided into two groups either receiving a normal chowdiet (NCD) or HFD (60% energy as fat; D12492; ResearchDiets, New Brunswick, NJ) until 20 weeks (Fig. 1A).

In Vivo Metabolic TestingIntraperitoneal glucose tolerance tests (2 g/kg body wt)and insulin tolerance tests (ITT) (0.8 unit/kg body wt) wereperformed in unrestrained conscious mice after a 16- and4 h-fast, respectively. Serum insulin level was assessed atovernight fasted state and 30 min after the glucose in-jection (Crystal Chem, Downers Grove, IL).

Serum AnalysisSerum cholesterol, triglyceride, nonesterified fatty acids,HDL, and LDL were assayed using a biochemical analyzer(TBA120FR; Toshiba, Tokyo, Japan). Serum leptin wasdetermined by radioimmunoassay (North Institute, Bei-jing, China). HOMA-insulin resistance (IR) was calculatedas follows: fasting serum insulin concentration (mU/mL)multiplied by fasting blood glucose level (mg/dL) dividedby 405 (25).

Islet Isolation and In Vitro Insulin SecretionPancreatic islets were isolated from 20-week-old mice aspreviously described (26). For the indicated experiment,10 islets (size-matched for each batches) were incubatedfor 1 h at 37°C in modified Krebs-Ringer bicarbonatebuffer containing 2.8 mmol/L glucose and then incu-bated in the modified Krebs-Ringer bicarbonate buffer

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containing indicated glucose or various chemical com-pounds, including 200 mmol/L tolbutamide (Sigma-Aldrich),250 mmol/L diazoxide (Sigma-Aldrich), 2 mmol/L BayK8644 (Sigma-Aldrich), and 10 mmol/L nifedipine (Sigma-Aldrich). The supernatant was collected for insulin contentassay (Abnova, Taipei, China).

The pancreata of fetal mice at embryonic day 17 wasdirectly digested in 2 mg/mL collagenase with shaking in-cubation at 37°C for 25 min. After recovering overnightin RPMI medium, fetal islets were handpicked under a ste-reomicroscope and randomly separated into 5.6 mmol/L,16.7/5.6 mmol/L, and 16.7 mmol/L glucose groups (Fig. 6A).

Figure 1—Experimental design, offspring growth curves, and glucose tolerance. A: Experimental design. B: Maternal blood glucose levelduring pregnancy (n = 6 mice per group). C: Postnatal growth curves for male offspring (nCtrl-F1_NCD = 8, nINS-F1_NCD = 10, nGDM-F1_NCD = 10,nCtrl-F1_HFD = 8, nINS-F1_HFD = 8, nGDM-F1_HFD = 10). AUC, area under the curve; a.u. arbitrary units. D: Postnatal growth curves for femaleoffspring (nCtrl-F1_NCD = 7, nINS-F1_NCD = 7, nGDM-F1_NCD = 8, nCtrl-F1_HFD = 6, nINS-F1_HFD = 9, nGDM-F1_HFD = 7). E: Glucose tolerance test andAUC of 20-week-old F1 male offspring (nCtrl-F1_NCD = 6, nINS-F1_NCD = 7, nGDM-F1_NCD = 7, nCtrl-F1_HFD = 6, nINS-F1_HFD = 7, nGDM-F1_HFD = 6). F:Glucose tolerance test and AUC of 20-week-old F1 female offspring (nCtrl-F1_NCD = 5, nINS-F1_NCD = 5, nGDM-F1_NCD = 6, nCtrl-F1_HFD = 5, nINS-F1_HFD=6, nGDM-F1_HFD = 6). G: ITT in 20-week-old F1 male offspring (nCtrl-F1_NCD = 6, nINS-F1_NCD = 8, nGDM-F1_NCD = 6, nCtrl-F1_HFD = 8, nINS-F1_HFD = 9,nGDM-F1_HFD = 10). H: ITT in 20-week-old F1 female offspring (nCtrl-F1_NCD = 5, nINS-F1_NCD = 6, nGDM-F1_NCD = 5, nCtrl-F1_HFD = 6, nINS-F1_HFD = 7,nGDM-F1_HFD = 8). Data are expressed as mean 6 SEM. *P , 0.05 vs. Ctrl-F1; **P , 0.01 vs. Ctrl-F1. #P , 0.05 vs. INS-F1; ##P , 0.01vs. INS-F1 (ANOVA).

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Methylated DNA Immunoprecipitation SequencingMale offspring fed the NCDwere chosen at 20 weeks of agefor methylated DNA immunoprecipitation sequencing(MeDIP-seq) analysis. Genomic DNA of pancreatic isletswas extracted from nine mice per group and pooled by eachof the three. The MeDIP-seq was performed as describedpreviously (27). Briefly, DNA was sonicated to obtain theDNA fragments (200–700 base pairs). Sonicated genomicDNA was denatured and immunoprecipitated with anti–5-methylcytosine antibody (#28692; Cell Signaling, Danvers,MA). Illumina libraries were then created from the cap-tured DNA by TruSeq DNA LT Sample Prep Kit (Illumina,San Diego, CA). The samples were sequenced on theIllumina HiSeq 3000 system (Illumina).

Gene ExpressionTotal RNA from isolated pancreatic islets was extracted byusing the RNeasy Micro Kit (Qiagen, Valencia, CA). cDNAwas synthesized using oligo-deoxythymidylic acid and ran-dom primers (TaKaRa, Dalian, China) for real-time quanti-tative PCR with SYBR green detection (TaKaRa). The primersequences are provided in Supplementary Table 1.

DNA MethylationGenomic DNA was extracted from islets by using theTIANmap Micro DNA kit (Tiangen, Beijing, China). Bi-sulfite was converted using the EpiTect bisulfite kit (Qia-gen) according to the manufacturer’s instructions. Themethylation status of gene promoter regions was analyzedby pyrosequencing (28). In brief, pyrosequencing primerswere designed by Qiagen PyroMark Assay Design 2.0software (Qiagen) (Supplementary Table 2). The specificityof each PCR product was checked by agarose gel analysis.Pyrosequencing was conducted on a PyroMark Q24 pyro-sequencer (Qiagen) by using PyroMark Gold Q24 reagents(Qiagen), and quantification of methylated and unmethylatedalleles were performed by PyroMark Q24 software (Qiagen).

Immunofluorescence AnalysisFetal islets were identified by detecting insulin with immu-nofluorescence as previously described (29). Isolated fetalislets were incubated with antibody against insulin. Allimmunofluorescence images were acquired by laser scan-ning confocal microscope (Zeiss, Jena, Germany). The anti-bodies used are described in Supplementary Table 3.

Western BlotsThe protein was extracted from mouse islets as describedbefore (29). Samples were separated using 8% SDS-PAGE.Western blots were performed using polyvinylidene fluo-ride membrane and the antibodies listed in SupplementaryTable 3. Protein bands were visualized by the enhancedchemiluminescence system (Pierce, Rockford, IL).

STZ-Injected Nondiabetic ModelThe identical amount of STZ was injected in pregnant miceas described above. Only a glucose level of ,7.5 mmol/Lwas considered as an STZ-injected nondiabetic model.

Offspring were fostered by normal females until 3 weeksold. Mice were fed the HFD, and glucose tolerance test, andITT were performed as described above. Islets from theSTZ-injected nondiabetic offspring were isolated for fur-ther analysis.

Statistical AnalysisAll data are shown as mean 6 SEM. Statistical analysiswere performed by one-way ANOVA, followed by leastsignificant difference post hoc test and two-tailed Studentt test, as described in the table and figure legends, usingSPSS 17.0 software. P , 0.05 was considered statisticallysignificant.

RESULTS

Insulin Therapy for GDM Conferred Offspring a PartialProtection From Metabolic DisordersWe established a mousemodel of hyperglycemia during themidlate stage of pregnancy. GDM dams were averaginga 328.6mg/dL plasma glucose level after two STZ injections.Blood glucose level declined and averaged 129.5 mg/dLduring insulin therapy in the INS group (Fig. 1B). Gesta-tional length, litter size, and birth weight were similaramong the three groups (Supplementary Table 4 and Sup-plementary Fig. 1).

Notably, increased body weight and most metabolicabnormalities were seen in GDM-F1 adult offspring(Fig. 1C and D and Table 1). Insulin therapy was associatedwith normal weight trajectory, serum glucose, and lipidmetabolism in adult offspring (Fig. 1C and D and Table 1).However, these associations were observed only in NCDoffspring. When fed the HFD after 8 weeks of age, a sig-nificant increase in body weight (Fig. 1C), fasting insulinlevels, and lipid levels was seen in INS-F1 males (Table1).Females in HFD INS-F1 group only exhibited increasedbody weight compared with HFD Ctrl-F1 females (Fig. 1D).

Insulin Therapy–Mediated Protection Against GlucoseIntolerance in Offspring Was Abolished by HFDExposure in AdulthoodInsulin therapy for GDM resulted in a distinct rescue ofglucose intolerance in INS-F1 male offspring, with only anincrease in glucose levels at 60 min after injection (Fig. 1E).But strikingly, the HFD abolished this protection (Fig.1E). Results of the ITT showed only GDM-F1 male miceexhibited significant impaired insulin sensitivity with ag-ing in the NCD group (Supplementary Fig. 2B and Fig. 1G).However, when challenged with the HFD, not only GDM-F1 males developed much more serious insulin intolerance,but INS-F1 males also exhibited a pronounced impairmentof insulin tolerance compared with controls (Fig. 1G). Infemale offspring, only GDM-F1 females showed an eleva-tion of glucose level at 30 and 120 min after the insulininjection (Fig. 1H).

Insulin secretion defects could also contribute to glu-cose intolerance. We assessed glucose-stimulated insulinsecretion (GSIS) in vivo and in vitro. In vivo, GSISwas reducedin both male and female GDM-F1 offspring (Fig. 2A–D).

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In the INS-F1 group, only males exhibited lower insulinlevels in response to the glucose injection (Fig. 2A and B).In vitro, insulin secretory response to 5.6 mmol/L glucosewas similar among all groups (Fig. 2E and F); however,islets of GDM-F1 males or females showed impairedinsulin secretion when exposed to 16.7 mmol/L glucose(Fig. 2E and F). Defective insulin response to high glucose(16.7 mmol/L) was also seen in INS-F1 males (Fig. 2E). Nosignificant difference was found in INS-F1 females inglucose tolerance, insulin sensitivity, or GSIS (Figs. 1Fand H and 2C, D, and F).

Insulin Therapy for GDM Did Not Restore the AlteredDNA Methylation in Offspring Pancreatic IsletsGDM altered the methylation of 220 upstream2k (8.11%),201 downstream2k (7.41%), 77 59 untranslated region(UTR) (2.84%), 55 39UTR (2.03%), 711 coding sequence(26.2%), and 1,448 intron element-associated genes(53.37%) (Fig. 3B and Supplementary Table 5) in isletsof GDM-F1 male offspring, respectively. Global cytosinemethylation status was also altered in INS-F1 off-spring, and the methylation of 258 upstream2k (8.37%),237 downstream2k (7.69%), 81 59UTR (2.63%), 70 39UTR(2.27%), 745 coding sequence (24.17%), and 1,690 intronelement-associated genes (54.83%) was changed, respec-tively (Fig. 3B and Supplementary Table 6). In GDM-F1,there were 1,326 element-associated hypermethylatedgenes, and 326 were also hypermethylated in INS-F1 islets(Fig. 3C), and there were 1,350 element-associated hypo-methylated genes, with 493 also hypomethylated in INS-F1 (Fig. 3D).

KEGG (Kyoto Encyclopedia of Genes and Genomes)analysis showed that the differentially methylated genesin GDM-F1 and INS-F1 mainly encoded the ion channels inislet b-cell and are involved in insulin secretion. Thesegenes selected for validation were Abcc8 (encoding sulfo-nylurea receptor 1, Sur1, belonged to ATP-binding cassettesuperfamily of transporters), Cacna1c (Cav1.2, encodingone subunit of L-type Ca2+ channels with widespreadexpression in mouse, rat, and human islets b-cells), Cac-na1e (Cav2.3, encoding the R-type Ca2+ channel andexpressed in rodent and human), and Cacna1g (Cav3.1,encoding T-type Ca2+ current and mainly expressed inNOD mouse, rat, and human) (Fig. 3E). MeDIP-seq datashowed that these candidate genes displayed hypermethy-lation status in GDM and INS offspring islets comparedwith controls.

Insulin Therapy for GDM Did Not Restore the AlteredExpression of Ion Channels and the Defective InsulinSecretion in Offspring Pancreatic IsletsThe mRNA and protein levels of Abcc8, Cav1.2, and Cav2.3were all significantly lower in GDM-F1 and INS-F1 males(Fig. 4A–G). In addition, HFD exposure downregulatedCav1.2 expression in all groups, but GDM-F1 and INS-F1males showed dramatically decreased expression of Cav1.2after HFD feeding (Fig. 4B, D, and F). The similar alter-ation of gene expression was also seen in GDM-F1 females(Supplementary Fig. 3A–C). But in INS-F1 females, onlyCav2.3 showed decreased expression (Supplementary Fig.3C). There was no significant difference in Cav3.1 expres-sion among the groups (data not shown).

Table 1—Metabolic parameters in F1 offspring

NCD HFD

Ctrl-F1 INS-F1 GDM-F1 Ctrl-F1 INS-F1 GDM-F1

Male (n) 8 10 9 8 11 11TC (mmol/L) 2.58 6 0.09 2.86 6 0.10 3.56 6 0.15**## 2.74 6 0.05 3.55 6 0.13* 3.82 6 0.37**TG (mmol/L) 1.14 6 0.13 1.04 6 0.13 1.61 6 0.11*## 1.35 6 0.10 1.78 6 0.13 2.34 6 0.21**#NEFA (mmol/L) 1.08 6 0.11 1.13 6 0.08 1.38 6 0.07*# 1.18 6 0.05 1.42 6 0.09* 1.6 6 0.08**HDL (mmol/L) 1.74 6 0.10 1.80 6 0.11 1.98 6 0.08 1.61 6 0.18 1.52 6 0.09 1.78 6 0.14LDL (mmol/L) 0.99 6 0.08 1.02 6 0.06 1.28 6 0.08*# 0.90 6 0.17 1.51 6 0.10* 1.57 6 0.24*Leptin (ng/mL) 3.55 6 0.31 3.25 6 0.28 2.95 6 0.19 2.77 6 0.19 3.27 6 0.21 2.89 6 0.19Glucose (mg/dL) 64.00 6 1.08 60.12 6 1.22 67.68 6 3.20# 71 6 5.82 77.38 6 3.30 83.69 6 3.77*Insulin (ng/mL) 0.20 6 0.03 0.21 6 0.03 0.30 6 0.02**## 0.29 6 0.02 0.71 6 0.06** 0.85 6 0.12**HOMA-IR 0.67 6 0.11 0.67 6 0.05 1.07 6 0.11**## 1.04 6 0.05 2.87 6 0.25** 3.74 6 0.55**

Female (n) 8 8 10 8 10 10TC (mmol/L) 1.78 6 0.07 2.1 6 0.20 2.98 6 0.22**## 2.48 6 0.20 2.57 6 0.05 3.19 6 0.22*##TG (mmol/L) 1.23 6 0.07 1.35 6 0.17 1.55 6 0.12 1.25 6 0.03 1.70 6 0.13 1.86 6 0.14*NEFA (mmol/L) 1.33 6 0.09 1.37 6 0.08 1.76 6 0.12**## 1.40 6 0.06 1.52 6 0.11 2.03 6 0.15**##HDL (mmol/L) 0.94 6 0.04 1.06 6 0.16 1.45 6 0.11**# 1.36 6 0.20 1.49 6 0.33 1.49 6 0.10LDL (mmol/L) 0.48 6 0.04 0.59 6 0.10 0.93 6 0.15**# 0.94 6 0.12 1.29 6 0.09 1.33 6 0.12Leptin (ng/mL) 2.97 6 0.19 2.79 6 0.25 2.65 6 0.30 2.91 6 0.30 3.10 6 0.13 3.10 6 0.26Glucose (mg/dL) 57.43 6 1.00 53.95 6 1.00 55.70 6 2.55 59.60 6 2.57 58.56 6 3.05 59.16 6 1.34Insulin (ng/mL) 0.26 6 0.03 0.29 6 0.04 0.33 6 0.03 0.29 6 0.02 0.34 6 0.03 0.38 6 0.02*HOMA-IR 0.79 6 0.08 0.84 6 0.13 0.97 6 0.11 0.91 6 0.08 1.02 6 0.10 1.19 6 0.08*

Data are expressed as mean 6 SEM. NEFA, nonesterified fatty acids; TC, total cholesterol; TG, triacylglycerol. All parameters weremeasured at 20 weeks of age. *P, 0.05 vs. Ctrl-F1; **P, 0.01 vs. Ctrl-F1. #P, 0.05 vs. INS-F1; ##P, 0.01 vs. INS-F1. Significancewasdetermined by ANOVA.

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Further, insulin secretion responses to ion channelagonists and inhibitors were tested in the isolated pan-creatic islets from male offspring. Tolbutamide, a KATP

channel blocker, stimulated insulin release by 5.52-fold inNCD control islets and 5.05-fold in HFD control islets,respectively, but tolbutamide responses were significantlyreduced in GDM-F1 and INS-F1 groups (Fig. 4H). Diazo-xide, a KATP channel agonist, significantly inhibited insulinsecretion in control islets but did not have such a signif-icant effect on GDM-F1 and INS-F1 (Fig. 4I). Bay K8644,an L-type Ca2+ channels agonist, stimulated insulin secre-tion by 25.18-fold in NCD controls, but islets from GDM-F1 and INS-F1 mice showed decreased insulin secretion by13.13-fold and 16.35-fold in response to Bay K8644, re-spectively (Fig. 4J). In addition, HFD exposure reducedthe responses to Bay K8644 in control islets, but a moresignificant decreased insulin secretion was observed inHFD-fed GDM-F1 and INS-F1 islets (Fig. 4J). Nifedipine,

an L-type Ca2+ channels inhibitor, significantly suppressedinsulin secretion in control islets, but nifedipine revealeda minor decrement of insulin secretion in INS-F1, andaminimal suppression in GDM-F1 (Fig. 4K). The inhibitoryeffect of nifedipine on insulin secretion was not signifi-cantly affected by HFD exposure, although insulin secre-tion showed trends to increased levels in GDM-F1 andINS-F1 HFD males (Fig. 4K).

Insulin Therapy for GDM Did Not Reverse the AlteredDNAMethylation Status in Abcc8,Cav1.2, andCav2.3 inOffspring Pancreatic IsletsPancreatic islets of 20-week-old offspring were isolated,and pyrosequencing was used to analyze the methylationstatus of 10 cytosine phosphate guanine (CpGs) of theAbcc8 promoter, CpGs of Cav 1.2 promoter, and 11 CpGsof the Cav 2.3 promoter. The CpGs of Abcc8, Cav1.2, andCav2.3 showed significantly higher DNA methylation

Figure 2—GSIS. In vivo: serum insulin levels at fasting state and 30 min after glucose injection (A) and the fold change in serum insulin afterglucose loading (B) in male offspring (nCtrl-F1_NCD = 7, nINS-F1_NCD = 7, nGDM-F1_NCD = 7, nCtrl-F1_HFD = 7, nINS-F1_HFD = 9, nGDM-F1_HFD = 12). Seruminsulin levels at fasting state and 30 min after glucose injection (C) and the fold change in serum insulin after glucose loading (D) in femaleoffspring (nCtrl-F1_NCD = 7, nINS-F1_NCD = 8, nGDM-F1_NCD = 10, nCtrl-F1_HFD = 6, nINS-F1_HFD = 6, nGDM-F1_HFD = 7). E: In vitro GSIS in isolated isletsfrom 20-week-old male offspring (n = 5mice per group). F: GSIS in isolated islets from 20-week-old female offspring (n = 5mice per group). Dataare expressed as mean 6 SEM. *P , 0.05 vs. Ctrl-F1, **P , 0.01 vs. Ctrl-F1. #P , 0.05 vs. INS-F1; ##P , 0.01 vs. INS-F1 (ANOVA).

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status in islets of GDM-F1 and INS-F1 males (Fig. 5A–C).Compared with GDM-F1 males, altered DNA methyla-tion status in Cav1.2 and Cav2.3 was improved to a dif-ferent degree in INS-F1 males (Fig. 5B and C). Further,we found that the effect of GDM treatment on DNAmethylation in the three target genes with a sex-specificdifference (Fig. 5 and Supplementary Fig. 3). Notably,maternal glycemic control was associated with a distinctrestoration of DNA methylation levels in Abcc8 andCav1.2 in INS-F1 females (Supplementary Fig. 3D andE) and a moderate hypermethylated level in Cav2.3promoter regions (Supplementary Fig. 3F). In addition,HFD feeding caused a significantly higher DNA methyl-ated level at Cav1.2 promoter regions in GDM and INS

offspring (Fig. 4B and Supplementary Fig. 3E), but theeffect of the HFD on DNA methylation was not found inAbcc8 and Cav2.3.

Effect of High Glucose on Fetal Islets Gene Expressionand DNA MethylationWe collected islets from normal mice at embryonic day 17to verify whether the high-glucose environment directlyaffected islet gene expression and DNA methylation. Treat-ing fetal islets with high glucose (16.7 mmol/L) forthe entire 6 days significantly increased Abcc8, Cav1.2,and Cav2.3 promoter DNA methylation levels and re-duced their gene expression compared with physiologicalglucose level (5.6 mmol/L) (Fig. 6C and F–H). In the

Figure 3—DNA methylation patterns in pancreatic islets of male offspring. A: Heat map of differentially methylated regions between Ctrl-F1(C) and GDM-F1 (G), Ctrl-F1 (C), and INS-F1 (I). B: Distribution of differentially methylated peaks within the genome in G vs. C and I vs. C. C:Venn diagram of hypermethylated genes overlapped between G vs. C and I vs. C. D: Venn diagram of hypomethylated genes overlappedbetween G vs. C and I vs. C. E: KEGG analysis of differentially methylated genes associated with type 2 diabetes.

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16.7/5.6 mmol/L group, the increased Abcc8, Cav1.2, andCav2.3 DNA methylation levels and reduced gene transcrip-tion persisted during subsequent culture at 5.6 mmol/Lglucose (Fig. 6C and F–H). Moreover, we examined DNAmethylation enzyme (Dnmts) and demethylation enzyme(TETs) expression in fetal islets after high glucose expo-sure. We found that the Dnmt1, but not Dnmt3a/3b,expression level was upregulated in the 16.7 mmol/L and16.7/5.6 mmol/L groups (Fig. 6D) and that TET2 and TET3,but not TET1, expression levels were also downregulated in16.7 mmol/L and 16.7/5.6 mmol/L groups (Fig. 6E).

Assessment of STZ’s Effect on Offspring Metabolismand Gene ExpressionSTZ, a cytotoxic agent, is targeted to islet b-cells to inducediabetes in specific species (30). In contrast to adultpancreatic b-cells, STZ had no cytotoxic effect on fetalproislets (31). Although STZ does cross the placenta toa limited extent, it is evident that the fetal pancreas does

not concentrate this cytotoxic agent (32). Furthermore,STZ’s half-life is very short (5–15 min) in vivo (30), butdifferentiation of pancreatic endocrine cells occurs afterday 15 of gestation in the mouse (33), implying that a lowdose of STZ may not directly act for offspring metabolicdysfunction and alterations in gene expression. To furtherevaluate STZ’s effect on offspring, we collected the non-diabetic pregnant mice administered STZ alone. No sig-nificant difference was found with respect to glucosetolerance or insulin sensitivity in NCD or HFD offspringbetween mice administered STZ and the control group(Supplementary Fig. 4). Quantitative PCR analysis alsoshowed no significant changes in ion channel expressionin the group injected with STZ (Supplementary Fig. 5).

DISCUSSION

In this study, we provide the first experimental evi-dence addressing the effects of insulin therapy on thelong-term metabolic health of GDM offspring. Insulin

Figure 4—Ion channels expression and insulin secretion in the pancreatic islets of male offspring. Representative mRNA levels of Abcc8(A), Cav1.2 (B), and Cav2.3 (C) in islets of 20-week-old male offspring (nCtrl-F1_NCD = 7, nINS-F1_NCD = 6, nGDM-F1_NCD = 8, nCtrl-F1_HFD = 5,nINS-F1_HFD = 6, nGDM-F1_HFD = 6).D–G: Representative protein levels of Abcc8, Cav1.2, and Cav2.3 in islets of 20-week-oldmale offspring (n =4 mice per group). H: Tolbutamide (200 mmol/L) stimulated insulin secretion. I: Diazoxide (250 mmol/L) inhibited insulin secretion. J: BayK8644 (2 mmol/L) stimulated insulin secretion.K: Nifedipine (10mmol/L) inhibited insulin secretion. Isolated 20-week-old islets, n = 5mice pergroup. Data are expressed as mean6 SEM. *P, 0.05 vs. Ctrl-F1, **P, 0.01 vs. Ctrl-F1; #P, 0.05 vs. INS-F1; §P, 0.05 vs. Ctrl-F1_NCD,§§P , 0.01 vs. Ctrl-F1_NCD (ANOVA).

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Figure 5—Analysis of the DNA methylation level in male offspring pancreatic islets by pyrosequencing. A: Methylation status of Abcc8promoter regions, mean DNAmethylation, and average methylation in each CpG site in male offspring islets. B: Methylation status of Cav1.2promoter regions, mean DNAmethylation, and average methylation in each CpG site in male offspring islets.C: Methylation status ofCav2.3promoter regions, mean DNA methylation, and average methylation in each CpG site in male offspring islets. Data are expressed asmethylation percentage of each CpG site (nNCD = 6mice per group and nHFD = 4 mice per group). *P, 0.05 vs. Ctrl-F1, **P, 0.01 vs. Ctrl-F1;#P , 0.05 vs. INS-F1, ##P , 0.01 vs. INS-F1; §P , 0.05 vs. Ctrl-F1_NCD, §§P , 0.01 vs. Ctrl-F1_NCD (ANOVA).

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therapy resulted in a significant improvement of metabolicdisorders in offspring fed the NCD. But importantly, inresponse to the HFD, the offspring developed significantlyexacerbated glucose intolerance, obesity, and insulin resis-tance. These abnormalities weremore obvious inmales than

in females, suggesting that males might be more vulnerableto the adverse environment. The findings indicate thatpredisposition to metabolic disorders still persisted in off-spring even with efficient insulin therapy for GDM andwas significantly enhanced by the HFD challenge.

Figure 6—Fetal islets experiment in vitro. A: Schematic representation of experimental design. B: Fetal islets ex vivo were cultured overnightand identified by detecting insulin with immunofluorescence. Black scale bar, 200 mm; white scale bars, 50 mm. C–E: Expression levels oftarget genes, DNAmethyltransferase genes, and demethyltransferase genes in fetal islets (n = 3 replicates per group, and three independentisolation). F–H: Methylation status of Abcc8, Cav1.2, and Cav2.3 in fetal islets cultured in medium containing indicated glucose (n =3 replicates per group, and two independent isolation). Data are expressed as mean 6 SEM. *P , 0.05 vs. 5.6 mmol/L, **P , 0.01 vs.5.6 mmol/L; #P , 0.05 vs. 16.7/5.6 mmol/L, ##P , 0.01 vs. 16.7/5.6 mmol/L (ANOVA).

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For the offspring with insulin therapy for GDM, im-paired glucose tolerance (IGT) is an early key phenotypewith a major contribution from b-cell dysfunctions. Invivo and in vitro experiments confirm that the offspringexhibited reduced GSIS. Epigenetic modifications providea plausible link between the intrauterine environment andalterations in gene expression that may lead to a diseasephenotype (17). In accordance with the sex differences ofphenotypes, the alterations in gene expression and DNAmethylation were also more obvious in males than that infemales. In the male offspring, MeDIP-seq data of pan-creatic islets showed hypermethylated status of genes thatregulate insulin secretion, including ion channel genesAbcc8, Cav1.2, and Cav2.3. Consistently, downregulatedexpression of Abcc8, Cav1.2, and Cav2.3 and impairment ofKATP and L-type Ca2+ channels that mediate insulin secre-tion were observed. However, the female offspring onlyexhibited higher DNAmethylation and lower expression ofCav2.3, suggesting that at least at the promoter regions ofthe three target genes, the epigenetic modification of themale fetus might be more sensitive to the intrauterinehyperglycemia than the female fetus.

All of the Abcc8, Cav1.2, and Cav2.3 ion channel areimportant but with different functions in pancreatic islets.In agreement, Abcc8 encodes a regulatory subunit ofthe KATP channel, coupling the blood glucose level tomembrane electricity activity and insulin secretion (34).Abcc82/2 mice display moderate glucose intolerance,and isolated islets from Abcc82/2 mice show impairedfirst-phase insulin secretion and response to tolbutamide(35). Cav1.2, encoding a subunit of the L-type Ca2+channel,functions as an important role in the Ca2+ entry pathway(36). Pancreatic b-cell–selective Cav1.2 ablation decreasesthe whole-cell Ca2+ current by only 45% but almost abol-ishes first-phase insulin secretion and causes glucose in-tolerance (37). In contrast, Cav2.3 mediates the secondphase of insulin secretion, involved in recruiting insulingranules from pools that are not immediately available torelease insulin when glucose loaded (38,39). Isolated isletsfrom Cav2.32/2 mice exhibited reduced insulin contentbut normal GSIS, implying that the Cav2.3-deficiency maybe offset by other compensatory mechanisms (40). Thismay explain the sex-different phenotype in offspring afterinsulin therapy for GDM. Compared with the obviousglucose intolerance of male offspring, the female offspringwith decreased expression of Cav2.3 alone showed normalglucose tolerance and GSIS.

Further, in vitro culture confirmed the effect of shorthigh-glucose exposure on Abcc8, Cav1.2, and Cav2.3 geneexpression and DNAmethylation in fetal islets. Our animalmodel, together with in vitro culture, provides evidencethat early development is sensitive to the extrinsic factors(41) and that short exposure to intrauterine hyperglycemiais sufficient to persistently affect ion channel gene expres-sion and DNAmethylation. Although direct transfer of ourexperimental results to the human situation warrantscaution, it is important to recognize that freedom from

symptoms is one of the major difficulties with GDM, andthe pregnant woman is usually unaware that she has GDMuntil it is diagnosed at routine prenatal screening (42),suggesting that the fetus might already be exposed to theadverse intrauterine environment and exhibit adaptivechanges in the epigenome (43).

In addition, the in vitro experiment showed that thealtered gene expression of DNA methyl-writing andmethyl-erasing enzymes persisted during subsequent nor-mal glucose culture, indicating that other deleteriousfactors induced by hyperglycemia may also contribute tothe sustained epigenetic alterations. Maternal glucose canfreely permeate the placenta, and glucose excursions notonly cause fetal hyperglycemia but also induce fetal hyper-insulinemia and oxidative stress (44,45). Although insulintherapy for GDM normalized maternal blood glucose level,whether the insulin intervention reversed other adversefactors is unknown. If not, these factors may persist toaffect fetal development and epigenetic modifications (45).

Conditions in the postnatal environment are also im-portant cues for inducing adult metabolic diseases (46). Inour study, HFD exposure exacerbated glucose intolerance.But importantly, this HFD-induced prediabetic state inGDM offspring, whether with insulin therapy or not, wasmore severe than that observed in control offspring,suggesting that early fetal insult could impair the abilityto adapt to an HFD. Extrinsic factors can also affect theepigenome postnatally (46–48). We consistently foundthat the HFD caused a higher DNA methylation level ofCav1.2. However, compared with control offspring, thepre- and postnatal factors act synergistically to inducea more significant hypermethylated level of Cav1.2 in theoffspring with insulin therapy for GDM, which may partlycontribute to the exacerbated glucose intolerance afterHFD exposure.

An additional factor likely to be responsible for theexacerbated glucose intolerance is insulin resistance. De-fective insulin secretion and action are twomajor results ofdiabetes (49,50). Notably, even with insulin therapy forGDM, insulin resistance arose when the offspring werechallenged with the HFD in adulthood. Although the un-derlying mechanisms are still unknown, it is interesting tonote that these offspring in HFD group displayed signif-icant obese phenotypes, suggesting that insulin resistancemight be associated with the overweight and lipid metab-olism disorders.

In summary, our study provides novel experimentalevidence about the effects of insulin therapy for GDM onthe long-term health of the offspring, revealing that theseoffspring are still susceptible to metabolic disorders, es-pecially exposed to an adverse postnatal environment.Although our finding was generated in a mouse model,it is important to recognize that even with efficient insulintherapy for GDM, follow-up and lifestyle interventions arestill necessary to the offspring during postnatal life. Fur-ther, the results show that short exposure to maternaldiabetes during early development is sufficient to cause

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persistent alterations in DNA methylation and expressionof genes that regulate insulin secretion, suggesting amethylation-mediated epigenetic mechanism for GDM-induced intergenerational glucose intolerance. These alteredepigenetic markers not only partly explained the suscepti-bility in GDM offspring, but importantly, will also hopefullyallow for the early diagnosis and therapy for individualswith a propensity for adult-onset disease. In light of ourresults, efficacious screenings and more early interven-tions should be administrated in GDM patients. Moreimportantly, further elucidation of the molecular eventsthat enable, before glycemic control, to result in offspringprotection may lead to the development of new approachesfor reducing the fetal-originated adult diseases.

Funding. This work was supported by the Special Fund for the National KeyResearch and Development Plan grant (no. 2017YFC1001300), the NationalNatural Science Foundation of China (no. 31671569, no. 81490742, no.31471405, and no. 31571556), the Municipal Human Resources DevelopmentProgram for Outstanding Young Talents in Medical and Health Sciences inShanghai (no. 2017YQ047), and the Fundamental Research Funds for the CentralUniversities.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. H.Z. and B.C. designed and performed experi-ments and analyzed data. H.Z. and G.-L.D. wrote and edited the manuscript. Y.C.,Y.Z., and Y.-S.Y. contributed to study design, conducted experiments, and assistedwith the data analysis. Q.L. and Y.J. contributed to the discussion and edited themanuscript. J.-Z.S. contributed to the study design and discussion and edited themanuscript. G.-L.D. and H.-F.H. designed and supervised the research, contrib-uted to discussion, and edited the manuscript. H.-F.H. is the guarantor of this workand, as such, had full access to all data in the study and takes responsibility for theintegrity and accuracy of data analysis.

References1. Chiefari E, Arcidiacono B, Foti D, Brunetti A. Gestational diabetes mellitus: anupdated overview. J Endocrinol Invest 2017;40:899–9092. Rosenn B, Tsang RC. The effects of maternal diabetes on the fetus andneonate. Ann Clin Lab Sci 1991;21:153–1703. Catalano PM, Kirwan JP, Haugel-de Mouzon S, King J. Gestational diabetesand insulin resistance: role in short- and long-term implications for mother andfetus. J Nutr 2003;133(Suppl. 2):1674S–1683S4. Dabelea D. The predisposition to obesity and diabetes in offspring of diabeticmothers. Diabetes Care 2007;30(Suppl. 2):S169–S1745. Clausen TD, Mathiesen ER, Hansen T, et al. High prevalence of type 2 di-abetes and pre-diabetes in adult offspring of women with gestational diabetesmellitus or type 1 diabetes: the role of intrauterine hyperglycemia. Diabetes Care2008;31:340–3466. Ding GL, Wang FF, Shu J, et al. Transgenerational glucose intolerance withIgf2/H19 epigenetic alterations in mouse islet induced by intrauterine hypergly-cemia. Diabetes 2012;61:1133–11427. Clausen TD, Mathiesen ER, Hansen T, et al. Overweight and the metabolicsyndrome in adult offspring of women with diet-treated gestational diabetesmellitus or type 1 diabetes. J Clin Endocrinol Metab 2009;94:2464–24708. Aerts L, Vercruysse L, Van Assche FA. The endocrine pancreas in virgin andpregnant offspring of diabetic pregnant rats. Diabetes Res Clin Pract 1997;38:9–199. Gilmartin AB, Ural SH, Repke JT. Gestational diabetes mellitus. Rev ObstetGynecol 2008;1:129–13410. Crowther CA, Hiller JE, Moss JR, McPhee AJ, Jeffries WS, Robinson JS;Australian Carbohydrate Intolerance Study in Pregnant Women (ACHOIS) Trial

Group. Effect of treatment of gestational diabetes mellitus on pregnancy outcomes.N Engl J Med 2005;352:2477–248611. Landon MB, Spong CY, Thom E, et al.; Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development Maternal-Fetal Medicine UnitsNetwork. A multicenter, randomized trial of treatment for mild gestational diabetes.N Engl J Med 2009;361:1339–134812. Gillman MW, Oakey H, Baghurst PA, Volkmer RE, Robinson JS, Crowther CA.Effect of treatment of gestational diabetes mellitus on obesity in the next gen-eration. Diabetes Care 2010;33:964–96813. Landon MB, Rice MM, Varner MW, et al.; Eunice Kennedy Shriver NationalInstitute of Child Health and Human Development Maternal-Fetal Medicine Units(MFMU) Network. Mild gestational diabetes mellitus and long-term child health.Diabetes Care 2015;38:445–45214. Dabelea D, Crume T. Maternal environment and the transgenerational cycleof obesity and diabetes. Diabetes 2011;60:1849–185515. Sundrani DP, Roy SS, Jadhav AT, Joshi SR. Sex-specific differences anddevelopmental programming for diseases in later life. Reprod Fertil Dev 2017;29:2085–209916. Lingwood BE, Henry AM, d’Emden MC, et al. Determinants of body fat ininfants of women with gestational diabetes mellitus differ with fetal sex. DiabetesCare 2011;34:2581–258517. Jirtle RL, Skinner MK. Environmental epigenomics and disease susceptibility.Nat Rev Genet 2007;8:253–26218. Klose RJ, Bird AP. Genomic DNA methylation: the mark and its mediators.Trends Biochem Sci 2006;31:89–9719. Finer S, Mathews C, Lowe R, et al. Maternal gestational diabetes is as-sociated with genome-wide DNA methylation variation in placenta and cord bloodof exposed offspring. Hum Mol Genet 2015;24:3021–302920. Ruchat SM, Hivert MF, Bouchard L. Epigenetic programming of obesity anddiabetes by in utero exposure to gestational diabetes mellitus. Nutr Rev 2013;71(Suppl. 1):S88–S9421. Buchanan TA, Xiang AH. Gestational diabetes mellitus. J Clin Invest 2005;115:485–49122. Chia R, Achilli F, Festing MFW, Fisher EMC. The origins and uses of mouseoutbred stocks. Nat Genet 2005;37:1181–118623. Zhang M, Lv XY, Li J, Xu ZG, Chen L. The characterization of high-fat diet andmultiple low-dose streptozotocin induced type 2 diabetes rat model. Exp DiabetesRes 2008;2008:70404524. Wang MY, Chen L, Clark GO, et al. Leptin therapy in insulin-deficient type Idiabetes. Proc Natl Acad Sci U S A 2010;107:4813–481925. Vogt MC, Paeger L, Hess S, et al. Neonatal insulin action impairs hypo-thalamic neurocircuit formation in response to maternal high-fat feeding. Cell2014;156:495–50926. Martinez SC, Tanabe K, Cras-Méneur C, Abumrad NA, Bernal-Mizrachi E,Permutt MA. Inhibition of Foxo1 protects pancreatic islet beta-cells against fattyacid and endoplasmic reticulum stress-induced apoptosis. Diabetes 2008;57:846–85927. Weber M, Hellmann I, Stadler MB, et al. Distribution, silencing potential andevolutionary impact of promoter DNA methylation in the human genome. Nat Genet2007;39:457–46628. Radford EJ, Ito M, Shi H, et al. In utero effects. In utero undernourishmentperturbs the adult sperm methylome and intergenerational metabolism. Science2014;345:125590329. Park SH, Ryu SY, YuWJ, et al. Leptin promotes K(ATP) channel trafficking by AMPKsignaling in pancreatic b-cells. Proc Natl Acad Sci U S A 2013;110:12673–1267830. Srinivasan K, Ramarao P. Animal models in type 2 diabetes research: anoverview. Indian J Med Res 2007;125:451–47231. Liu X, Hering BJ, Brendel MD, Bretzel RG. The effect of streptozotocin on thefunction of fetal porcine and rat pancreatic (pro-)islets. Exp Clin Endocrinol 1994;102:374–37932. Reynolds WA, Chez RA, Bhuyan BK, Neil GL. Placental transfer of strepto-zotocin in the rhesus monkey. Diabetes 1974;23:777–782

diabetes.diabetesjournals.org Zhu and Associates 707

33. Pictet RL, Clark WR, Williams RH, Rutter WJ. An ultrastructural analysis of thedeveloping embryonic pancreas. Dev Biol 1972;29:436–46734. Aguilar-Bryan L, Bryan J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 1999;20:101–13535. Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J. Sur1 knockoutmice. A model for K(ATP) channel-independent regulation of insulin secretion. JBiol Chem 2000;275:9270–927736. Yang SN, Berggren PO. The role of voltage-gated calcium channels inpancreatic b-cell physiology and pathophysiology. Endocr Rev 2006;27:621–67637. Schulla V, Renström E, Feil R, et al. Impaired insulin secretion and glucosetolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice. EMBO J 2003;22:3844–385438. Yang SN, Berggren PO. CaV2.3 channel and PKClambda: new players ininsulin secretion. J Clin Invest 2005;115:16–2039. Jing X, Li DQ, Olofsson CS, et al. CaV2.3 calcium channels control second-phase insulin release. J Clin Invest 2005;115:146–15440. Drobinskaya I, Neumaier F, Pereverzev A, Hescheler J, Schneider T. Di-ethyldithiocarbamate-mediated zinc ion chelation reveals role of Cav2.3 channelsin glucagon secretion. Biochim Biophys Acta 2015;1853:953–96441. Tobi EW, Lumey LH, Talens RP, et al. DNA methylation differences afterexposure to prenatal famine are common and timing- and sex-specific. Hum MolGenet 2009;18:4046–4053

42. Carolan-OIah MC. Educational and intervention programmes for gestationaldiabetes mellitus (GDM) management: an integrative review. Collegian 2016;23:103–11443. Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero andearly-life conditions on adult health and disease. N Engl J Med 2008;359:61–7344. HAPO Study Cooperative Research Group,Metzger BE, Lowe LP, Dyer AR,et al. Hyperglycemia and adverse pregnancy outcomes. N Engl J Med 2008;358:1991–200245. Luo ZC, Fraser WD, Julien P, et al. Tracing the origins of “fetal origins” ofadult diseases: programming by oxidative stress? Med Hypotheses 2006;66:38–4446. Srinivasan M, Patel MS. Metabolic programming in the immediate postnatalperiod. Trends Endocrinol Metab 2008;19:146–15247. Feil R, Fraga MF. Epigenetics and the environment: emerging patterns andimplications. Nat Rev Genet 2012;13:97–10948. Newnham JP, Moss TJ, Nitsos I, Sloboda DM, Challis JR. Nutrition and theearly origins of adult disease. Asia Pac J Clin Nutr 2002;11(Suppl. 3):S537–S54249. Muoio DM, Newgard CB. Mechanisms of disease: molecular and metabolicmechanisms of insulin resistance and b-cell failure in type 2 diabetes. Nat Rev MolCell Biol 2008;9:193–20550. White MF. Insulin signaling in health and disease. Science 2003;302:1710–1711

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