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Thyroid Hormone Promotes Postnatal Rat Pancreatic β-Cell Development and Glucose-Responsive Insulin Secretion Through MAFA

Neonatal β cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of β cells, but the physiological regulators of this process are unknown. Here we show that postnatal rat β cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of β cells via its induction of Mafa.

Neonatal b cells do not secrete glucose-responsive insulin and are considered immature. We previously showed the transcription factor MAFA is key for the functional maturation of b cells, but the physiological regulators of this process are unknown.
Here we show that postnatal rat b cells express thyroid hormone (TH) receptor isoforms and deiodinases in an age-dependent pattern as glucose responsiveness develops. In vivo neonatal triiodothyronine supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to triiodothyronine enhanced the expression of Mafa, the secretion of glucose-responsive insulin, and the proportion of responsive cells, all of which are effects that were abolished in the presence of dominant-negative Mafa. Using chromatin immunoprecipitation and electrophoretic mobility shift assay, we show that TH has a direct receptor-ligand interaction with the Mafa promoter and, using a luciferase reporter, that this interaction was functional. Thus, TH can be considered a physiological regulator of functional maturation of b cells via its induction of Mafa. Diabetes 62:1569-1580, 2013 b -Cell replacement therapy is a major goal of diabetes research. Insulin-positive cells have been successfully derived from stem cells (1)(2)(3)(4), but these cells have not been responsive to glucose in vitro and must be considered functionally immature. Fetal and neonatal rodent b cells also lack glucose responsiveness (5) and therefore provide a good model to study the acquisition of glucose responsiveness and b-cell maturation.
Neonatal b cells are characterized by low expression of many key genes of the specialized b-cell phenotype (6). We previously showed that in neonatal islets, Mafa is expressed at ;10% of the adult level and that its adenoviral-mediated reconstitution to adult levels in P2 islets induced secretion of glucose-responsive insulin (7). But what regulates Mafa in vivo? To identify physiological regulators, we considered the changes that normally occur in the physiological milieu during the neonatal period (8). During the second postnatal week, serum thyroxine (T 4 ) (9) and corticosterone (10) surge and prolactin levels rise by postnatal day 20 (11).
We hypothesized that thyroid hormone (TH) could physiologically regulate Mafa, enhancing its expression and driving the maturation of the insulin secretory response to glucose in neonatal b cells. TH serum levels start increasing in parallel with Mafa expression increases, and TH is important in the postnatal development of the central nervous system and the digestive track (8). Inhibition of TH synthesis prevents or delays maturation of these systems, and TH administration results in precocious development. Triiodothyronine (T 3 ) has been shown to enhance the differentiation of a human pancreatic duct cell line toward a b-cell phenotype (12). Moreover, thyrotoxicosis leads to hyperinsulinemia with increased hepatic glucose production and insulin resistance (13); hypothyroidism reduces production of hepatic glucose and insulin resistance (14).
The effects of TH are mediated by TH receptors (THRs), which are members of the nuclear receptor superfamily. Three major isoforms-THRA1, THRB1, and THRB2exhibit similar ligand-dependent regulation of gene activity, whereas a fourth isoform, THRA2, lacks the ligand-binding and transactivation domains (15). Different isoforms have been identified in whole pancreas during development (16), and THRA1 has been identified in adult islet cells (17); however, little is known about their expression in b cells during the postnatal period.
The active TH bound to receptors is 3,5,39-T 3 , and available T 3 is derived from the thyroid gland or from conversion from thyroxine (T 4 ) by type 1 or type 2 iodothyronine deiodinases (D1 and D2). A third deiodinase, type 3 (or D3), inactivates T 3 . D3-null animals, which had higher levels of TH during development, were glucose intolerant with impaired secretion of glucose-stimulated insulin, suggesting that early exposure to high amounts of T 3 might be deleterious to developing b cells (18). However, the role of TH in b-cell development under physiological conditions as well as the mechanisms involved are still unclear.
Herein we show that postnatal rat b cells express THR isoforms and deiodinases in an age-dependent pattern and therefore have the ability to respond to the rapidly rising T 4 concentration that peaks at about postnatal day 15. In vivo neonatal T 3 supplementation and TH inhibition, respectively, accelerated and delayed metabolic development. In vitro exposure of immature islets to T 3 enhanced Mafa expression and increased glucose responsiveness, effects that were abolished in the presence of dominantnegative (DN) Mafa. Using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) we show that TH has a direct receptor-ligand interaction with the Mafa promoter; using a luciferase reporter, we then showed that this interaction is functional. Thus, TH is a physiological stimulus for the postnatal maturation of functional b cells.

RESEARCH DESIGN AND METHODS
Animals. Female Sprague-Dawley rats with litters of various ages (P0 is day of birth) were purchased from Taconic Farms (Germantown, NY) and kept under conventional conditions with free access to water and food. Animals were killed under anesthesia at postnatal days 2-28 or as an adult; blood was collected by cardiac puncture, and pancreas was excised for histology or islet isolation. Islets were isolated (19), cultured overnight in RPMI-1640 medium, and were handpicked to ensure purity. For each sample from postnatal days 2 or 7, islets from 10 pups were pooled; for samples from postnatal days 9 to 28, islets from 2 or 3 pups were pooled; and for the adult sample, islets from one animal were used. Three to six samples per age group were included. For immunostaining, excised pancreas was either fixed for 2 h in 4% paraformaldehyde for paraffin embedding or embedded in optimal cutting temperature medium (Tissue Tek) and was frozen in chilled isopentane. The Joslin Institutional Animal Care and Use Committee approved all animal procedures. Plasma insulin and T 4 levels. Plasma insulin and T 4 levels were measured using enzyme-linked immunoassay (ALPCO, Windham NH) and COAT-A-COUNT total T 4 kit (DPC, Los Angeles, CA), as previously described (20). Neonatal models of T 3 supplementation and inhibition of TH synthesis. Timed-pregnant Sprague-Dawley rats were randomized into one of three groups: 1) Control pups received subcutaneous injections of 0.9% NaCl daily for 7 days, starting at postnatal day 1. 2) For inhibition of TH synthesis, rats were given tap water ad libitum with 20 mg methimazole (MMI)/100 mL water (21) from birth to postnatal days 15 or 21, when the pups were killed. 3) For T 3 supplementation, pups received subcutaneous injections of T 3 (0.05 mg/g body weight) daily for 7 days starting on postnatal day 1 (22).
Body weight and fed glucose levels were measured weekly. For intraperitoneal glucose tolerance tests, glucose solution was injected intraperitoneally (2 g/kg) after fasting for 4 h, and blood glucose levels were determined at 0, 30, and 120 min. Islets were isolated from T 3 -treated and control rats at postnatal day 7 and reported as in vivo.
The effectiveness of treatments was evaluated by growth parameters, T 4 levels, and deiodinase activity when the animal was killed (23) ( Table 1). Hepatic D1 activity was measured in 5-to 20-mg liver homogenate with 10 mmol/L dithiothreitol and 1 mmol/L 125 IrT 3 for 60 min. Background levels were determined by the addition of 1 mmol/L propylthiouracil (20). Islet culture: Hormone treatment and adenoviral infection. At postnatal day 7 or 9, islets were cultured for 4 days in RPMI-1640 medium (20 mmol/L glucose and 10% charcoal-stripped [CS] FBS) with T 3 (150 pmol/L, equivalent to 7.5 pmol/L free T 3 in 10% CS-FBS [24]) for RNA or insulin secretion assays. As an alternative, islets at postnatal day 9 were cultured for 2 days in RPMI-1640 medium in 10% FBS with dexamethasone (50 pmol/L) for RNA.
To test the specificity of MAFA as the mechanism of T 3 effect, islets at postnatal day 7 were totally dispersed and cultured in RPMI-1640 medium (20 mmol/L glucose, 10% CS-FBS) with or without T 3 (7.5 pmol/L free T 3 ) and with or without adeno-CMV-DN Mafa-Ires-Gfp (DN-Mafa) (25,26) (MOI 2) or control adeno-CMV-Ires-Gfp (Ad-Gfp). Reaggregated islets were cultured for 4 days for secretion or RNA. Insulin secretion in vitro. Insulin secretion was measured by static sequential incubation in 2.6 mmol/L and 16.8 mmol/L glucose in Krebs-Ringer bicarbonate buffer (16 mmol/L HEPES and 0.1% BSA; pH 7.4), as previously described (27). Supernatants and cells were frozen until assayed. As an alternative, we measured insulin secretion of single b cells using the reverse hemolytic plaque assay (7,28) in which secreted insulin is revealed by the presence of hemolytic plaques around secreting cells. The percentage of insulin-secreting cells forming plaques and the area of the plaques were measured and multiplied to calculate the secretion index, a measure of the overall secretory activity of b cells. Quantitative real-time PCR. Total RNA was isolated with a PicoRNA extraction kit (Arcturus) and reverse transcribed (SuperScript reverse transcriptase, Invitrogen, Grand Island, NY). Real-time quantitative PCR used SYBR green detection and specific primers (Supplementary Table 1). Samples were normalized to a control gene (S25), and the comparative threshold cycle method used to calculate levels of gene expression. Immunostaining. Paraffin sections were incubated at 4°C overnight with primary antibodies listed in Supplementary For each antigen, all images were taken with the same settings in confocal mode using a Zeiss LSM 410 or LSM 710 microscope and were handled similarly in Adobe Photoshop; at least two animals per age were examined. Morphometric evaluation. Images covering entire whole pancreatic sections were collected using the LSM 710 tile-scan system. For MAFA nuclear staining, insulin-positive cells were scored as high, low, or undetected nuclear MAFA. Data from four or five individual animals were averaged; between 36 and 58 islets were sampled for each age. For proliferation and apoptosis, insulinpositive and Ki-67-positive or TUNEL-positive cells were counted in 160-327 islets and 2,764-6,189 cells per group; data from individual animals (n = 3-5) were averaged. b-Cell mass was calculated by multiplying the relative area of b cells by the pancreatic weight (29). The densitometric mean of the intensity of MAFA staining was calculated using the AxioVision Measure feature and were considered a reflection of protein levels (n = 6-7 animals per group). Cross-sectional area of the b cell was used as an indicator for cell size and was determined by dividing the number of nuclei in a given insulin-positive area; 7,000-8,794 nuclei were counted among four animals in each group. Cell line culture. INS-1 cells maintained in RPMI-1640 medium containing 11 mmol/L glucose + 10% FCS, 10 mmol/L HEPES, 2 mmol/L L-glutamine, penicillin/ streptomycin, 1 mmol/L sodium pyruvate, and 20 mmol/L b-mercaptoethanol were switched to RPMI-1640 medium containing 1.6 mmol/L glucose + 10% CS-FBS 24 h before treatment and then were kept for 14 h in T 3 (150 pmol/L) and actinomycin D (5 mg/mL). MIN-6 cells, maintained in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% FBS, were switched to high-glucose DMEM with 15% CS-FBS and T 3 (10 nmol/L) for 24 h; they then were harvested for ChIP or to assess the effect of MMI (1.5 mmol/L, 0.15 mmol/L, and 0.015 mmol/L) on gene transcription. For the luciferase reporter construct, a 10-Kb Nhe1-Fse1 fragment from the BAC mouse genomic library clone 128L24 spanning the mouse Mafa promoter to downstream of the transcription start site was cloned into HindIII-BglII cut -238 wild-type luciferase plasmid (30). Renilla luciferase in a SacI backbone was used as a transfection control. For transient transfections, MIN-6 cells were transfected using lipofectamine and 7 mg of final DNA and grown in highglucose DMEM with/without 100 nmol/L T 3 for 24 h. ChIP assay. Rabbit anti-THR (TRa/b, Santa Cruz FL-4083) was used with the Imprint ChIP kit (Sigma Aldrich, St. Louis, MO; CHP1) by following manufacturer's instructions. DNA from 250,000 cells was used for each condition in four independent experiments. Samples were analyzed by quantitative PCR using specific primers for three putative thyroid response elements (TREs; S1, S2, and S3) (Supplementary Table 1) and were run on gels. Gel-mobility shift assay (EMSA). Nuclear extracts were obtained from HEK1 cells transfected with Thrb, or untransfected cells as controls, using the NucBuster Protein Extraction Kit (Novagen, EMD Biosciences, San Diego, CA) following the manufacturer's instructions. Twenty micrograms of nuclear extract were incubated with 80 pmol/L of double-stranded oligonucleotides, reproducing the potential TRE S3 in the rat Mafa gene or the reported TRE of D1 (31) at room temperature for 30 min. The reaction buffer for binding was 75 mmol/L Tris (pH 7.8), 264 mmol/L potassium chloride, 1.5 mmol/L EDTA, 30 mmol/L b-mercaptoethanol, 30% glycerol, and 1.2 mg/mL BSA. In some of the binding assays, anti-THRB antiserum (Affinity Bioreagents, Golden, CO) or a random antibody were added 1 h before addition of the DNA probes. After the binding reactions, samples were analyzed by separation on 10% polyacrylamide gel in 13 Tris-acetate-EDTA buffer followed by 40 min of staining with SYBR green EMSA nucleic acid gel stain (Molecular Probes). Data analysis. Data are shown as mean 6 SEM. For statistical analysis, unpaired Student t tests were used to compare two groups, and one-way ANOVA followed by a Bonferroni post hoc test were used to compare more than two groups. A P value ,0.05 was considered statistically significant.

RESULTS
Postnatal glucose and insulin levels change concurrently with expression of TH, deiodinases, and THR. Blood glucose levels at postnatal day 2 were significantly lower than levels in adults (Fig. 1A), but they increased, peaking at postnatal day 21 at a level 20% higher than that of adults. Perhaps as a result of rising plasma glucose levels and as part of the functional maturation process of b cells, plasma insulin levels peaked at postnatal day 11, with levels threefold higher than in newborns or adults (Fig. 1B). During the first postnatal month (Fig.  1C), the rate of glucose disposal evaluated by the area under the curve (AUC) of intraperitoneal glucose tolerance test was progressively enhanced; that at postnatal days 7 and 11 were slower (high AUC values) than at later ages. Adult values were not yet achieved by postnatal day 21.
For TH to physiologically regulate the functional development of b cells, its serum level and the tissue regulation of biologically active T 3 must be sufficient and its receptors must be expressed by b cells. Serum T 4 , the principal circulating TH, steadily increased over the first 2 weeks, peaking at about twice adult levels at postnatal day 15 before falling to adult levels by postnatal day 21 (Fig.  1D). Because tissue concentrations of T 3 are determined by deiodinases as well as by serum T 3 , we measured the islet expression of deiodinases Dio1, Dio2, and Dio3 transcripts during postnatal development (Fig. 1E). Dio1 mRNA in islets increased throughout the postnatal period; its positive regulation by TH provides an increased paracrine supply of T 3 as the islet develops. In contrast, expression of both Dio2 and Dio3 fell during the postnatal period.
D1 and D3 proteins were examined by immunostaining ( Supplementary Fig. 1) in pancreas from animals at postnatal day 7 and adult animals. The small number of islets obtained from rats at postnatal day 7 precludes protein quantification using Western blots. However, careful titration of antibody, parallel staining, and confocal imaging allowed assessment of protein levels by differences in intensity. Islet D1 protein was much lower at postnatal day 7 than in adults ( Supplementary Fig. 1A), although its mRNA level did not differ. This discrepancy might reflect developmental differences in the synthesis of selenoproteins that occur downstream of transcription (32). In contrast, D3 ( Supplementary Fig. 1B) had very strong cytoplasmic localization in islets at postnatal day 7 and became almost undetectable in adult islets.
As serum T 4 increased, islet expression of thyroid receptors increased at both mRNA and protein levels. Thra mRNA increased sharply between postnatal days 7 and 9; they were sixfold higher in islets at postnatal day 9 than in adults (Fig. 1F) and decreased to adult levels by postnatal day 15. By immunostaining ( Fig. 2A), nuclear THRA protein (both the T 3 -binding THRA1 and the non-T 3binding THRA2 isoforms are recognized by the antibody) increased from postnatal day 7 to day 10 but became much lower by adulthood (Fig. 2C). Similarly, Thrb mRNA increased between postnatal days 7 and 9 and decreased to adult levels by postnatal day 15 (Fig. 1F). THRB protein also increased by postnatal day 7 but was mainly cytoplasmic; nuclear THRB localization was seen only by postnatal day 15 and increased in the adult ( Fig. 2B and C). Comparison of mRNA expression of Thra and Thrb in islets ( Supplementary Fig. 2) shows a change in predominant receptor isoform through development: Thra predominates at early ages, Thra and Thrb are equal from postnatal day 9 to 15, after which Thrb becomes the predominant isoform in islets. T 3 supplementation until postnatal day 7 accelerated metabolic development. To analyze the direct effects of TH, newborn rats were injected with T 3 from postnatal days 1 to 7. As expected, this supplementation increased body and pancreatic weights, reduced T 4 levels (because of suppression of thyrotropin by T 3 ), and increased D1 activity in the liver ( Table 1). The pancreas of animals treated with T 3 had greater density of both acinar and islet cells (Fig. 3A), greater b-cell proliferation (20% vs. 8% Ki-67 + insulin + cells in untreated animals) ( Fig. 3B and C), and no change in the frequency of apoptotic b cells (Fig.  3D). Their b-cell mass was unchanged (Fig. 3E), but their b cells were smaller (Fig. 3F), with no significant change in the number of cells (Fig. 3G). Fasting glucose levels were lower (Fig. 3H) and fed plasma insulin levels elevated (Fig.  3I), but their response to intraperitoneal glucose tolerance tests were not different (data not shown). These results show that T 3 supplementation enhanced the functional development of b cells. Inhibition of TH synthesis delayed pancreatic development. Animals treated with MMI from birth until postnatal day 15 or 21 were confirmed as having hypothyroidism because of delayed growth with lower body and pancreatic weights, decreased circulating T 4 (due to inhibition of TH synthesis from the thyroid gland), and decreased activity of D1 in the liver (Table 1). At postnatal day 15 there was no change in pancreatic cell density ( Supplementary Fig. 3A), b-cell proliferation ( Supplementary  Fig. 3C) or b-cell mass (Supplementary Fig. 3B). However, animals treated with MMI at postnatal days 15 and 21 had lower fasting blood glucose levels than their untreated agematched controls (Supplementary Figs. 3D and 4A), similar to that of younger animals (Fig. 1A). Compared with untreated controls, plasma insulin levels of animals treated with MMI were lower at postnatal day 15 ( Supplementary  Fig. 3E) but were fivefold higher at postnatal day 21 (Supplementary Fig. 4B); values were similar to those at postnatal day 11, when plasma insulin levels peak (Fig. 1B). The Values shown as means 6 SEM. A: Fed blood glucose (n = 5-27 animals per age; *P < 0.0001); B: plasma insulin (n = 4 animals per age; *P < 0.04). AUC for intraperitoneal glucose tolerance test (C) and serum T 4 (D) seen at different ages; n = 5-27 animals per age, *P < 0.0001 with respect to the previous age in C and D. Deiodinase (Dio1, type 1; Dio2, type 2; Dio3, type 3) (E) and thyroid receptor isoform mRNA (F) over the same time course by quantitative PCR; the same samples are used for both. Data are expressed as -fold change with respect to adult levels (10 weeks old) using S25 as the internal control gene (n = 4-6 samples per age, each pooled from 3-10 animals; *P £ 0.0001).
gradual postnatal development of glucose clearance was delayed by inhibiting T 4 synthesis ( Supplementary Fig. 3G and Fig. 1C). Animals treated with MMI at postnatal day 21 had glucose clearance similar to untreated animals at postnatal day 7 (Fig. 1C). Animals treated with MMI at postnatal day 15 were not as affected, possibly because of shorter treatment and differences in T 3 sensitivity of other glucosedisposing tissues. Overall, the lack of TH during the postnatal period delayed the development of efficient glucose disposal. Local regulation of TH action in postnatal rat islets by T 3 or MMI treatment. Systemic changes in TH status can be regulated by local changes in T 3 concentrations by deiodinases in target tissues. Changing levels and isoforms of thyroid receptors also potentially regulate local TH effects. Islets from animals treated with T 3 at postnatal day 7 had increased nuclear THRA (Fig. 4A) and cytoplasmic THRB protein (Fig. 4C), suggesting more functional receptors. In animals treated with MMI at postnatal day 21 ( Fig. 4B and D), both THRA and THRB had the nonfunctional cytoplasmic localization typical of younger ages. Deiodinase changes also reflected the hormonal status, with increased Dio1 and Dio3 mRNAs in islets after T 3 treatment (Fig. 4E) and reduced Dio1 and Dio3 and elevated Dio2 mRNA levels ( Supplementary Fig. 3F) after MMI treatment until postnatal day 21. This confirmation that normal neonatal changes in deiodinases and thyroid receptors were replicated by external manipulation of TH status suggests that the endogenous events are causally related to the TH status. In vivo T 3 modulation affected Mafa gene expression and its protein nuclear translocation. Islet transcriptional profile changed with in vivo T 3 treatment (Fig. 4E and F): Mafa, Mafb, Pgc1a (peroxisome proliferatoractivated receptor g coactivator-1 a), and Thrb mRNA increased; Pdx1 and Neurod1 mRNA were unchanged; and Rest (RE1-silencing transcription factor) mRNA decreased. In contrast, Mafa decreased at postnatal day 15 with MMI treatment (Supplementary Fig. 5D and Supplementary Table 3); partial T 3 supplementation reversed this decrease. Mafb, preproinsulin, Ins2, Glp1r, and Thra mRNAs increased and Rest mRNA decreased in P15 islets (Supplementary Table 3). Rest, Thra, and Thrb mRNAs  Table 3). Insulin staining was more intense in the T 3 -treated group and less intense in MMI-treated animals (Fig. 5A).
T 3 supplementation increased MAFA protein both in nuclear location (Fig. 5A) and amount (Fig. 5B). In pups treated with T 3 at postnatal day 7, 93% of b cells had nuclear MAFA compared with only 62% in untreated pups (Figs. 5A and C); 51% of T 3 -treated pups had MAFA high nuclear staining compared with 10% of untreated animals. The observed effect of T 3 on MAFA translocation could be an indirect effect mediated by altering the redox state of the cell (33) through enhancement of mitochondrial function following increased expression of Pgc1a (Fig. 4C). It is surprising that even with the increased nuclear expression of MAFA, glucose-stimulated insulin secretion was not increased in islets from T 3 -treated animals (Fig. 5D). In contrast, hypothyroidism at postnatal day 15 decreased Mafa mRNA levels ( Supplementary Fig. 5D) and protein ( Supplementary Fig. 5A and B) without changes in location ( Supplementary Fig. 5C). MMI treatment also decreased b-cell function as estimated by homeostasis model assessment-B index (Supplementary Fig. 5E) (18). The direct effect of MMI on transcription was ruled out because no significant changes were found in the above genes after culturing MIN-6 cells in the presence of MMI (Supplementary Fig. 5F). In vitro T 3 specifically increased Mafa mRNA and improved glucose-stimulated insulin secretion. Because the in vivo data may be confounded by the effects on insulin sensitivity by thyrotoxicosis or hypothyroidism (14), we developed an in vitro system to directly test whether increasing Mafa was the mechanism mediating the T 3 -maturation effect. DN MAFA lacks an N-terminal transactivation domain and can form heterodimers with endogenous MAFA, impairing its ability to activate target genes (25). In T 3 -treated Ad-Gfp transduced islets, Mafa mRNA increased significantly (Fig. 6A), confirming the in vivo T 3 results (Fig. 4F); glucokinase mRNA also increased. It is important to note that these increases were absent in T 3 -treated islets infected with Ad-DN-Mafa, indicating a specific role of MAFA in T 3 -mediated effects. Interestingly, the T 3 -induced Mafa increase was inhibited in the presence of DN-Mafa, suggesting a positive feedback mechanism of MAFA upon its own expression. In vitro T 3 -treatment induced glucose-stimulated insulin secretion. In static incubations at postnatal day 7, islets, whether untreated or Ad-Gfp infected, had little response to 16.8 mmol/L glucose (Fig. 6B), whereas T 3treated islets, both uninfected and Ad-Gfp infected, had insulin secretion increased 5.5-fold (Fig. 6B). This effect also was abolished in the presence of Ad-DN-Mafa. Using the reverse hemolytic plaque assay to evaluate insulin secretion from individual b cells (Fig. 6C), the effect of T 3 on glucose responsiveness was shown to result from an increased proportion of insulin-secreting cells (Fig. 6D).
Glucocorticoids inhibit the effects of T 3 on glucosestimulated insulin secretion. Paradoxically, glucosestimulated insulin secretion was not increased in islets from animals treated with T 3 in vivo (Fig. 5D), as was expected from our in vitro studies (7) and those described earlier. However, T 3 affects the maturation of other tissues, including the adrenal gland (34), so the blunted in vivo glucose responsiveness may have resulted from increased glucocorticoids. In untreated animals, circulating corticosterone was quite low after birth and increased only modestly by postnatal day 15 and then surged to adult levels ( Supplementary Fig. 6A). Glucocorticoid receptor FIG. 4. T 3 treatment results in differential changes of thyroid receptor proteins and islet gene expression. Islets from T 3 -treated and untreated animals at postnatal day (P) 7 immunostained for THRA (A) and THRB (C) or from MMI-treated rats at P21 stained for THRA (B) and THRB (D). Representative confocal images were taken at the same settings for each protein so differences in intensity reflect differences in protein. At least three animals each group. Changes in Dio1, Dio2, and Dio3 (n = 3) (E) and key islet gene (n = 18) (F) mRNA in islets isolated from T 3 -treated and untreated control pups by quantitative PCR at P7. Values shown as mean 6 SEM; *P < 0.05 with respect to control animals at P7.
FIG. 5. In vivo T 3 treatment increased Mafa expression and enhanced its nuclear localization but did not increase glucose-stimulated insulin secretion. A: Representative images from T 3 -treated and control animals at postnatal day (P) 7 immunostained for insulin (green) and MAFA (red); bottom panels show only the red channel for MAFA visualization. B: Intensity was quantified as densitometric mean of MAFA staining from at least three animals per group. C: Nuclear localization of MAFA in islets of animals treated with T 3 increased compared with untreated controls at P7. Quantification of 600-2,200 insulin + cells from four or five animals for each group. D: Glucose-stimulated insulin secretion in static incubations of islets freshly isolated from in vivo T 3 -treated animals at P7 or control animals (n = 5-6 experiments). Values shown as mean 6 SEM; *P < 0.02 with respect to controls. mRNA in islets remained unchanged from embryonic day 20.5 to postnatal day 28 ( Supplementary Fig. 6B). However, with T 3 supplementation, corticosterone levels at postnatal day 7 were twice the normal values and were comparable with those at postnatal day 11 ( Supplementary  Fig. 6C). In contrast, after MMI treatment, corticosterone levels were reduced at postnatal day 21 ( Supplementary  Fig. 6D). These data indicate a rapid T 3 -dependent change in adrenal function that could affect in vivo islet function over the neonatal period.
To assess whether increased glucocorticoids explained the difference in functional maturation of in vitro and in vivo T 3 treatments, islets at postnatal day 9 were cultured for 48 h with T 3 , dexamethasone, or both. In islets cultured with T 3 alone, Mafa mRNA was increased significantly ( Supplementary Fig. 6E), consistent with islets from animals treated with T 3 at postnatal day 7 (Figs. 4F and 6A). However, in islets cultured in the presence of dexamethasone, with or without T 3 , Mafa levels remained unchanged and Pdx1 mRNA was significantly suppressed compared with untreated controls (Supplementary Fig.  6E). Insulin secretion was blunted in similarly cultured islets at postnatal day 9 ( Supplementary Fig. 6F). These results suggest that islets from animals treated with T 3 in vivo lacked enhanced glucose-stimulated insulin secretion due to the counter effects of increased glucocorticoids. T 3 directly enhanced Mafa transcription. To analyze the mechanism by which T 3 regulates Mafa expression, we used b-cell lines (INS-1 and MIN-6), both of which express THRA and THRB (data not shown). T 3 increased Mafa mRNA transcriptionally rather than by enhancing its stability, as shown by a 90% inhibition of Mafa mRNA in the presence of transcription inhibitor actinomycin D (Fig.  7A).
For T 3 to have direct effect on Mafa transcription, THR must bind to TREs. Using AliBaba 2.1 software, we found potential TRE motifs in the proximal promoter and coding sequence of mouse Mafa and designed quantitative PCR primers for them (Supplementary Table 1). In ChIP assay with MIN-6 cells (Fig. 7B-D), S2 had 2-fold and S3 had 10fold enrichment with respect to IgG, indicating both were TREs. Further evidence of binding of THR to S3 is provided by EMSA, in which a band observed in the presence of HEK1 nuclear extract from cells transfected with Thr expression plasmid was inhibited upon incubation with antibody against THR (Fig. 7E), suggesting a specific disruption of the THR:S3 interaction. In addition, in a luciferase assay using a reporter construct containing the Mafa gene 59-promoter region (S3 was not included) (Fig. 7F), T 3 induced a significant increase of luciferase activity, indicating S2 is active in the presence of T 3 and increases Mafa transcription. The modest size of the increase in luciferase activity is primarily the result of the lack of the S3 site in the construct and the high basal Mafa/luciferase expression in the MIN-6 cells, limiting the impact of T 3 on the transcription of Mafa/luc gene. Overall, these results indicate that T 3 regulates Mafa transcription through direct receptor-ligand interaction.

DISCUSSION
Previously we showed that overexpression of Mafa in neonatal islets to approximately adult Mafa mRNA levels induced glucose-responsive insulin secretion and thus the functional maturation of b cells (7). The data presented here support TH as a physiological regulator of Mafa FIG. 6. In vitro T 3 selectively increased Mafa mRNA and enhanced glucose-stimulated insulin secretion. A: Culture for 4 days with T 3 (7.5 pmol/L free T 3 ) induced changes in key islet mRNA levels in islets isolated from animals at postnatal day (P) 7 and infected with either control Ad-Gfp (black bars) or DN Mafa (white bars) as normalized to those of Ad-Gfp without T 3 treatment. (*P < 0.04 respect to Ad-Gfp; +P < 0.05 respect to Ad-Gfp+T 3; n = 4-7). B: Insulin secretion from similarly infected and cultured islets at P7 in response to 2.6 mmol/L glucose (black bars) and 16.8 mmol/L glucose (white bars) in sequential static incubations (seven experiments for Ad-Gfp +T 3 and four for Ad-Gfp +T 3 +DN-Mafa; *P < 0.04 with respect to Ad-Gfp). C: T 3 -enhanced glucose-responsive insulin secretion was confirmed by reverse hemolytic plaque assay for individual cell secretion, showing an increased percentage of insulin-secreting cells (D) (three experiments; *P £ 0.006 with respect to 2.6 mmol/L glucose). Values shown as mean 6 SEM.
expression and postnatal functional maturation of b cells. Increased expression of THR and increased serum T 4 levels accompanied the increased Mafa mRNA between postnatal days 7 and 9. Islets treated with T 3 in vitro at postnatal day 7 showed increased glucose responsiveness with a greater proportion of responsive b cells. Importantly, expression of DN-MAFA blocked secretion of glucose-responsive insulin and the increased Mafa mRNA. These data, along with our previous work (7), indicate that T 3 induction of glucose responsiveness was dependent on MAFA. Moreover, the thyroid receptor directly interacts with two putative TREs in the Mafa gene. In vivo manipulation of TH (treatment with T 3 or MMI) resulted in the expected change in Mafa expression, demonstrating the importance of this mechanism during normal physiological development.
TH is a known regulator of development in different tissues, yet its developmental effects on b-cell function have not been described. Previous studies showed that the T 3 -THRA complex enhanced proliferation of RINm5f cells (35) and that THRA knockout mice had greater wholebody insulin sensitivity (36). We now show that Mafa is a direct target of TH in b cells. The physiological importance of T 3 regulation of islet development is underscored by our in vivo models of T 3 modulation.
Although a postnatal switch of thyroid receptor isoforms has been described in the heart, dorsal root ganglia, and sciatic nerve with Thra characteristic of immature tissues and Thrb characteristic of functionally mature tissues (37,38), such an isoform switch had not been previously described for b cells. Comparison of the relative mRNA expression of the isoforms shows that Thra predominates B: Schema of the murine Mafa promoter and coding sequence. The top arrow indicates the transcription start site. The black ovals indicate the experimentally tested TRE sites by ChIP: site 1 (S1), site 2 (S2), and site 3 (S3). The TREs are localized at 22,342/-2,354, 21,927/-1,946 and +647/ +659. The amplified sequences are shown as is their conservation in other species. S1 is not conserved in humans or rats, whereas S2 is conserved in rats but not in humans. C: In ChIP studies of MIN-6 cells using an antibody against THR, putative TREs from S2 and S3 showed direct binding of the THR, but no binding was evident at S1. Gel representative of three or four experiments. D: Quantitative PCR products from ChIP-amplified DNA with corresponding IgG, RPII, and input. (values shown as mean 6 SEM; *P < 0.03; n = 3-4 controls). E: Electrophoretic mobility shift assay showing a band observed in the presence of HEK1 nuclear extract from cells transfected with Thr expression plasmid that was inhibited upon incubation with antibody against. S3, potential TRE Site 3 in Mafa cds; NE, nontransfected nuclear extract; D1 TRE, known TRE in Dio1 promoter; IgG, unspecific antibody; a-THR, antibody against Thr. F: A dual luciferase reporter assay using a firefly luciferase reporter construct with the 59 Mafa promoter region in MIN-6 cells grown in high-glucose DMEM +/2 100 nmol/L T3 for 24h. Renilla luciferase in a SacI backbone was used as a transfection control (values shown as mean 6 SEM; *P < 0.01; n = 3). (A high-quality color representation of this figure is available in the online issue.) at early ages, that both Thra and Thrb are equal from postnatal days 9 to 15, and that Thrb becomes the predominant isoform in adult islets. At the protein level, b cells expressed both THRA and THRB at postnatal day 7, but the "immature" isoform THRA had nuclear localization between postnatal days 7 and 15, whereas the THRB isoform was mainly cytoplasmic, suggesting that THRA mediates TH effects on b cells during the early postnatal period. THRB showed nuclear translocation only at postnatal day 15, which was when T 3 plasma levels peaked. Nucleocytoplasmic shuttling of both THRA and THRB have been described in other cell types (39)(40)(41) and can be mediated by T 3 in an energy-dependent process (39); their nuclear export is considered passive (40), but it is unclear why there is differential shuttling of the two isoforms.
We also showed that the effects T 3 has on other tissues could influence the observed phenotype. The surprising absence of glucose-stimulated insulin secretion in islets from pups treated with T 3 until postnatal day 7 was likely due to the accelerated maturation of the adrenal gland induced by T 3 . At birth the adrenal lacks full function, and circulating corticosterone increases only gradually after postnatal day 7 until about postnatal day 15, when a surge occurs (10). Size of the adrenal gland and circulating corticosterone levels previously have been directly related to circulating T 3 levels (42). In our studies, corticosterone levels doubled in animals that received T 3 supplementation and significantly decreased in those in which T 4 synthesis was inhibited. Even low concentrations of dexamethasone decreased Pdx1 mRNA and blocked the stimulatory effects of T 3 on Mafa mRNA and insulin secretion.
In conclusion, we have shown that TH is a physiological regulator of b-cell maturation, a process mediated through direct interaction of THR and the Mafa promoter. In vitro, the active hormone T 3 increased glucose-stimulated insulin secretion and potentially could have a similar maturation role for in vitro stem cell-derived b cells. Identification of additional physiological regulators that drive b-cell maturation and glucose responsiveness should lead to effective strategies for developing fully mature in vitro-derived b cells for replacement therapy for diabetes.