Page 1 of 45

Insulin therapy of does not fully protect offspring from

diet-induced metabolic disorders

Hong Zhu1#, Bin Chen1#, Yi Cheng1,2, Yin Zhou1, Yi-Shang Yan1, Qiong Luo3, Ying

Jiang3, Jian-Zhong Sheng1,2, Guo-Lian Ding4,5*, He-Feng Huang1,4,5*

1. The Key Laboratory of Reproductive Genetics (Zhejiang University), Ministry of

Education, Zhejiang university school of medicine, Hangzhou, Zhejiang, China

2. Department of Pathology and Pathophysiology, Zhejiang University school of

medicine, Hangzhou, Zhejiang, China

3. Department of Obstetrics, Women’s Hospital, Zhejiang University school of

medicine, Hangzhou, China

4. The International Peace Maternity and Child Health Hospital, Institute of

Embryo-Fetal Original Adult Disease, School of Medicine, Shanghai Jiao Tong

University, Shanghai, China

5. Shanghai Key Laboratory of Embryo Original Diseases, Shanghai, China.

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

*Corresponding author: He-Feng Huang, [email protected], (86)02164073897 or

Guo-Lian Ding, [email protected], (86)18521321298.

Diabetes Publish Ahead of Print, published online January 29, 2019 Diabetes Page 2 of 45

Abstract

Gestational diabetes mellitus (GDM) is associated with increased risk of metabolic

disorders in offspring in later life. Although mounting evidence suggests that therapy

for GDM could improve neonatal health, it is not known whether the therapy confers

long-term metabolic benefits to offspring in their later adult lives. Here, using a

mouse model of diabetes in the latter half of pregnancy to mimic human GDM, we

find that the efficient therapy of GDM confers significant protection against

glucose intolerance and obesity in offspring fed on normal chow diet. However, the

therapy fails to protect offspring when challenged with a high fat diet, especially for

male offspring. Genome-wide DNA methylation profiling of pancreatic islets from

male offspring identified hypermethylated regions in several genes that regulate

2+ insulin secretion, including Abcc8, Cav1.2 and Cav2.3 that encode KATP or Ca channels, which is associated with reduced gene expression and impaired insulin secretion. This finding suggests a methylation-mediated epigenetic mechanism for

GDM-induced intergenerational glucose intolerance. It highlights that even efficient insulin therapy of GDM is insufficient to fully protect adult offspring from diet-induced metabolic disorders.

Keyword: Gestational diabetes mellitus; Insulin therapy; Offspring; Ion channel,

DNA methylation Page 3 of 45 Diabetes

Introduction

Gestational diabetes mellitus (GDM), defined as glucose intolerance first diagnosed

during pregnancy, affects up to 15% of pregnancies in the world (1). GDM is

associated with adverse consequences not only during fetal development such as

stillbirth, visceromegaly, and macrosomia but also later in life (2, 3). Accumulating

evidence suggests that GDM, independent of maternal obesity and genetic

background, predispose offspring to metabolic disorders in later life, such as obesity,

impaired glucose tolerance and diabetes (4-6). Longitudinal studies of GDM offspring

indicate that the maternal glucose level is a strong predictor of altered carbohydrate

metabolism during childhood, which can be extended into adulthood (7, 8).

The therapeutic management of GDM is critical to minimize these complications.

Glycemic control is the cornerstone of GDM management (9). Randomized trials

have confirmed that the therapy of GDM confers immediate benefits such as

reduction of perinatal complications and prevalence of macrosomia (10, 11).

However, it is still unclear whether the therapy of GDM confers long-term metabolic

benefits to offspring (12, 13). Importantly, the appropriate offspring follow-up period

for assessing the effects of GDM therapy is still debatable. Offspring enrolled in most

follow-ups were prepubertal (age 5-10 years), yet the long-term effect of GDM on

offspring metabolic disorders or its reduction through therapy might not be evident

until adolescence or adulthood (12, 14). Diabetes Page 4 of 45

Additionally, offspring gender also has a profound effect. Sexual dimorphism in the response to insult in utero presents with an uneven disease susceptibility: although both genders can be affected, one is more susceptible (15). Metabolic differences exist between male and female fetus (15), differing sensitivities to maternal may result in sex-specific disease risks later in life (6, 16). Evidence from epidemiological studies demonstrate that the effect of therapy for GDM also differs with fetal sex in infancy and childhood (11, 16). Therefore, fetal sex may influence the impact that therapy for GDM have on offspring long-term health. Moreover, social and environmental factors could also confound the follow-up results. Thus, it remains unknown whether therapy of GDM is a modifiable risk factor for offspring metabolic disorders.

Mechanistically, epigenetic modifications such as DNA methylation, histone modification, and non-coding RNAs provide a plausible link between environment exposures early in development and the susceptibility to diseases later in life (17).

DNA methylation, the most studied epigenetic modification, can alter status of gene expression and be inherited mitotically in somatic cells (17, 18), which provides a potential mechanism by which environment effects on epigenome could have long-term effects on gene expression. Results from animal and human studies support that intrauterine hyperglycemia could result in altered fetal DNA methylation patterns and subsequent changes in the risk of developing disease (6, 19). In epidemiological Page 5 of 45 Diabetes

and experimental studies, glycemic control improved GDM neonatal outcomes.

However, none of studies investigated whether these positive effects were

accompanied by a favorable restoration of DNA methylation (20), which may be

critically important for the susceptibility to disease later in life. Thus, the possibility

that fetal metabolic programming in GDM can have long-term effect on health that

may in turn be modified by therapy of GDM remains open to question.

Since it is difficult to exclude the confounders and analyze the underlying

mechanisms in humans, we established a mouse model of diabetes in the latter half of

pregnancy to mimic human GDM with high incidence during the third trimester of

gestation (21). We treated maternal hyperglycemia with insulin and evaluated the

pancreatic islet β-cell function in offspring. We also addressed whether lifestyle

factors in the adulthood, such as high fat diet, may increase the risk of developing

metabolic disorders in offspring. Finally, we carried out genome-wide DNA

methylation sequencing in offspring pancreatic islets and assessed the alterations of

candidate genes which may contribute to metabolic phenotypes in offspring. Diabetes Page 6 of 45

Research Design and Methods Animal care

All animal protocols were approved by the Zhejiang University Animal Care and Use

Committee. All experiments were performed with Institute of Cancer Research (ICR) mice (22). ICR mice were purchased from Shanghai SLAC Laboratory Animal Co.

(Shanghai, China). Virgin ICR females (age, 6-8 weeks; weight, 26-28g) were mated with normal ICR males. Pregnancy was dated by the presence of a vaginal plug (day

0.5). Pregnant females were randomly assigned to “Control” (Ctrl), “Gestational diabetes mellitus” (GDM) or “GDM + Insulin therapy” (INS) groups. On day 6 and day 12 of pregnancy, GDM and INS dams fasting 8 hours received i.p. injection of streptozotocin (STZ; 100 mg/kg; Sigma, St. Louis, MO), respectively (23, 24) (Fig.

1A). Control pregnant mice received an equal volume of citrate buffer. Blood glucose level was measured via the tail vein 48-72 hours after the second STZ injection, and diabetes was defined as a blood glucose level between 14 and 19 mmol/L (6).

INS dams were treated with recombinant insulin (Novolin® R, Novo Nordisk,

Bagsvaerd, Denmark) by mini-osmotic pumps (Alzet model 1007D, Durect,

Cupertino, CA) at a dose of 0.35 IU/day. Pumps were implanted in INS dams on Day

14.5 or Day 15 of pregnancy. Pumps were implanted posterior to the scapulae under avertin (Sigma) anesthesia (0.1ml per 20 g body wt). Control and GDM dams were implanted with pumps containing normal saline. In order to maintain glycemic level stable, INS dams received another injection of 0.1 units of long acting insulin Page 7 of 45 Diabetes

(Levemir®, Novo Nordisk) about 1 hour prior to the fed state (darkness) during late

gestation. Only mice with near-normal blood glucose level in INS group were

included in the further study.

At birth, litter size was equalized to eight. Pups from GDM and INS group were

fostered by normal females until the age of 3 weeks. Offspring were designated as

Ctrl-F1, GDM-F1 and INS-F1. At 8 weeks of age, offspring were divided into two

groups either receiving normal chow diet (NCD) or high fat diet (HFD; 60% energy

as fat; D12492, Research Diets, New Brunswick, NJ) until 20 weeks (Fig. 1A).

In vivo metabolic testing

Intraperitoneal glucose (2 g/kg body wt.) and insulin (0.8 unit/kg body wt.) tolerance

tests were performed in unrestrained conscious mice after a 16- and 4h-fast,

respectively. Serum insulin level was assessed at overnight fasted state (time 0) and

30 min after glucose injection (Crystal Chem, Downers Grove, IL).

Serum analysis

Serum cholesterol, triglyceride, non-esterified fatty acids, high- and low-density

lipoprotein were assayed using a biochemical analyzer (TBA120FR, Toshiba, Tokyo,

Japan). Serum leptin was determined by radioimmunoassay (North Institute, Beijing,

China). HOMA-IR was calculated as follows: fasting serum insulin concentration

(μU/ml) multiplied by fasting blood glucose level (mg/dl) divided by 405 (25).

Islet isolation and in vitro insulin secretion Diabetes Page 8 of 45

Pancreatic islets were isolated from 20-week-old mice as previously described (26)

For indicated experiment, ten islets (size-matched for each batches) were incubated

for 1 h at 37 °C in KRB buffer containing 2.8 mM glucose, and then incubated in the

KRB buffer containing indicated glucose or various chemical compounds including

200 μM tolbutamide (Sigma), 250 μM diazoxide (Sigma), 2 μM Bay K8644 (Sigma)

and 10 μM nifedipine (Sigma). The supernatant was collected for insulin content assay (Abnova, Taiwan, China).

In fetal mice of embryonic day 17 (E17), the pancreas was directly digested in 2 mg/mL collagenase with shaking incubation at 37°C for 25 min. After recover overnight in RPMI medium, fetal islets were handpicked under a stereomicroscope and randomly separated into 5.6 mM, 16.7/5.6 mM and 16.7 mM glucose groups (Fig.

6A).

Methylated DNA Immunoprecipitaton Sequencing (MeDIP-seq)

Male offspring receiving NCD at 20 week of age were chosen for MeDIP-seq analysis. Genomic DNA of pancreatic islets was extracted from nine mice per groups and pooled by each of the three. The MeDIP-seq was performed as described previously (27). Briefly, DNA was sonicated to obtain the DNA fragments (200-700 bp). Sonicated genomic DNA was denatured and immunoprecipitated with anti-5-methylcytosine antibody (#28692, Cell Signaling, Danvers, MA). Illumina libraries were then created from the captured DNA by TruSeq® DNA LT Sample Page 9 of 45 Diabetes

Prep Kit (Illumina, San Diego, CA). The samples were sequenced on Illumina

HiSeq3000 (Illumina).

Gene expression

Total RNA from isolated pancreatic islets was extracted by using RNeasy Micro Kit

(Qiagen, Valencia, CA). cDNA was synthesized using oligo-dT and random primers

(TaKaRa, Dalian, China) for real-time quantitative PCR with SYBR Green detection

(TaKaRa). All primer sequences are shown in Table s1.

DNA methylation

Genomic DNA was extracted from islets by using TIANmap Micro DNA kit

(Tiangen, Beijing, China). Bisulfite was converted using the EpiTect bisulfite kit

(Qiagen) according to the manufacturer’s instructions. Methylation status of gene

promoter regions was analyzed by pyrosequencing (28). In brief, pyrosequencing

primers were designed by Qiagen PyroMark Assay Design software 2.0 (Qiagen)

(Table s2). Specificity of each PCR product was checked by agarose gel analysis.

Pyrosequencing was conducted on a PyroMark Q24 pyrosequencer (Qiagen) by using

PyroMark Gold Q24 reagents (Qiagen), and quantification of methylated and

unmethylated alleles were performed by Pyro Q24 software (Qiagen).

Immunofluorescence Analysis

Fetal islets were identified by detecting insulin with Immunofluorescence as Diabetes Page 10 of 45

previously described (29). Isolated fetal islets were incubated with antibody against insulin. All immunofluorescence images were acquired by laser scanning confocal microscope (Zeiss, Jena, Germany). A description of the antibodies used is shown in

Table s3.

Western Blots

The protein was extracted from mouse islets as described before (29). Samples were separated using 8% SDS/PAGE. Western blots were performed using PVDF membrane and the antibodies which were summarized in the Table s3. Protein bands were visualized by ECL system (Pierce, Rockford, IL).

STZ-treated non-diabetic Model

The identical amount of STZ was injected in pregnant mice as described above. Only glucose level less than 7.5 mM was considered as an STZ-treated nondiabetic model.

Offspring were fostered by normal females until 3-week-old. HFD, GTT and ITT were performed as described above. Islets from the STZ-treated non-diabetic offspring were isolated for further analysis.

Statistically Analysis

All data are shown as mean ± SEM. Statistical analysis were performed by one-way

ANOVA followed by LSD post hoc test, and two-tailed Student test as described in Page 11 of 45 Diabetes

the Table and Figure legends (SPSS 17.0). P < 0.05 was considered statistically

significant. Diabetes Page 12 of 45

Results

Insulin therapy of GDM conferred offspring a partial protection from metabolic disorders.

We established a mouse model of hyperglycemia during the mid-late stage of pregnancy. GDM dams were averaging 328.6 mg/dl of plasma glucose level after twice STZ injection. Blood glucose level declined and averaged 129.5 mg/dl during insulin therapy in INS group (Fig. 1B). Gestational length, litter size and birth weight were similar among the three groups (Table s4, Fig. s1).

Notably, increased body weight and most metabolic abnormalities were seen in

GDM-F1 adult offspring (Fig. 1C and D, Table 1). Insulin therapy was associated with normal weight trajectory, serum glucose and lipid metabolism in adult offspring

(Fig. 1C and D, Table 1). However, these associations were observed only in NCD offspring. When fed with HFD after 8 weeks of age, a significant increase in body weight (Fig. 1C), fasting insulin levels, and lipid levels were seen in INS-F1 males

(Table1). Females in HFD INS-F1 group only exhibited increased body weight compared with HFD Ctrl-F1 females (Fig. 1D).

Insulin therapy-mediated protection against glucose intolerance in offspring was abolished by an exposure to high-fat diet in adulthood

Insulin therapy of GDM resulted in a distinct rescue of glucose intolerance in INS-F1 Page 13 of 45 Diabetes

male offspring, with only an increase in glucose levels at 60 min after injection (Fig.

1E). But strikingly, HFD abolished this protection (Fig. 1E). When subjected to

insulin tolerance test (ITT), only GDM-F1 male mice exhibited significant impaired

insulin sensitivity with aging in NCD group (Fig. s2B, Fig. 1G). However, when

challenged with HFD, not only GDM-F1 males developed much more serious insulin

intolerance, INS-F1 males also exhibited a pronounced impairment of insulin

tolerance compared with controls (Fig. 1G). In female offspring, only GDM-F1

females showed an elevation of glucose level at 30 and 120 min after insulin injection

(Fig. 1H).

Defect of insulin secretion could also contribute to glucose intolerance. We assessed

glucose-stimulated-insulin-secretion (GSIS) in vivo and in vitro. In vivo,

glucose-stimulating insulin secretion was reduced in both male and female GDM-F1

offspring (Fig. 2A - D). In INS-F1 group, only males exhibited lower insulin levels in

response to glucose injection (Fig. 2A and B). In vitro, insulin secretory response to

5.6 mM glucose was similar among all groups (Fig. 2E and F). However, islets of

GDM-F1 males or females showed impaired insulin secretion when exposure to 16.7

mM glucose (Fig. 2E and F). Defective insulin response to high glucose (16.7 mM)

was also seen in INS-F1 males (Fig. 2E). No significant difference was found in

INS-F1 females in terms of glucose tolerance, insulin sensitivity, or glucose

stimulated insulin secretion (Fig. 1F and H, Fig. 2C, D and F). Diabetes Page 14 of 45

Insulin therapy of GDM did not restore the altered DNA methylation in

offspring pancreatic islets

Gestational diabetes altered the methylation of 220 (8.11 %) upstream 2k, 201

(7.41 %) downstream 2k, 77 (2.84 %) 5′UTR, 55 (2.03 %) 3′UTR, 711 (26.2 %) CDS,

and 1448 (53.37 %) intron element-associated genes (Fig. 3B, Table s5) in islets of

GDM-F1 male offspring, respectively. Global cytosine methylation status was also

altered in INS-F1 offspring and the methylation of 258 (8.37 %) upstream2k, 237

(7.69 %) downstream2k, 81 (2.63 %) 5′UTR, 70 (2.27 %) 3′UTR, 745 (24.17 %)

CDS, and 1690 (54.83 %) intron element-associated genes were changed, respectively

(Fig. 3B, Table s6). In GDM-F1, 1326 element-associated hypermethylated genes,

326 were also hypermethylated in INS-F1 islets (Fig. 3C), and 1350

element-associated hypomethylated genes, 493 were also hypomethylated in INS-F1

(Fig. 3D).

KEGG (Kyoto Encydopedia of gens and Genomes) analysis showed that the

differentially methylated genes in GDM-F1 and INS-F1 mainly encoded the ion

channels in islet β-cell, and are involved in insulin secretion. These genes selected for

validation were the following: Abcc8 (encoding sulfonylurea receptor 1, Sur1, belonged to ATP-binding cassette superfamily of transporters), Cacna1c (Cav1.2,

encoding one subunit of L-type Ca2+ channels with widespread expression in mouse, rat and human islets β-cell), Cacna1e (Cav2.3, encoding R-type Ca2+ channel and

expressed in rodent and human), and Cacna1g (Cav3.1, encoding T-type Ca2+ current Page 15 of 45 Diabetes

and mainly expressed in NOD mouse, rat and human) (Fig. 3E). MeDIP-seq data

showed that candidate genes above displayed hypermethylation status in GDM and

INS offspring islets compared with controls.

Insulin therapy of GDM did not restore the altered expression of ion channels

and the defective insulin secretion in offspring pancreatic islets.

The mRNA and protein levels of Abcc8, Cav1.2 and Cav2.3 were all significantly

lower in GDM-F1 and INS-F1 males (Fig.4A-G). Additionally, HFD exposure

down-regulated Cav1.2 expression in all groups, but GDM-F1 and INS-F1 males

showed dramatically decreased expression of Cav1.2 after HFD-feeding (Fig.4B, D

and F). The similar alteration of gene expression was also seen in GDM-F1 females

(Fig. s3A-C). But in INS-F1 females, only Cav2.3 showed decreased expression (Fig.

s3C). There was no significant difference in Cav3.1 expression among groups (data

not shown).

Further, insulin secretion responses to ion channel agonists and inhibitors were tested

in the isolated pancreatic islets from male offspring. Tolbutamine, a KATP channel

blocker, stimulated insulin release by 5.52 fold in NCD control islets and 5.05 fold in

HFD control islets, respectively, but tolbutamine responses were significantly reduced

in GDM-F1 and INS-F1 groups (Fig. 4H). Diazoxide, a KATP channel agonist,

significantly inhibited insulin secretion in control islets, but did not have such a

significant effect on GDM-F1 and INS-F1 (Fig. 4I). Bay K8644, a L-type Ca2+ Diabetes Page 16 of 45

channels agonist, stimulated insulin secretion by 25.18 fold in NCD controls, but

islets from GDM-F1 and INS-F1 mice showed decreased insulin secretion by 13.13

fold and 16.35 fold in response to Bay K8644, respectively (Fig. 4J). Additionally,

HFD exposure reduced the responses to Bay K8644 in control islets, but a more

significant decreased insulin secretion was observed in HFD-fed GDM-F1 and

INS-F1 islets (Fig. 4J). Nifedipine, a L-type Ca2+ channels inhibitor, significantly suppressed insulin secretion in control islets, but nifedipine revealed a minor decrement of insulin secretion in INS-F1, and a minimal suppression in GDM-F1

(Fig. 4K). The inhibitory effect of nifedipine on insulin secretion was not significantly affected by HFD exposure, although insulin secretion showed trends to increased levels in GDM-F1 and INS-F1 HFD males (Fig. 4K).

Insulin therapy of GDM did not reverse the altered DNA methylation status in

Abcc8, Cav1.2, and Cav2.3 in offspring pancreatic islets.

Pancreatic islets of 20-week-old offspring were isolated and analyze the methylation

status of 10 cytosine phosphate guanine (CpGs) of Abcc8 promoter, 10 CpGs of Cav

1.2 promoter, and 11 CpGs of Cav 2.3 promoter by pyrosequencing. The CpGs of

Abcc8, Cav1.2 and Cav2.3 showed significant higher DNA methylation status in islets

of both GDM-F1 and INS-F1 males (Fig. 5A-C). Compared with GDM-F1 males,

altered DNA methylation status in Cav1.2 and Cav2.3 were improved to different

degree in INS-F1 males (Fig. 5B and C). Further, we found that the effect of GDM

treatment on DNA methylation in the three targets genes with sex-specific difference Page 17 of 45 Diabetes

(Fig.5 and Fig. s3). Notably, maternal glycemic control was associated with a distinct

restoration of DNA methylation levels in Abcc8 and Cav1.2 in INS-F1 females (Fig.

s3D and E), and a moderate hypermethylated level in Cav2.3 promoter regions (Fig.

s3F). Additionally, HFD-feeding caused a significantly higher DNA methylated level

at Cav1.2 promoter regions in GDM and INS offspring (Fig. 4B, Fig. s3E), but the

effect of HFD on DNA methylation was not found in Abcc8 and Cav2.3.

Effect of high glucose on fetal islets gene expression and DNA methylation

We collected islets from normal E17 mice to verify whether high glucose

environment directly affected islet gene expression and DNA methylation. Treating

fetal islets with high glucose (16.7 mM) for the entire 6-day significantly increased

Abcc8, Cav1.2 and Cav2.3 promoter DNA methylation levels and reduced their gene

expression compared to physiological glucose level (5.6 mM) (Fig. 6C, F-H). In

16.7/5.6 mM group, the increased Abcc8, Cav1.2 and Cav2.3 DNA methylation levels

and reduced gene transcription persisted during subsequent culture at 5.6 mM glucose

(Fig. 6C, F-H). Moreover, we examined DNA methylation enzyme (Dnmts) and

demethylation enzyme (TETs) expression in fetal islet after high glucose exposure.

We found that Dnmt1, not Dnmt3a/3b, expression level was up-regulated in both 16.7

mM and 16.7/5.6 mM groups (Fig. 6D), and TET2 and TET3, not TET1, expression

levels were also down-regulated in 16.7 mM and 16.7/5.6 mM groups (Fig. 6E).

Assessment of STZ's effect on offspring metabolism and gene expression Diabetes Page 18 of 45

STZ, a cytotoxic agent, is targeted to islet β cells to induce diabetes in specific species

(30). In contrast to adult pancreatic β cells, STZ had no cytotoxic effect on fetal pro-islets (31). Although STZ does cross the placenta to a limited extent, it is evident that fetal pancreas does not concentrate this cytotoxic agent (32). Furthermore, STZ's half-life is very short (5-15min) in vivo (30), but differentiation of pancreatic endocrine cells occurs after the Day 15 of gestation in mouse (33), implying that low dose of STZ may not directly act for offspring metabolic dysfunction and alterations in gene expression. To further evaluate STZ's effect on offspring, we collected the

STZ-alone-treated non-diabetic pregnant mice. No significant difference was found with respect to glucose tolerance, insulin sensitivity in NCD or HFD offspring between STZ-treated and control group (Fig s4). qPCR analysis also showed that no significant changes in ion channel expression in STZ-treated group (Fig s5). Page 19 of 45 Diabetes

Discussion

In this study, we provide the first experimental evidence addressing the effects of

insulin therapy on GDM offspring long-term metabolic health. Insulin therapy

resulted in a significant improvement of metabolic disorders in offspring fed with

NCD. But importantly, in response to HFD, the offspring developed significantly

exacerbated glucose intolerance, obesity, and . These abnormalities

were more obvious in males than in females, suggesting that males might be more

vulnerable to the adverse environment. The findings indicate that predisposition to

metabolic disorders still persisted in offspring even with efficient insulin therapy of

GDM, and was significantly enhanced by HFD challenge.

For the offspring with insulin therapy of GDM, impaired glucose tolerance (IGT) is

an early key phenotype with a major contribution from β-cell dysfunctions. Both in

vivo and in vitro experiments confirm that the offspring exhibited reduced

glucose-stimulated insulin secretion. Epigenetic modifications provide a plausible link

between intrauterine environment and alterations in gene expression that may lead to

disease phenotype (17). In accordance with the sex differences of phenotypes, the

alterations in gene expression and DNA methylation were also more obvious in males

than that in females. In the male offspring, Medip-seq data of pancreatic islets showed

hypermethylated status of insulin secretion regulating genes, including ion channel

genes Abcc8, Cav1.2 and Cav2.3. Consistently, downregulated expression of Abcc8, Diabetes Page 20 of 45

2+ Cav1.2 and Cav2.3, and impairment of KATP and L-type Ca channels mediated insulin secretion were observed. However, the female offspring only exhibited higher

DNA methylation and lower expression of Cav2.3, suggesting that at least at the

promoter regions of the three target genes, the epigenetic modification of male fetus

might be more sensitive to the intrauterine hyperglycemia than female.

The Abcc8, Cav1.2 and Cav2.3 are all important ion channel genes but with different

functions in pancreatic islets. In agreement, Abcc8 encodes a regulatory subunit of

KATP channel, coupling the blood glucose level to membrane electricity activity and

insulin secretion (34). Abcc8-/- mice display moderate glucose intolerance, isolated

islets from Abcc8-/- mice show impaired first-phase insulin secretion and response to

tolbutamide (35). Cav1.2, encoding a subunit of L-type calcium channel, functions as

an important role in Ca2+ entry pathway (36). Pancreatic β-cell selective Cav1.2

ablation decreases whole cell Ca2+ current by only 45%, but almost abolishes

first-phase insulin secretion and causes glucose intolerance (37). In contrast, Cav2.3

mediates the second phase of insulin secretion, involving in recruiting insulin granules

from pools which are not immediately available to release insulin when glucose

loaded (38, 39). Isolated islets from Cav2.3-/- mice exhibited reduced insulin content

but normal GSIS, implying that the Cav2.3-deficiency may be offset by other

compensatory mechanisms (40). This may explain the sex different phenotype in

offspring after insulin therapy of GDM. Compare with the obvious glucose

intolerance of male offspring, the female offspring with decreased expression of Page 21 of 45 Diabetes

Cav2.3 alone showed normal glucose tolerance and GSIS.

Further, in vitro culture confirmed the effect of short high glucose exposure on Abcc8,

Cav1.2 and Cav2.3 gene expression and DNA methylation in fetal islets. Our animal

model, together with in vitro culture, provide evidence that early development is

sensitive to the extrinsic factors (41), and short exposure to intrauterine

hyperglycemia is sufficient to persistently affect ion channel gene expression and

DNA methylation. Although direct transfer of our experimental results to the human

situation warrants caution, it is important to recognize that one of the major

difficulties with GDM is symptoms-free, and the pregnant woman is usually unaware

that she has GDM until it is diagnosed at routine prenatal screening (42), suggesting

that the fetus might already be exposed to the adverse intrauterine environment and

exhibit adaptive changes in epigenome (43). Additionally, in vitro experiment showed

that the altered gene expression of DNA methyl-writing and methyl-erasing enzymes

persisted during subsequent normal glucose culture, indicating that other deleterious

factors induced by hyperglycemia may also contribute to the sustained epigenetic

alterations. Maternal glucose can freely permeate the placenta, and glucose excursions

not only cause fetal hyperglycemia, but also induce fetal hyperinsulinemia and

oxidative stress (44, 45). Although insulin therapy of GDM normalized maternal

blood glucose level, it was unknown whether insulin intervention reversed other

adverse factors. If not, they may persist to affect fetal development and epigenetic

modifications (45). Diabetes Page 22 of 45

Postnatal environment conditions are also important cues for inducing adult metabolic diseases (46). In our study, HFD exposure exacerbated glucose intolerance. But importantly, this HFD-induced prediabetic state in GDM offspring, whether with insulin therapy or not, was more severe than that observed in control offspring, suggesting that early fetal insult could impair the ability to adapt HFD. In postnatal life, extrinsic factors can affect the epigenome postnatally as well (46-48).

Consistently, we found that HFD caused a higher DNA methylation level of Cav1.2.

However, compared with control offspring, the pre- and postnatal factors act synergistically to induce a more significant hypermethylated level of Cav1.2 in the offspring with insulin therapy for GDM, which may partly contribute to the exacerbated glucose intolerance after HFD exposure. An additional factor likely to be responsible for the exacerbated glucose intolerance is insulin resistance. Defective insulin secretion and action are two major causes to diabetes mellitus (49, 50).

Notably, even with insulin therapy of GDM, insulin resistance arises when the offspring were challenged with HFD in adulthood. While the underlying mechanisms are still unknown, it is interesting to note that these offspring in HFD group displayed significant obese phenotypes, suggesting that insulin resistance might be associated with the overweight and lipid metabolism disorders.

In summary, our study provides novel experimental evidence about the effects of insulin therapy of GDM on offspring long-term health, revealing that these offspring Page 23 of 45 Diabetes

are still susceptible to metabolic disorders, especially exposed to adverse postnatal

environment. Although our finding was generated in mouse model, it is important to

recognize that even with efficient insulin therapy of GDM, the follow-ups and

lifestyle interventions are still necessary to the offspring during postnatal life. Further,

the results show that short exposure to maternal diabetes during early development is

sufficient to cause persistent alterations in DNA methylation and expression of genes

that regulate insulin secretion, suggesting a methylation-mediated epigenetic

mechanism for GDM-induced intergenerational glucose intolerance. These altered

epigenetic markers not only partly explained the susceptibility in GDM offspring, but

importantly, will hopefully allow for the early diagnosis and therapy of individuals

with a propensity for adult-onset disease. In light of our results, efficacious screenings

and more early interventions should be administrated in GDM patients. More

importantly, further elucidation of the molecular events that enable prior to glycemic

control to result in offspring protection may lead to the development of new

approaches for reducing the fetal originated adult diseases. Diabetes Page 24 of 45

Acknowledgements

Funding

This work was supported by the Special Fund for the National Key Research and

Development Plan Grant (No. 2017YFC1001300), the National Natural Science

Foundation of China (No. 31671569, No. 81490742, No. 31471405 and No.

31571556), the Municipal Human Resources Development Program for Outstanding

Young Talents in Medical and Health Sciences in Shanghai (No. 2017YQ047) and the

Fundamental Research Funds for the Central Universities.

Author contributions

H.Z. and B.C. designed and performed experiments, analyzed data. Y.C., Y.Z. and

Y.-S.Y. contributed to study design, conducted experiments and assisted with the data analysis. H.Z., G.-L.D. wrote and edited the manuscript. J.-Z.S. contributed to the study design and discussion, and edited the manuscript. Q.L. and Y.J. contributed to the discussion and edited the manuscript. G.-L.D. and H.-F.H. designed and supervised the research, contributed to discussion and edited the manuscript. H.-F.H. is the guarantor of this work and, as such, had full access to all data in the study and takes responsibility for the integrity and accuracy of data analysis.

Conflict of Interest

Authors have no potential conflicts of interest related to this article. Page 25 of 45 Diabetes

References:

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SH, Berggren PO, Jeon JH, Ho WK: Leptin promotes KATP channel trafficking by AMPK signaling in pancreatic β-cells. P Natl Acad Sci USA 2013; 110:12673-12678. 30.Srinivasan K, Ramarao P: Animal models in type 2 diabetes research: An overview. Indian J Med Res 2007; 3:451-472. 31.Liu X, Hering BJ, Brendel MD, Bretzel RG: The effect of streptozotocin on the function of fetal porcine and rat pancreatic (pro-) islets. Exp Clin Endocrinol 1994; 102:374-379. 32.Reynolds WA, Chez RA, Bhuyan BK, Neil GL: Placental transfer of streptozotocin in the rhesus monkey. Diabetes 1974; 23:777-782. 33.Pictet RL, Clark WR, Williams RH, Rutter WJ: An Ultrastructural Analysis of the Developing Embryonic Pancreas. Dev Biol 1972; 29:436-476. 34.Aguilar-Bryan L, Bryan J: Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 1999; 20:101-135. Page 27 of 45 Diabetes

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A Model For KATP Channel-Independent Regulation Of Insulin Secretion. J Biol Chem 2000; 275:9270-9277. 36.Yang S, Berggren P: The Role of Voltage-Gated Calcium Channels in Pancreatic β -Cell Physiology and Pathophysiology. Endocr Rev 2006; 27:621-676. 37.Schulla V, Renström E, Feil R, Feil S, Franklin I, Gjinovci A, Jing X, Laux D, Ludquist I, Msgnuson MA, Obermüller S, Olofsson CS, Salehi A, Wendt A, Klugbauer N, Woollheim CB, Rorsman P, Hofmann F: Impaired insulin secretion and glucose tolerance in b cell-selective CaV1.2 Ca2+ channel null mice. The EMBO Journal 2003; 22:3844-3854. 38.Yang S, Berggren P: CaV2.3 channel and PKCλ: new players in insulin secretion. J Clin Invest 2005; 115:16-20. 39.Jing X, Li D, Olofsson CS, Salehi A, Surve VV, Caballero J, Ivarsson R, Lundquist I, Pereverzev A, Schneider T, Rorsman P, Renström E: CaV2.3 calcium channels control second-phase insulin release. J Clin Invest 2005; 115:146-154. 40.Drobinskaya I, Neumaier F, Pereverzev A, Hescheler J, Schneider T: Diethyldithiocarbamate-mediated zinc ion chelation reveals role of Cav2.3 channels in secretion. BBA-Mol Cell Res 2015; 1853:953-964. 41.Tobi EW, Lumey LH, Talens RP, Kremer D, Putter H, Stein AD, Slagboom PE, Heijmans BT: DNA methylation differences after exposure to prenatal famine are common and timing- and sex-specific. Hum Mol Genet 2009; 18:4046-4053. 42.Carolan-Olah MC: Educational and intervention programmes for gestational diabetes mellitus (GDM) management: An integrative review. Collegian 2016; 23:103-114. 43.Gluckman PD, Hanson MA, Cooper C, Thornburg KL: Effect of In Utero and Early-Life Conditions on Adult Health and Disease. N Engl J Med 2008; 359:61-73. 44.Metzger BELL, D. R. HDRM: Hyperglycemia and adverse pregnancy outcomes. N Engl J Med. 2008; 33:115-122. 45.Luo ZC, Fraser WD, Julien P, Deal CL, Audibert F, Smith GN, Xiong X, Walker M: Tracing the origins of “ fetal origins ” of adult diseases: Programming by oxidative stress? Med Hypotheses 2006; 66:38-44. 46.Srinivasan M, Patel MS: Metabolic programming in the immediate postnatal period. Trends Endocrin Met 2008; 19:146-152. 47.Feil R, Fraga MF: Epigenetics and the environment: emerging patterns and implications. Nat Rev Genet 2012; 13:97-109. 48.Newnham JP, Moss TJ, Nitsos I, Sloboda DM, Challis JR: Nutrition and the early origins of adult disease. Asia Pac J Clin Nutr 2002; 11 Suppl 3: S537-S542. 49.Muoio DM, Newgard CB: Molecular and metabolic mechanisms of insulin resistance and β-cell failure in type 2 diabetes. Nat Rev Mol Cell Bio 2008; 9:193-205. 50.White MF: Insulin Signaling in Health and Disease. Science 2003; 302:1710-1711. Diabetes Page 28 of 45

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 11 TC (mM) 2.58±0.09 2.86±0.10 3.56±0.15**## 2.74±0.05 3.55±0.13* 3.82±0.37** TG (mM) 1.14±0.13 1.04±0.13 1.61±0.11*## 1.35±0.10 1.78±0.13 2.34±0.21**# NEFA(mM) 1.08±0.11 1.13±0.08 1.38±0.07*# 1.18±0.05 1.42±0.09* 1.6±0.08** HDL (mM) 1.74±0.10 1.80±0.11 1.98±0.08 1.61±0.18 1.52±0.09 1.78±0.14 LDL (mM) 0.99±0.08 1.02±0.06 1.28±0.08*# 0.90±0.17 1.51±0.10* 1.57±0.24* Leptin (ng/mL) 3.55±0.31 3.25±0.28 2.95±0.19 2.77±0.19 3.27±0.21 2.89±0.19 Glucose (mg/dL) 64.00±1.08 60.12±1.22 67.68±3.20# 71±5.82 77.38±3.30 83.69±3.77* Insulin (ng/mL) 0.20±0.03 0.21±0.03 0.30±0.02**## 0.29±0.02 0.71±0.06** 0.85±0.12** HOMA-IR 0.67±0.11 0.67±0.05 1.07±0.11**## 1.04±0.05 2.87±0.25** 3.74±0.55** Female (n) 8 8 10 8 10 10 TC (mM) 1.78±0.07 2.1±0.20 2.98±0.22**## 2.48±0.20 2.57±0.05 3.19±0.22*## TG (mM) 1.23±0.07 1.35±0.17 1.55±0.12 1.25±0.03 1.70±0.13 1.86±0.14* NEFA(mM) 1.33±0.09 1.37±0.08 1.76±0.12**## 1.40±0.06 1.52±0.11 2.03±0.15**## HDL (mM) 0.94±0.04 1.06±0.16 1.45±0.11**# 1.36±0.20 1.49±0.33 1.49±0.10 LDL (mM) 0.48±0.04 0.59±0.10 0.93±0.15**# 0.94±0.12 1.29±0.09 1.33±0.12 Leptin (ng/mL) 2.97±0.19 2.79±0.25 2.65±0.30 2.91±0.30 3.10±0.13 3.10±0.26 Glucose (mg/dL) 57.43±1.00 53.95±1.00 55.70±2.55 59.60±2.57 58.56±3.05 59.16±1.34 Insulin (ng/mL) 0.26±0.03 0.29±0.04 0.33±0.03 0.29±0.02 0.34±0.03 0.38±0.02* HOMA-IR 0.79±0.08 0.84±0.13 0.97±0.11 0.91±0.08 1.02±0.10 1.19±0.08* TC total cholesterol; TG triacylglycerol; HDL high-density lipoprotein; LDL low-density lipoprotein; NEFA non-esterified fatty acids. All parameters were measured at 20 weeks of age. All data were expressed as mean±S.E.M. *P <0.05 vs. Ctrl-F1, ** P <0.01 vs. Ctrl-F1. # P <0.05 vs. INS-F1, ## P <0.01 vs. INS-F1. Significance was determined by ANOVA. Page 29 of 45 Diabetes

Figure legends

Figure 1. Experimental design, offspring growth curves and glucose tolerance. (A) Experimental design. (B) Maternal blood glucose level during 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). (D) Postnatal growth curves for female offspring (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) and AUC 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) Insulin tolerance test 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) Insulin tolerance test 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). All data were expressed as mean±S.E.M. * 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).

Figure 2. Glucose-stimulated insulin secretion. In vivo: (A) Serum insulin levels at fasting state and 30 min after glucose injection and (B) the fold change in serum insulin after glucose loaded 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). (C) Serum insulin levels at fasting state and 30 min after glucose injection and (D) the fold change in serum insulin after glucose loaded in female

offspring (nCtrl-F1_NCD=7, nINS-F1_NCD=8, nGDM-F1_NCD=10, nCtrl-F1_HFD=6, nINS-F1_HFD=6, nGDM-F1_HFD=7). In vitro: (E) Glucose stimulated insulin secretion in isolated islets from 20-week-old male offspring (n=5 mice per group). (F) Glucose stimulated insulin secretion in isolated islets from 20-week-old female offspring (n=5 mice per group). All data were expressed as mean±S.E.M. * 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).

Figure 3. DNA methylation patterns in pancreatic islets of male offspring. (A) Heat map of differentially methylated DMRs 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 diagrams of hypermethylated genes overlapped between G vs. C and I vs. C. (D) Venn diagrams of hypomethylated genes overlapped between G vs. C and I vs. C. (E) KEGG analysis of differentially methylated genes associated with type 2 diabetes.

Figure 4. Ion channels expression and insulin secretion in the pancreatic islets of male offspring. (A-C) Representative mRNA levels of Abcc8, Cav1.2 and Cav2.3 in islets of Diabetes Page 30 of 45

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-old male offspring (n=4 mice per group). (H) Tolbutamide (200 µM) - stimulated insulin secretion. (I) Diazoxide (250 µM) inhibit insulin secretion. (J) Bay K8644 (2 µM)-stimulated insulin secretion. (K) Nifedipine (10 µM) inhibit insulin secretion. Isolated 20-week-old islets, n=5 mice per group. All data were expressed as mean±S.E.M. * 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).

Figure 5. Analysis of DNA methylation level in male offspring pancreatic islets by pyrosequencing. (A) Methylation status of Abcc8 promoter regions, mean DNA methylation and average methylation in each CpG site in male offspring islets. (B) Methylation status of Cav1.2 promoter regions, mean DNA methylation and average methylation in each CpG site in male offspring islets. (C) Methylation status of Cav2.3 promoter regions, mean DNA methylation and average methylation in each CpG site in male offspring

islets. Data are expressed as methylation percentage of each CpG sites (nNCD=6 mice 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).

Figure 6. Fetal islets experiment in vitro. (A) Schematic representation of experimental design. (B) Fetal islets ex vivo were culture overnight and identified by detecting insulin with immunofluorescence. Black scale, 200 µm; White scale bar, 50 µm. (C-E): Expression levels of target genes, DNA methyltransferase genes and demethyltransferase genes in fetal islets (n=3 replicates per group, and 3 independent isolation). (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 2 independent isolation). All data were expressed as mean±S.E.M. * P < 0.05 vs. 5.6 mM, ** P < 0.01 vs. 5.6 mM; # P < 0.05 vs. 16.7/5.6 mM, ## P < 0.01 vs. 16.7/5.6 mM (ANOVA). Page 31 of 45 Diabetes

Figure 1 Diabetes Page 32 of 45

Figure 2 Page 33 of 45 Diabetes

Figure 3 Diabetes Page 34 of 45

Figure 4 Page 35 of 45 Diabetes

Figure 5 Diabetes Page 36 of 45

Figure 6 Page 37 of 45 Diabetes

Supplementary Tables and Figures

Table s1 Primers are used for RT-qPCR. Sequences are printed in the 5’ to 3’direction. Transcript Primers Abcc8 F: GGAGTGGACAGGACTGAAGG R: AGTCAAGGCGGAGACACAGA Cav1.2 F: CAGGAGGTGATGGAGAAGCCA R: CTGCAGGCGGAACCTGTTGTT Cav1.3 F: GGGGTCCAGCTGTTCAAGGGGAA R: GCATGATGAGGACGAACATCATG Cav2.3 F: CCGATGTCTGCTCCCAACATG R: CCTCCGATAAAGGCTGGGGTG Cav3.1 F: CACCAAGTCTGAGTCAGAGC R: TGATTTCATCTCATGATGGGC β-actin F: AGTGTGACGTTGACATCCGT R: GCAGCTCAGTAACAGTCCGC Dnmt1 F: ACCTGGAGAGCAGAAATGGC R:TCCTCGTAGCCACGGAACTA Dnmt3a F: CAGAGCCGCCTGAAGCC R: TCTTCCTTGCCACGGTTCTC Dnmt3b F: TCAGAAGGCTGGAGACCTCCCTCTT R:TTCAGTGACCAGTCCTCAGACACGAA TET1 F: AATGGGCCAACCAGGAAGAG R:GTTGTGTGAACCTGATTTATTGTGG TET2 F: ATATTGATGCGGAGGCGAGG R:CAAATCCTACAGGGCAGCCA TET3 F: TCCGGGAACTCATGGAGGAT R:GAACTCTTCCCCTCCTTGCC Diabetes Page 38 of 45

Table s2 Pyrosequencing primers. (Primers used at 100 nM)

Gene Primers Annealing [Btn] indicates biotinylated primer temp ℃ primer1 F: GGGGATTTTTTTGATTTATATTTAGTTGAG 55 R: [Btn]AACCCCCCCCTTCCAACTACTCTTATA S: TTTGGTTATTATTTTTATTTAGATA primer2 F: AGTGTAGAGATTAGTGAGGAATAGTG 56 Abcc8 R:[Btn] ACCCCTTCTAAAAAAAAATATAAAATTCCA S: TTTTTAGAGAGTTTGTATTTTAAGG primer3 F: AGAAGGGGTTTTTTTAGTTTAGAGGT 55 R: [Btn]AAATTATCAACATCTCCATCCATACT S: ATTTATAGTTTTTTAAGGTTGTG Primer1 F: ATGAAGAGTGTAAAGGGAGATG 55 R:[Btn] AATCCTAAATCCCACACACAAT S: TTAAGGAAAATGAGAATATTAATG Primer2 F: TTGTAAGGGTAGGATGTTAAGGAGTT 55 R: [Btn] ACCCCTATTTCTTCATTCTACTTTAACACT S: GTTAAGGAGTTGTTGG Primer3 F: GGTTATTAAAAGGAGTATTAAGATAGAGG 55 Cav1.2 R: [Btn] CACCTCCTAAAAACTTAATAAACTCT S: AAAATAGTTTTGTAGATTAGTAGTA Primer4 F: TTTTAGTTTAGTTTAATAGTTGGGGAGAGT 56 R: [Btn] AACCTTATCACCTTCTCCCTATCT S: ATTGGTTATAGTTTAGAGTTGTA Primer5 F: GAATAGGGGGGTTGGTGGTA 56 R: [Btn] CCATCTCAAAAACACCCTAATATATCTCC S: GAAGTTTGAGATGATTAAGT Primer1 F: TGAGGAAGTTAGGTATGTGGATTAAG 55 R: [Btn] AAAACAACCAAACCCAAATACCAACATC Cav2.3 S: TTGTATGTTGATTTTGGGA Primer2 F: GATTTAATAGTTTAGAGTAGGGTTGAAGA 55 R: [Btn] CACAAACAAACTCAAAACCTTATCC S: AGTTTGTAGGGGTTTTTA Page 39 of 45 Diabetes

Table s3 Information about antibodies for immunostaining.

Primary Antibody

1st Ab Company Cat. No. Dilution Application

Insulin Cell Signal #4590 1:100 IF

Abcc8 Abcam ab134292 1:2000 WB

Cav1.2 Abcam ab58552 1:200 WB

Cav2.3 Alomone Lab Acc-006 1:200 WB

Potassium ATPase Abcam ab76020 1:100000 WB

Second Antibody

2 nd Ab Company Cat. No. Dilution Application

Donkey anti-rabbit-Alexa 594 Jackson 711-585-192 1:200 IF

Goat anti-mouse H&L (HRP) Abcam ab6789 1:10000 WB

Goat anti-rabbit H&L (HRP) Abcam ab6721 1:10000 WB Diabetes Page 40 of 45

Table s4 Gestational length and litter size in F0 mice. Control-F0 INS-F0 GDM-F0 Gestational length 19.63 ± 0.48 19.00 ± 0.00 19.25 ± 0.48 (days) Litter size 10.83 ± 0.75 10.00 ± 0.37 10.33 ± 0.42 (n) All data were expressed as mean±S.E.M. (n=6 mice per group).

Table s5 Differentially methylated genes in pancreatic islets of GDM-F1 male offspring. (https://www.dropbox.com/s/t68tfyyp9pi1ms0/Table_s5.xlsx?dl=0)

Table s6 Differentially methylated genes in pancreatic islets of INS-F1 male offspring. (https://www.dropbox.com/s/clmb34q42fyhe6k/Table_s6.xlsx?dl=0) Page 41 of 45 Diabetes

Supplementary figures

Figure s1. Body weight at birth in F1 offspring. All data were expressed as mean±S.E.M. (n=20 mice per group, three litters) (ANOVA). Diabetes Page 42 of 45

Figure s2. GTT and ITT in F1 offspring at 8 weeks. (A) Glucose tolerance test (n=8 mice per group). (B) Insulin tolerance test in 8-week-old F1 offspring (in male offspring, nCtrl-F1=15, nINS-F1=11, nGDM-F1=15; in female offspring, nCtrl-F1=9, nINS-F1=7, nGDM-F1=10). All data were expressed as mean±S.E.M. * 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). Page 43 of 45 Diabetes

Figure s3. Ion channels gene expression and DNA methylation status in female F1 offspring. (A-C) Ion channel gene expression in islets of GDM-F1 and INS- females offspring (n=5 mice per group). (D-F) Methylation status of Abcc8, Cav1.2

and Cav2.3 promoter regions in female offspring islets (nNCD=5 mice per group and nHFD=4 mice per group). All data were expressed as mean±S.E.M. * 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). Diabetes Page 44 of 45

Figure s4. GTT and ITT in F1 offspring of STZ-treated non-diabetic group. (A) Glucose tolerance test and AUC in 20-week-old offspring fed with NCD (in male

offspring, nControl=5, nSTZ-alone=7; in female offspring, nControl=6, nSTZ-alone=6). (B) Glucose tolerance test and AUC in 20-week-old offspring fed with HFD (in male

offspring, nControl=6, nSTZ-alone=6; in female offspring, nControl=5, nSTZ-alone=5). (C): Insulin tolerance test in 20-week-old offspring fed with NCD (in male offspring,

nControl=5, nSTZ-alone=7; in female offspring, nControl=6, nSTZ-alone=6). (D) Insulin tolerance test in 20-week-old offspring fed with HFD (in male offspring, nControl=6, nSTZ-alone=6; in female offspring, nControl=5, nSTZ-alone=5). All data were expressed as mean±S.E.M. Page 45 of 45 Diabetes

Figure s5. The expression levels of ion channel genes in STZ-treated non-diabetic offspring. (A) Expression levels of ion channel genes in STZ-treated non-diabetic offspring fed with NCD (n=5 mice per group). (B) Expression levels of ion channel genes in STZ-treated non-diabetic offspring fed with HFD (n=5 mice per group). All data were expressed as mean±S.E.M.