Liganded Thyroid Hormone Receptor-α Enhances Proliferation of Pancreatic β-Cells

Failure of the functional pancreatic β-cell mass to expand in response to increased metabolic demand is a hallmark of type 2 diabetes. Lineage tracing studies indicate that replication of existing β-cells is important for β-cell proliferation in adult animals. In rat pancreatic β-cell lines (RIN5F), treatment with 100 nm thyroid hormone (triiodothyronine, T3) enhances cell proliferation. This result suggests that T3 is required for β-cell proliferation or replication. To identify the role of thyroid hormone receptor α (TRα) in the processes of β-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTRα. Infection with AdTRα to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTRα-infected cells led to activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E2F pathway, along with cell cycle progression and cell proliferation following treatment with 100 nm T3. Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTRα-infected cells led to down-regulation of the cyclin D1/CDK/Rb/E2F pathway and inhibited cell proliferation. Furthermore, in immunodeficient mice with streptozotocin-induced diabetes, intrapancreatic injection of AdTRα led to the restoration of islet function and to an increase in the β-cell mass. These results support the hypothesis that liganded TRα plays a critical role in β-cell replication and in expansion of the β-cell mass during postnatal development. Thus, liganded TRα may be a target for therapeutic strategies that can induce the expansion and regeneration of β-cells.

The pancreatic ␤-cell mass plays an essential role in determining the amount of insulin that is secreted to maintain blood glucose levels within a narrow range. Elucidation of the mechanisms that control the size of the ␤-cell mass is essential to allow development of regenerative therapy for both type 1 and type 2 diabetes, which are diseases characterized by an insufficient ␤-cell mass (1). Several mechanisms have been proposed to explain the process of adult ␤-cell mass expansion, including neogenesis from pancreatic duct cells or hematopoietic tissues, replication of highly active ␤-cell progenitors within the islets, and simple ␤-cell proliferation. The size of the ␤-cell mass is governed by the balance between the growth (differentiation and replication) and death (apoptosis) of these cells, but the mechanisms that sense the ␤-cell mass and maintain its home-ostasis are largely unknown (2). Several recent reports have indicated that proliferation or replication of existing ␤-cells rather than differentiation of new ␤-cells from stem cells, is one mechanism involved in maintenance of the ␤-cell mass in adults (3,4).
To clarify the mechanisms involved in the replication of ␤-cells, we focused on molecular regulation of the cell cycle in mature pancreatic ␤-cells. Terminally differentiated cells often make the decision to replicate at the interface of the G 0 /G 1 phase of the cell cycle (5). Mitotic stimulation can induce entry into the cell cycle during the G 1 phase through the assembly of cyclin D by cyclin-dependent kinase (CDK) 2 4/6. The activated cyclin D-CDK 4/6 complex then phosphorylates retinoblastoma protein (pRb). This initial phosphorylation of pRb is followed by additional phosphorylation mediated by the cyclin E-CDK 2 complex. Once pRb has been phosphorylated, it releases the tethered transcription factor E2F and irreversibly commits the cell to progression through the cell cycle. It has been reported that complexes of CDK 4 with cyclin D1 or D2 are critical regulators of ␤-cell proliferation and ␤-cell mass homeostasis after birth (6). Although cyclin D plays a key role in regulating the transition of ␤-cells from a quiescent to a proliferative state, cyclin D alone is unlikely to be sufficient for committing cells to division.
Thyroid hormone nuclear receptors (TRs) belong to the superfamily of ligand-inducible transcription factors (7). Two TR genes located on two different chromosomes encode four isoforms that bind triiodothyronine (T 3 ) and these isoforms are designated as ␣1, ␤1, ␤2, and ␤3. TRs bind to specific DNA sequences (thyroid hormone response elements) on promoters to regulate target gene transcription, and TR-mediated transcription is regulated at multiple levels. In addition to the role of T 3 and the influence of the thyroid hormone response elements, TR transcription is modulated by tissue-and development-dependent TR isoform expression and by numerous co-repressors and co-activators. Thyroid hormone influences various physiological processes, including cell cycle progression and cell differentiation/development in the vertebrate nervous system. We previously reported that ligand binding to TR regulates the phosphatidylinositol 3-kinase/AKT pathway upstream of cyclin D1 (8,9). It was also reported that ligand-binding TR regulates the transcription of cyclin D1 (10). Moreover, it was recently shown that TR interacts with cell cycle regulators such as cyclin D1 and p53 (11).
Pancreatic ␤-cells contain a range of nuclear receptors, many of which are implicated in the regulation of insulin secretion (12). Normally, there is a hyperbolic relationship between whole body insulin sensitivity and insulin secretion so that any change in insulin sensitivity elicits a reciprocal and proportional change in insulin secretion. However, the mechanisms by which ligand binding to TRs induce the proliferation of pancreatic ␤-cells are not clear, although it was recently reported that activation of the phosphatidylinositol 3-kinase pathway by liganded TR mediates pancreatic ␤-cell proliferation (13).
Insufficient understanding of the signals that regulate the growth and survival of adult ␤-cells remains one of the main challenges in diabetes research. However, new advances in identification of the factors involved in cell cycle progression by ␤-cells suggest that the molecular basis of this process may ultimately be revealed. In the present study, we investigated the influence of TR␣ on cell cycle progression by pancreatic ␤-cells. We explored the role of liganded TR␣ in ␤-cell replication and in expansion of the ␤-cell mass during postnatal development. The effects of TR␣ gene transfer into rat pancreatic cell lines or into the in vivo mouse pancreas were investigated using adenoviral vectors. Our findings led to the hypothesis that TR␣ might be involved in a positive regulatory mechanism that controls the maintenance of the pancreatic ␤-cell mass.

EXPERIMENTAL PROCEDURES
Construction of Recombinant Adenoviral Vectors-AdTR␣ is a recombinant adenoviral vector that expresses human TR␣1 under the control of the cytomegalovirus immediate early promoter. The FLAG-TR␣1 plasmid (14) was used as the template for cloning human TR␣1 into pShuttle2 (Clontech, Mountain View, CA) using the polymerase chain reaction. The PCR primers were: FLAG-ApaI-5Ј (AAGGGCCCGCCGCCATGGAC-TACAAAGACGATGACGAC), and TR␣-3Ј KpnI (TTAGG-TACCTTAGACTTCCTGATCCTCAAAG) in which the underlines indicate restriction sites. All of the plasmid constructs were confirmed by DNA sequencing. An adenovirus vector was then constructed using the Adeno X Expressing System (Clontech) according to the manufacturer's protocol. AdLacZ, which contains the lacZ gene controlled by the cytomegalovirus promoter, was provided by Quantum Biotechnologies (Montréal, Canada) and was used as a control. A doublestranded short hairpin RNA, encoding sense and antisense siRNA sequences against mouse TR␣ (5Ј-CGCTCTTCCTG-GAGGTCTT-3Ј) (15), separated by a loop sequence (TTCAA-GAGA), was cloned into the pENTR/U6 vector. A recombinant adenovirus expressing this short hairpin RNA (AdshTR␣) was generated using the pAd/BLOCK-iT-DEST vector kit (Invitrogen) according to the manufacturer's protocol. Recombinant adenoviruses were purified using a plaque-forming assay, were harvested 48 h after infection of 293 cells, and were further purified using double cesium chloride gradient ultracentrifuga-tion (16). Viral titers were determined using a plaque-forming assay and cultured 293 cells, as described previously (17).
The number of cells treated with T 3 was measured using a nonradioactive cell proliferation assay (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). One day after plating, the cells were incubated with 10, 30, or 100 nM T 3 in resin-stripped (T 3depleted) FBS (18). Cell viability was determined after 48 h of incubation by counting the cells according to the manufacturer's protocol.
Cells were synchronized in the G 0 /G 1 phase by culture for 48 h in RPMI 1640 medium supplemented with 0.5% (v/v) FBS (19). The medium was then replaced with 10% (v/v) FBS-containing medium and the cells were then incubated with 30 multiplicities of infection (m.o.i.) of adenovirus for 12 h and 50 nM siRNA were transfected. After 24 h, cells were subsequently arrested in G 0 /G 1 . Arrest of the cells was confirmed by flow cytometry. For analysis, the cells were stained with 20 g/ml of propidium iodide (Invitrogen) and analyzed with a BD Biosciences FACS Calibur (Franklin Lakes, NJ) using Cell Quest software version 3.0. The percentage of cells in the G 1 , S, or G 2 /M phase was calculated using ModFitLT version 3.1.
In the apoptosis studies, 30 m.o.i. of adenovirus-infected cells were incubated with or without T 3 for 12 h, followed by transfection of 50 nM siRNA. After 24 h, the cells were plated on glass coverslips (Microscope coverglass, catalog number 12-545-84 18CIR-1D; Fisherbrand) at a density of 1 ϫ 10 5 cells per coverslip. After 24 h, the cells were exposed to streptozotocin (STZ) (Sigma) (15 mM) for 2 h in the presence or absence of T 3 . Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using the DeadEnd Fluorometric TUNEL system (Promega, Madison, WI) according to the manufacturer's instructions.
Cell proliferation was also analyzed by a 5-bromo-2Ј-deoxyuridine (BrdUrd) uptake assay using a BrdUrd Labeling and Detection Kit (catalog number 11296736001; Roche Applied Science). Adenovirus-infected cells were incubated with or without T 3 for 12 h followed by transfection of 50 nM siRNA. The cells were then passaged on glass coverslips at 1 ϫ 10 5 cells per coverslip. After 24 h, the cells were exposed to 10 M BrdUrd labeling medium for 30 min. The cells were then washed twice with phosphate-buffered saline, fixed with 70% (v/v) ethanol, and incubated with anti-BrdUrd antibody (0.5 g/ml) overnight at 4°C. Cells that had incorporated BrdUrd were detected using fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1 g/ml). Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA) and laser scanning images were captured using an Olympus FV-1000 microscope (Olympus Corp., Tokyo, Japan).
Quantitative Real Time Reverse Transcriptase (RT)-PCR-RNA was extracted from tissues or cells using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. After quantification by spectrophotometry, 5 g of total RNA was reverse transcribed to obtain cDNA using 160 M deoxynucleotide triphosphate, 50 ng of random hexamer primers, and 200 units of SuperScript II according to the manufacturer's recommendations (Invitrogen). TaqMan probes for rat TR␣ (catalog number Rn00579692), rat TR␤ (catalog number Rn00562044), rat cyclin D1 (catalog number Rn00432359), and 18 S rRNA (catalog number Hs99999901) were purchased from Applied Biosystems (Foster City, CA). Determination of mRNA levels by real time RT-PCR was carried out as described previously by Furuya et al. (20).
Animal Experiments and Vector Injection-Immunodeficient, 4-week-old nude mice (BALB/cAJc1-nu/nu) were purchased from Clea Japan Inc. (Tokyo, Japan). Diabetes was induced using STZ (Sigma), which was injected intraperitoneally at a single dose of 200 mg/kg (21). Depletion of ␤-cells was confirmed by immunohistochemical staining of the pancreas, as well as by detection of severe hyperglycemia. At 7 days after STZ administration, mice that showed hyperglycemia were randomly divided into experimental groups that received 6 ϫ 10 10 plaque-forming units/mouse of AdTR␣, AdshTR␣, or control, AdLacZ groups I, II, and III, respectively. Mice were anesthetized by intraperitoneal injection of pentobarbitone (40 mg/kg). The pancreas was identified and mobilized at laparotomy. The recombinant adenoviral suspension (a total volume of 100 l) was directly injected into 2-3 sites in the distal pancreas using a 27-gauge needle (21). The efficiency of viral infec-tion was confirmed by immunostaining to determine maximal viral expression in pancreatic tissue. The abdomen was closed using 2 layers of 4-0 vicryl (Ethicon, Somerville, NJ). T 3 (5 g/20 g body weight) was injected intraperitoneally for 3 days (group I). Adenovirus administration was repeated in the mice in groups II or III, on day 21 or 35, respectively, following T 3 treatment.
Cell proliferation in AdTR␣-infected pancreas tissue was also analyzed using the BrdUrd Labeling and Detection Kit (Roche Applied Science). STZ-treated mice that showed hyperglycemia were injected with 6 ϫ 10 10 plaque-forming units/ mouse of viral vector. Adenovirus-injected mice were then treated with T 3 (5 g/20 g body weight) for 3 days. Adenovirus administration was repeated on days 21 and 28 following T 3 treatment. On day 35, mice were intravenously injected with a BrdUrd labeling reagent and BrdUrd incorporation was analyzed according to the manufacturer's protocol.
For histological studies of islet apoptosis, TUNEL staining was performed using the DeadEnd Fluorometric TUNEL system (Promega) according to the manufacturer's instruction. Adenovirus (6 ϫ 10 10 plaque-forming units) was injected 2 days before (200 mg/kg), and 3 days after STZ administration. The mice were sacrificed 5 days after STZ treatment. This pretreatment regimen was selected because it has been reported that pretreatment of mice with a glucagon-like peptide significantly reduces apoptosis in STZ-treated mice (22). The institutional biosafety committee of the University of Yamanashi approved the animal procedures and the use of recombinant DNA.
Measurement of Glucose and Insulin, and Glucose Tolerance Test-Serial blood glucose levels were monitored using a One-Touch Blood glucose meter (Johnson and Johnson, Milpitas, CA) after appropriate calibration. Blood was collected from the tail vein on day 0 (before injection of streptozotocin), on day 7 (before injection of the recombinant adenoviral vector), and during the intraperitoneal glucose tolerance test that was done according to the protocol of the animal models of diabetic complications consortium (niddk.nih.gov). On day 13 (day 6 after vector administration), mice were fasted overnight with free access to water. Glucose was injected intraperitoneally at a dose of 500 mg/kg of body weight in a volume of 1 ml. Blood glucose was measured before and 5, 15, 30, 60, and 120 min after glucose injection using an OneTouch Blood glucose meter (Johnson and Johnson, New Brunswick, NJ). Plasma insulin concentrations were measured by insulin enzyme-linked immunosorbent assay kit (Morinaga, Yokohama, Japan) using mouse insulin as a standard. Mice were exsanguinated by bleeding from the retro-orbital plexus at 0, 5, 15, 30, 60, and 120 min after glucose injection for the insulin assay.
Immunostaining-Organs were removed from mice, fixed in 10% buffered formalin, and subsequently embedded in paraffin. Then 3-m sections were prepared and stained with hematoxylin and eosin, whereas other sections were processed for immunohistochemistry. Sections were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. Nonspecific binding of the antibodies was blocked with 3% bovine serum albumin before incubation overnight at 4°C with primary antibody. Paraffin sections were stained for ␤-cells with guinea pig anti-insulin antibody (Dako, Glostrup, Denmark), for ␣-cells with anti-glucagon antibody (Dako), or for non-␤-cells with a mixture of antisera (anti-glucagon, anti-somatostatin, and anti-pancreatic polypeptide). The sections were subsequently incubated with 1.0 g/ml of rhodamine-conjugated goat anti-guinea pig IgG (Jackson ImmunoResearch, West Grove, PA) or fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Nuclei were stained with DAPI (Vector Laboratories) and laser scanning images were captured using an Olympus FV-1000 microscope (Olympus Corp.).
The relative ␤-cell area was determined on insulin-stained sections and estimated by the following formula: ␤-cell mass (g) ϭ the area of islets/the whole pancreatic area ϫ pancreas weight (10 sections in each group). The area of insulin-stained pancreatic islets was measured using image analysis software (Image J). Using Image J, these ␤-cell areas were divided by the number of ␤-cell nuclei in the total area to calculate the mean area of the individual ␤-cells.
Statistics-Data are expressed as the mean Ϯ S.D. Statistical analysis was performed using one-way analysis of variance or the unpaired 2-tailed Student's t test and probability values of less than 0.05 were considered to be significant.

T 3 Enhances Pancreatic ␤-Cell
Proliferation-To investigate the effect of T 3 on pancreatic ␤-cell proliferation, we used a rat pancreatic ␤-cell line (RIN5F) that expresses endogenous nuclear TRs, TR␣ and TR␤, that mediate the actions of T 3 (23). TRs function as ligand-dependent transcription factors to increase or decrease the expression of various target genes (24,25). Recent reports indicate that liganded TRs have a critical role in the development of the central nervous system and skeletal muscles (26,27). Our preliminary experiments indicated that T 3 treatment significantly increased the number of RIN5F cells (data not shown).
To further explore the role of TR␣ in the processes of ␤-cell proliferation, we determined the effect of overexpression of TR␣ using a recombinant adenovirus vector that expresses TR␣ (AdTR␣). RIN5F cells were incubated in T 3 -depleted medium for 24 h and then infected with 30 m.o.i. of AdTR␣ or control AdLacZ. After 12 h of incubation in adenovirus-containing medium, the cells were cultured with or without T 3 for a further 24 h. T 3 treatment enhanced the cell number in AdLacZ-infected cells that express endogenous TR␣ (Fig. 1). The cell number of AdTR␣-infected cells was significantly increased by treatment with 10, 30, or 100 nM T 3 , compared with the cell number of AdLacZ cells treated with 10, 30, or 100 nM T 3 , respectively (Fig. 1). These results supported the concept that T 3 has a critical role in the growth or proliferation of ␤-cells and that infection with AdTR␣ enhances T 3 -induced cell proliferation.
Liganded TR␣ Specifically Induces Cell Cycle Progression of Pancreatic ␤-Cells via Induction of Cyclin D1 Expression-To explore the molecular mechanisms through which cell proliferation was induced by T 3 treatment, we analyzed the effect of T 3 on the expression of cyclin D1 mRNA, a cell cycle regulator. RIN5F cells were incubated in T 3 -depleted medium for 24 h and then infected with 30 m.o.i. of AdTR␣ or AdLacZ. After 12 h of incubation in adenovirus-containing medium, the cells were cultured with or without T 3 (100 nM) for a further 24 h. Cyclin D1 mRNA expression was analyzed by quantitative RT-PCR and normalized to 18 S rRNA. As expected, treatment with T 3 slightly enhanced the expression of cyclin D1 mRNA in AdLacZ-infected RIN5F cells (1.5-fold) because these cells constitutively express TR␣ (Fig. 2). However, in AdTR␣-infected RIN5F cells, T 3 treatment markedly enhanced cyclin D1 expression (5.6-fold).
Enhanced expression of the cyclin D1 protein is known to be accompanied by concurrent activation of the CDK/Rb/E2F pathway and enhanced proliferation of pancreatic ␤-cells (6). To explore if the cyclin D1/CDK/Rb/E2F pathway was activated in AdTR␣-infected ␤-cells, we analyzed the expression levels of CDK4, CDK6, Rb, and p21 proteins by Western blot analysis. The expression levels of cyclin D1 and Rb protein are enhanced in AdLacZ-infected RIN5F cells with T 3 treatment, compared with no T 3 treatment (Fig. 3A). T 3 treatment of AdTR␣-infected cells enhances cyclin D1 protein levels. p21 is a CDK inhibitor that binds to and inhibits the activity of cyclin-CDK complexes. The expression levels of CDK4, CDK6, or p21 proteins are not affected by the presence of liganded TR␣. The abundance of the cyclin D2 protein is not affected by AdTR␣ infection (data not shown). Furthermore, hyperphosphorylated Rb protein is clearly detected in TR␣-transfected RIN5F cells following T 3 treatment (Fig. 3A). These results indicated that the cyclin D1/CDK/Rb/E2F pathway is activated in the pancreatic ␤-cell line in response to overexpression of liganded TR␣. Endogenous TR is insufficient to induce obvious activation of the cyclin D1/CDK/Rb/E2F pathway.
We next used a siRNA approach to ascertain the effect of knocking down cyclin D1 expression on the TR␣-induced cyclin D1/CDK/Rb/E2F pathway. Knockdown of the cyclin D1 protein using specific siRNA was observed in AdTR␣-infected cells with or without T 3 treatment, as determined by Western blot analysis (Fig. 3A, lanes 5 and 6). Consistent with the reduced cyclin D1 protein, phosphorylation of its downstream effector, Rb, was decreased in AdTR␣-infected RIN5F cells. The combined results suggested that enhancement of cyclin D1 by TR␣, followed by activation of the Rb/E2F pathway, induces the proliferation of pancreatic ␤-cells.
DNA synthesis in AdTR␣-infected cells was analyzed using a BrdUrd labeling. RIN5F cells were incubated with resinstripped medium for 24 h and infected with 30 m.o.i. of adenovirus, followed by incubation with or without T 3 (100 nM) for an additional 24 h. The ratio of BrdUrd incorporating nuclei to DAPI-stained nuclei is shown in Fig. 3B. When RIN5F cells were infected with AdTR␣, BrdUrd accumulation was significantly increased (5.2-fold) in T 3 -treated cells compared with cells cultured in T 3 -depleted medium (p Ͻ 0.01) (Fig. 3B). This TR␣-induced BrdUrd accumulation was not observed in cells in which cyclin D1 protein expression was inhibited by transfection with siRNA. These findings indicated that overexpression of liganded TR␣ induces cell cycle progression, DNA synthesis, and proliferation in pancreatic ␤-cells.
Liganded TR␣ Specifically Enhances Cell Proliferation of Pancreatic ␤-Cells-To confirm that the effect of TR␣ on cyclin D1 levels was reflected in an effect on cell cycle progression, we compared the effect of TR␣ overexpression with that of TR␣ knockdown on cell cycle progression. Western blot analysis of 30 m.o.i.-infected AdLacZ or AdTR␣ RINF cells, or TR␣-specific siRNA-transfected cells are indicated as low, high, and inhibited expression of TR␣ protein, respectively (Fig. 4A). FACS analysis of cell cycle stages was then performed on these synchronized ␤-cells (Fig. 4B). Most of the AdLacZ-infected ␤-cells had proceeded to the S/G 2 phase within 12 to 24 h after release from serum starvation. There were no significant differences between the presence and absence of T 3 treatment. In AdTR␣-transfected cells, T 3 treatment accelerated an initial increment that progressed to the S/G 2 M phase compared with that in cells without T 3 treatment (Fig. 4B). We employed the mixture of 3 vectors expressing different siRNAs against TR␣ to address whether endogenous TR␣ had a causal effect on cell cycle progression. Most of the siTR␣-transfected cells had proceeded to the S/G 2 /M phase at 12 h after release from serum starvation. These results are consistent with previous results that the cyclin D1/CDK/Rb/E2F pathway is not activated in control adenovirus-infected cells (Fig. 3).
To assess whether liganded TR␣ could affect survival of ␤-cells exposed to a proapoptotic agent, apoptosis was induced using 15 mM STZ. As shown in Fig. 4C, the TUNEL assay, an indicator of apoptosis, was positive in 23% of control cells that were incubated with T3-depleted medium (lane 1) and STZ treatment enhanced apoptosis (3.3-fold) in these cells (lane 2). T 3 treatment caused a 31% decrease in STZ-associated apoptosis (lanes 2 and 4). In AdTR␣-infected cells, the TUNEL assay was positive in 11 or 6% of cells that were incubated in the absence or presence of T 3 , respectively (lanes 5 and 7). T 3 treat- ment also reduced STZ-induced apoptosis (lanes 6 and 8). Lanes 9 and 10 show that, when endogenous TR␣ is knocked down, the T 3 -induced reduction in STZ-induced apoptosis in ␤-cells is not observed. These results indicated that liganded TR␣ could be considered a survival factor that protects ␤-cells from apoptosis.
Adenoviral Vector-mediated TR␣ Gene Transfer into the Mouse Pancreas Corrects Streptozotocin-induced Hyperglycemia and Insulin Secretion-To analyze the effect of AdTR␣ on the replication of mature pancreatic ␤-cells in vivo, immunodeficient mice were made diabetic by injection of STZ. Hyperglycemic mice were injected with adenovirus (AdTR␣ or AdLacZ) into the distal pancreas on days 7, 14, or 21 after STZ injection, and then injected with T 3 intraperitoneally every day for 3 days (5 g/day). To further explore the role of endogenous TR␣, the expression of TR␣ was inhibited by infection with an adenovirus expressing double-stranded short hairpin RNA against mouse TR␣ (AdshTR␣). Mice treated with control adenovirus demonstrated hyperglycemia. In contrast, mice infected with AdTR␣ exhibited significantly lower blood glucose levels at 14, 21, and 28 days after STZ injection (Fig. 5A). AdshTR␣-infected mice developed progressive hyperglycemia, with levels of blood glucose rising steadily several days after STZ administration. Levels of plasma insulin after day 7 were significantly higher in AdTR␣-treated mice compared with those in control mice (Fig. 5B). Plasma insulin levels at day 28 in endogenous TR␣-expressing mice were significantly higher than those in AdshTR␣-infected mice. Body weight values were similar in each group. The improved glycemia values following AdTR␣ treatment were not due to decreased body weight secondary to an effect of TR␣ on appetite (Fig. 5C). To quantify the increase in ␤-cells, we analyzed the ␤-cell mass by immunostaining for insulin. AdTR␣ infection clearly enhanced ␤-cell mass on days 14, 21, and 28 (Fig. 5D). Following AdLacZ treatment, no obvious islet cell masses were observed 14 days after STZ administration and only small insulin-stained areas or scattered single or small clusters of insulin-positive cells were observed on days 21 and 28 (data not shown). An obvious ␤-cell mass was not observed in AdshTR␣-infected mice. These data suggested that exogenous TR␣ in ␤-cells is coupled to the induction of ␤-cell proliferation and enhances the values of plasma insulin levels resulting in a reduction in blood glucose levels.
We compared the glucose tolerance of AdTR␣and AdLacZinfected mice on day 14 (Fig. 6A). After an overnight fast, there were no differences in blood glucose levels between these two  5 and 6). The loading controls using ␥2-tubulin are shown in the lower panel. Western blot analysis was carried out using 20 g of nuclear protein. B, the cells were synchronized in the G 0 /G 1 phase by serum starvation. Synchronized cells were collected at time 0 and then released by culture in growth medium containing 10% fetal bovine serum with or without T 3 . Cells harvested at various times after release were subjected to FACS analysis to define the phase of the cell cycle. *, p Ͻ 0.05 compared with T 3 depletion. C, induction of apoptosis in RIN5F cells by STZ treatment. After transfection with adenovirus and siRNA, the cells were incubated with 15 mM STZ for 2 h. Apoptosis was evaluated using the TUNEL method. The ratio of TUNEL-positive to DAPI-stained cells is shown. Data are expressed as the mean Ϯ S.D. *, p Ͻ 0.05.

FIGURE 5. Effects of TR␣ on ␤-cell mass replication in STZ-treated mice.
Morning fed blood glucose levels, the levels of fed plasma insulin, and body weight over 28 days following STZ treatment of mice are shown in A-C, respectively. D, ␤-cell mass was calculated using the following formula: islet ␤-cell mass (mg) ϭ the area stained by insulin antibody/the area of the whole pancreas ϫ pancreas weight. All data are mean Ϯ S.D. (n ϭ 6 -10). Bars represent the mean Ϯ S.D. *, p Ͻ 0.05. compared with control.
groups of mice. Similar findings were observed in young mice treated with STZ, as reported recently (28). At 15 min after glucose injection, the blood glucose level was maximal (200 mg/dl) in mice without STZ treatment, at which point the response to intraperitoneal injection of glucose solution at 500 mg/kg body weight was assessed. AdLacZ-treated control mice had significantly higher glucose levels after 15 min compared with AdTR␣-injected mice (p Ͻ 0.05) (Fig. 6A). They showed peak glucose levels (407 mg/dl) after 30 min and a high glucose level (Ͼ300 mg/dl) still persisted after 60 min. In contrast, AdTR␣-injected mice had the highest glucose value after 5 min and showed normal blood glucose levels from 15 min onward. Basal insulin levels in the AdTR␣-treated mice were higher than those in control mice (2.3-fold). AdTR␣-infected mice demonstrated rapid secretion of plasma insulin levels by 5 min, which peaked at 30 min and subsequently dropped. Control AdLacZinfected mice did not exhibit a robust rise in levels of plasma insulin at 5 min or an increase in plasma insulin levels during the glucose tolerance test despite glycemic excursion. To evaluate the early phase of insulin secretion, we calculated the insulinogenic index between 0 and 30 min (⌬ insulin (IRI) 0 -30min /⌬ blood glucose (BG) 0 -30min ), which represents early phase insulin response. The values of ⌬IRI 0 -30min /⌬BG 0 -30min were significantly enhanced in AdTR␣-treated mice compared with that in AdLacZtreated mice (1.29 Ϯ 0.26 versus 0.35 Ϯ 0.06, p Ͻ 0.05). Insulin area under the blood concentration time curve was also enhanced in AdTR␣-treated mice compared with controls (1.85 Ϯ 0.46 versus 0.47 Ϯ 0.17, p Ͻ 0.05). These data suggested that AdTR␣ treatment enhances insulin secretion in the pancreas of STZ-induced diabetic mice and improves glucose levels.
Histological Assessment of Islet Proliferation in TR␣-treated Mice-We assessed the histological consequences of AdTR␣induced replication of ␤-cells. Islet architecture in STZ-treated mice was severely disrupted 7 days after treatment, with non-␤-cells located at the core of shriveled islets (Fig. 7A, a-c), whereas normally the islet core is occupied by ␤-cells. These observations are consistent with previous reports that STZ results in diabetes associated with ␤-cell destruction (29). The ␤-cell mass was clearly increased in AdTR␣-treated pancreas compared with AdLacZ-treated control (Fig. 7A, d and g). There were no significant differences in the glucagon-stained area between AdTR␣ and AdLacZ treatments (Fig. 7A, e and h). As assessed by point counting morphometrics on immunostained paraffin sections, the size of islet was increased 7.6-fold in the AdTR␣treated pancreas compared with AdLacZ-treated control (Fig. 7B).
Because increases in ␤-cell mass can result from either larger cells (hypertrophy) or more cells (replication), we measured the area of the individual ␤-cells. The mean size of individual ␤-cells of AdTR␣-treated mice did not differ from those of the control (Fig. 7C). Because the increase in ␤-cell mass after AdTR␣ treatment was not due to increased cell size, the increased ␤-cell mass must result from replication.
To further assess the process of AdTR␣-induced replication of pancreatic ␤-cells, we measured the uptake of BrdUrd into mouse islets. Islets treated with AdTR␣ showed a striking increase in BrdUrd incorporation (Fig. 8Ac) such that nearly all sections from islets overexpressing TR␣ had multiple BrdUrdpositive cells. In contrast, sections from AdLacZ-treated control islets had few BrdUrd-positive cells (Fig. 8A, a). The enhanced number of BrdUrd-positive cells in AdTR␣-treated islets correlated well with the results of the glucose tolerance test shown in Fig. 7 and clearly demonstrate a robust increase in the number of islet cells undergoing cell division in response to TR␣ overexpression (Fig. 8A, d).
Co-staining studies were carried out to further investigate the relationship between BrdUrd accumulation and TR expression. Treatment of islets with AdTR␣ caused the appearance of an obvious nuclear TR␣ (FLAG) signal determined by co-staining with an anti-FLAG antibody and DAPI. The infection efficiency was 54.8 Ϯ 3.1%. Importantly, overlay of BrdUrd and TR␣ nuclear staining (Fig. 8B) revealed that all BrdUrd-positive cells were also TR␣ positive. Conversely, a small proportion of TR␣-positive cells was BrdUrd negative (Fig. 8B). A likely explanation for this observation is that BrdUrd was only added during the last 18 h of the 98-h period following exposure of the islets to AdTR␣. Therefore, some TR␣-overexpressing cells may have completed the S phase prior to the addition of BrdUrd.
These results supported the hypothesis that AdTR␣ could lead to the formation of new ␤-cells through enhanced proliferation of pre-existing ␤-cells in the STZ-treated pancreas. Because STZ is known to induce ␤-cell destruction in part through activation of apoptotic pathways, we analyzed the possibility that TR␣ might also enhance ␤-cell mass via protection from cellular apoptosis. To test this hypothesis, mice were treated with STZ in the presence or absence of AdTR␣ that was administrated for 2 days before, and 3 days after, STZ treatment. Apoptosis of ␤-cells in the pancreas was studied using the TUNEL method. Only a rare apoptotic ␤-cell was detectable in histological sections from the pancreas of AdLacZ-or AdTR␣-treated mice in the absence of STZ. The number of TUNEL-positive apoptotic ␤-cells was markedly increased in STZ-treated mice, and was significantly reduced in mice administrated AdTR␣ (Fig. 9). The combined data indicate that treatment of islets with AdTR␣ preferentially stimulates ␤-cell proliferation and also facilitates reduced apoptosis in STZ-treated mice.

DISCUSSION
Pancreatic islet ␤-cell mass is controlled by a dynamic balance between cell proliferation and cell death. Diabetes occurs when this balance is disrupted by autoimmune-mediated ␤-cell destruction or by failure of the ␤-cell mass to compensate for metabolic demand. Gaining a better understanding of molecular mechanisms that regulate ␤-cell replication and survival is therefore of great relevance for development of new diabetic therapies. In this report, we demonstrated that: 1) thyroid hormone treatment enhances the numbers of pancreatic ␤-cells; 2) adenovirus-mediated transfer of the TR␣ gene enhances cell cycle progression and proliferation of a pancreatic ␤-cell line; 3) TR␣ gene transfer also enhances cyclin D1 expression and acti- FIGURE 7. Islet architecture in STZ-treated mice following AdTR␣ or AdLacZ infection. A, islet architecture 7 days after STZ treatment of noninfected control mice (a-c). Fourteen days following STZ treatment, AdTR␣ (d-f) or AdLacZ (g-i) mice were euthanized and the morphology of pancreatic islets was analyzed by immunohistochemistry. Insulin is stained red (a, d, and g) and glucagon is stained green (b, e, and h). Panels c, f, and i show the overlay of DAPI, insulin, and glucagon signals. B, the mean sizes Ϯ S.D. of 30 islets in AdLacZ-and AdTR␣-infected mice are shown. Relative quantification of islet size of circumference was determined by arbitrarily setting the control value from AdLacZ treatment to 1. All data are expressed as the mean Ϯ S.D. *, p Ͻ 0.05. C, the mean diameters Ϯ S.D. of ␤-cells or nuclei in 30 islets from AdLacZand AdTR␣-infected mice are shown. There are no differences between AdLacZ-and AdTR␣-infected mice. vates the cyclin D1/CDK/Rb pathway; 4) in vivo TR␣ gene transfer induces restoration of insulin secretion and improvement of glucose tolerance associated with an increase of the ␤-cell mass in diabetic mice; and 5) TR␣ gene transfer confers strong protection against STZ-mediated ␤-cell apoptosis. These data provide new insights into the molecular mechanisms regulating pancreatic ␤-cell proliferation induced by liganded TR␣.
Thyroid hormones reduce glucose tolerance in humans and other animals (30). Experimental hyperthyroidism induced by thyroid hormone treatment leads to a reduction in glucoseinduced insulin secretion from the isolated pancreas (31), and this is not due to impairment of the insulin-secreting capacity of individual ␤-cells (32). It was also reported that stimulated insulin secretion is significantly increased in patients with hyperthyroidism, possibly reflecting increased ␤-cell sensitivity to glucose (33). Additionally, recent reports indicate that the thyroid hormone receptor mediates up-regulation of protein synthesis and cell size, along with cell proliferation and survival (34). In the present study, we also demonstrated that thyroid hormone treatment induced the proliferation of rat pancreatic ␤-cell lines. These results support the concept that thyroid hormone has a role in the proliferation of pancreatic ␤-cells.
Little is known about the molecular mechanisms involved in thyroid hormone-induced proliferation of pancreatic ␤-cells. A gene silencing study revealed nongenomic activation of the phosphatidylinositol 3-kinase/AKT pathway by endogenous TR␤ in human pancreatic insulinoma cells (34). Our data suggested that liganded TR␣ also has an important role in the proliferation of pancreatic ␤-cells. Our in vitro studies also show that transfected TR␣ activates the cyclin D1/CDK/Rb pathway and cell cycle progression in a ligand-dependent manner (Fig.  3). Endogenous TR is insufficient for induction of expression of the cyclin D1 protein that activates the cyclin D1/CDK/Rb pathway. Previous reports have indicated that thyroid hormone enhances pancreatic acinar cell proliferation (35) and ␤-cell proliferation (36) in vitro. These results were compatible with our in vivo findings that overexpression of TR␣1 enhances ␤-cell proliferation in STZ-treated mice. There were no demonstrable abnormalities or changes of pancreatic exocrine cells with or without AdTR␣ treatment. TR␣ expression was not only observed in the islets, but also in other areas of the pancreas by staining with an anti-FLAG antibody (Fig. 8) and its expression was still detected up to 15 days after adenovirus injection (data not shown). In contrast, activation of pancreatic ␤-cells was not observed in the control adenovirus-infected pancreas (Fig. 8).
Previous findings have also suggested that modulation of the cyclin D-cdk4 complex has a major influence on cell cycle progression, proliferation, and survival of pancreatic ␤-cells (6). After birth, cyclin D1 or cyclin D2-cdk4 are critical regulators of ␤-cell proliferation and viability (37,38). Indeed, adenovirusmediated transfer of cyclin D1 was reported to induce ␤-cell proliferation and replication (39). These results indicate that cyclin D1, which is the major regulator of the gap-1/synthesis phase (G 1 /S) cell cycle checkpoint, has an important role in the cell cycle control of ␤-cells. Thyroid hormone may induce either growth stimulation or inhibition, depending on the tissue involved and the treatment regimen (40). Thyroid hormone has been reported to modulate the expression of several genes that have a key role in cell cycle regulation via stimulation of the cyclin family (11). In this study, we showed that the expression of cyclin D1 was stimulated by liganded TR␣, suggesting that T 3 transcriptional activity is one of the mechanisms by which cyclin D1 protein levels are increased. The overexpression of cyclin D1 protein was accompanied by hyperphosphorylated Rb. Phosphorylation of Rb is known to release the E2F transcription factor from Rb-E2F complexes to promote progression of the cell cycle from the G 1 to the S phase (Fig. 4).
Improved understanding of the signals regulating the growth and survival of adult ␤-cells remains one of the main challenges in diabetes research. Unfortunately, the molecular mechanisms regulating the pancreatic ␤-cell mass are poorly understood. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair (41). In contrast, Dor et al. (3) reported that pre-existing ␤-cells, rather than pluripotent stem cells, are the major source of new ␤-cells during adult life and after pancreatectomy in mice. Similarly, a study employing a novel DNA analog-based lineage-tracing technique that detects sequential cell division indicated that pancreatic ␤-cells are mainly produced by self-duplication (42). Our studies are the first to show that liganded TR␣ mediates ␤-cell proliferation via the regulation of cell cycle factors. We have further shown that these mechanisms actually exist and are operative in adult ␤-cells. The increased ␤-cell mass was accompanied by a corresponding increase in the number of BrdUrd-positive cells, suggesting that overexpression of TR␣ resulted in an increase in the capacity of ␤-cell precursors to proliferate or differentiate into insulin-expressing cells. The present study will explain these prior results by demonstrating that TR␣ can simultaneously enhance ␤-cell replication and function. Anti-apoptotic function of TR␣-mediated pathway was also suggested.
A better understanding of how the cell cycle progression of the ␤-cell is regulated could lead to new strategies for the therapy of diabetes. The TR␣ gene appears to have growth-promoting properties that also contribute to maintenance of the mature ␤-cell phenotype. These properties are an ideal combi- FIGURE 9. Islet apoptosis in AdTR␣-infected mice following vehicle or STZ administration. STZ was administered to AdLacZ and AdTR␣ pre-infected mice, after which mice were euthanized 5 days later for histological assessment of islet apoptosis using a TUNEL assay. The number of apoptotic cells normalized to the relative islet area is shown. Approximately 30 islets were assessed, in which a minimum of 10 slides were analyzed per mouse. All data are expressed as mean Ϯ S.D. *, p Ͻ 0.05.