Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
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1 Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA GGA GGT CAT C GCG TAG CCG CGG TTC TT-3 Mafa UTR 5 - GTC TTC AGG GTC GCC GTC TAG GAG GTT GGG ACG CAG AAC TG-3 INSULIN TCT TCT ACA CAC CCA TGT CCC GGT GCA GCA CTG ATC CAC-3 PDX1 5 - GAC ACA TCA AAA TCT GGT TCC AAA-3 5 -TCC CGC TAC TAC GTT TCT TAT CTT C-3 NeuroD 5 - ACT CCA AGA CCC AGA AAC TGT C ACT GGT AGG AGT AGG GAT GCA C-3 MafB 5 -GAA GCC CGC GAG GCA TAT GGC CCT GGC ACT CAC AAA -3 Glucagon 5 -GCC GAG CAA GGC GAG ACT-3 5 -CAT GTC TGC GCC CAA GTT C-3 GK 5 -AGC TCA GCG AAC CCC GGT CA TCC CCG GAG TCG CTC TCC AC-3 TR alpha 5 -TGG ACC TGG TTC TAG ACG ATT CA-3 5 -TCC CGG TTC TGC TCA ATC A-3 TR beta 5 -CTC TGT CGT CTT TCA ACC TGG AT-3 5 -TGG GCG ATC TGA AGA CAT CA-3 Nkx TCT TCT GGC CTG GGG TGA TG-3 5 -GGC TGC GTG CTT CTT TCT CCA-3 S1 5 -CCA AGT ACT GGG ATT AAA GGT ATA TGC-3 5 -TTG GGC AGT CTG CCT TCT CT-3 S2 5 - CTG TAG AGT CCC AAA GAT AGA CAT TCC-3 5 -AAC TCT CCC CTC AGC CTG TTC-3 S3 5 - CTT CAG CAA GGA GGA GGT CAT C-3 5 -GCG TAG CCG CGG TTC TT-3 Supplementary Table 2. Primary antibodies used for immunostaining. Name Species Vendor Dilution (method) THRA Rabbit ABR 1:800 (P, Bio-SA) THRB Mouse ABR 1:2000 (P, Bio-SA) D1 Rabbit Produced by PR Larsen (41) 1:1000 (P, Bio-SA) D3 Rabbit Novus 1:500 (P, Bio-SA) MAFA Rabbit Bethyl 1:1500 (C, Bio-SA) Ki67 Mouse BD Bioscience 1:50 (P, Bio-SA) Insulin Guinea-pig Linco 1:200 a (P/C) a The direct fluorescein-conjugated secondary antibody was applied for these primary antibodies. Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
2 Supplementary Table 3. Changes of mrna expression in islets from MMI-treated rats at P15 and P21 normalized to age matched controls. Mean SEM; n=3-18 animals; *p<0.05 with respect to untreated animals. Gene P15 + MMI (fold change to C P15) P21 + MMI (fold change to C P21) Mafa * Pdx * Nkx nd Mafb * NeuroD nd Rest * * Preproins * Ins * Glp1r * Thra * * Thrb *
3 Supplementary Figure 1. Changing patterns of deiodinase protein over the neonatal period as shown by immunostaining. D1 (red) (A) and D3 (red) (B) protein are expressed in both insulin-expressing (green) beta cells and non-beta endocrine cells in P7 and adult islets. Top panels show merged channels while the bottom ones show only the red channel for deiodinase visualization. Representative confocal images taken in parallel at same settings for each protein so differences in intensity reflect differences in protein, from at least 3 animals each group. Supplementary Figure 2. The dominant receptor isoform changes throughout development in beta cells as determined by relative mrna expression quantified by qpcr.
4 Supplementary Figure 3. In vivo inhibition of TH synthesis by methimazole (MMI) treatment for 15 or 21 days affects pancreatic structure, beta cell dynamics, and glucose homeostasis. A. Representative fields (hematoxylin stained) of pancreas from treated and untreated P15 animals. Beta cell mass (B)(n=5) and proliferation (% Ki67 + Insulin + cells) (C) (n=4), fasting blood glucose (D) (n=5) and fed plasma insulin levels (E) (n=4-5) (Controls 0.8 ± 0.3 vs. MMI 0.2 ± 0.1 ng/ml) shown for P15 control and MMI-treated animals. F. Dio1, Dio2 and Dio3 mrna in islets isolated from P21 control and MMI treated rats (n=3). G. Area under the curve for IPGTTs after 4 hr fast shown for control and MMI-treated P15 (n= 4-5) and P21 (n=6-10). Mean SEM; *p with respect to untreated animals.
5 Supplementary Figure 4. Inhibition of thyroid synthesis by MMI treatment for 21 days delayed carbohydrate metabolism. Blood glucose (A) and plasma insulin levels (B). Mean SEM, *p<0.05; n= 5-9 animals.
6 Supplementary Figure 5. After 15 days of MMI treatment, MAFA protein levels were lower than in controls. A. Representative images of islets from P15 MMI-treated and untreated age-matched controls animals immunostained for MAFA (green) and insulin (red). B. Quantification of MAFA staining as mean densitometric intensity on sections from at least 3 animals each group. Mean SEM, *p.001 C. No difference in nuclear localization of MAFA was found between groups insulin + cells counted over 4 animals of each group. D. After treatment with MMI for 15d, islets from treated rats had lower levels of MAFA than untreated groups. Supplementation with T 3 was able to reverse these effects. n=4-5, *p E. Change of HOMA-B as a measure of insulin secretion of islets with 100% being that of the control untreated rats. n=4-5, *p.0001 F. MMI had no direct effect upon MIN6 transcription after 24h culture;here shown data from mm MMI which is likely to be the in vivo exposure.; data from other concentrations in dose curve were similar n=3.
7 Supplementary Figure 6. In vivo T 3 treatment led to increased corticosterone levels, which can suppress the T 3 -enhanced expression of key islet genes. A. Circulating corticosterone changed over the postnatal period. n=13;* p=0.02 compared to adult. B. Glucocorticoid receptor mrna was stable throughout this period as normalized to S25 a housekeeping gene. (n= 3 each time point; n=2 for two conditions for NeuroD). C. Serum corticosterone levels after T 3 treatment in P7 animals compared to untreated littermates (n=9-10). D. Effects of MMI treatment during 21 days on circulating corticosterone levels (n= 7-9). E. Of key beta-cell transcription factors, only Pdx1 mrna was suppressed in P9 islets after 5d culture with dexamethasone (5 pm free dexamethasone) in the presence or absence of T 3 (7.5 pm free T 3 ). Additionally the increase of Mafa mrna seen with T 3 was blunted. (n=3). p 0.01 F. Similar culture with dexamethasone blunted glucosestimulated insulin secretion in the presence or absence of T 3 ; insulin secretion presented as fold increased at 16.8 mm glucose to that at 2.6 mm glucose for each group. Insulin values at 2.6 mm glucose were 21 9 pg/ng DNA control group, 5 ± 2 pg/ng DNA for T 3 ; 46±19 pg/ng DNA for DEX and 12±3 pg/ng DNA for DEX+T 3. Mean SEM; *p<0.04 to control; **p p<0.02. n= 4-6 experiments.
8
9 Supplementary Figure 7. In vivo T 3 treatment until P11 did not enhance glucose-stimulated insulin secretion in isolated islets compared to that of untreated age-matched controls. Mean SEM; n=6 animals for each group.
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c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
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