Liganded thyroid hormone receptor-alpha enhances proliferation of pancreatic beta-cells - PubMed (original) (raw)
Liganded thyroid hormone receptor-alpha enhances proliferation of pancreatic beta-cells
Fumihiko Furuya et al. J Biol Chem. 2010.
Abstract
Failure of the functional pancreatic beta-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 beta-cells is important for beta-cell proliferation in adult animals. In rat pancreatic beta-cell lines (RIN5F), treatment with 100 nM thyroid hormone (triiodothyronine, T(3)) enhances cell proliferation. This result suggests that T(3) is required for beta-cell proliferation or replication. To identify the role of thyroid hormone receptor alpha (TR(alpha)) in the processes of beta-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTR(alpha). Infection with AdTR(alpha) to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTR(alpha)-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 T(3). Conversely, lowering cellular cyclin D1 by small interfering RNA knockdown in AdTR(alpha)-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 AdTR(alpha) led to the restoration of islet function and to an increase in the beta-cell mass. These results support the hypothesis that liganded TR(alpha) plays a critical role in beta-cell replication and in expansion of the beta-cell mass during postnatal development. Thus, liganded TR(alpha) may be a target for therapeutic strategies that can induce the expansion and regeneration of beta-cells.
Figures
FIGURE 1.
Effect of T3 on pancreatic β-cell proliferation. RIN5F cells were incubated in T3-depleted medium for 24 h and then infected with 30 m.o.i. of AdTRα or control AdLacZ. The cells were incubated in medium with 10, 30, or 100 n
m
T3 for an additional 48 h. Relative cell numbers were determined by arbitrarily setting the value for control cultures incubated in T3-free medium to 1. Data are expressed as the mean ± S.D. (n = 6). *, p < 0.05. **, p < 0.01.
FIGURE 2.
Effect of T3 on cyclin D1 mRNA expression. AdLacZ- or AdTRα-infected RIN5F cells were incubated with 100 n
m
T3 for 24 h. Total RNA was then isolated from four independent samples. The level of cyclin D1 mRNA was determined in triplicate measurements using quantitative real time RT-PCR and 100 ng of cDNA. Relative quantification of the target cDNA was done by arbitrarily setting the control value (AdLacZ-infected cells cultured without T3) to 1. Data are expressed as the mean ± S.D. *, p < 0.05. **, p < 0.01.
FIGURE 3.
Activation of the cyclin D1/CDK/Rb pathway in AdTRα-infected RIN5F cells by T3 treatment. A, expression levels of the cyclin D1, Rb, CDK4, CDK6, and p21 proteins in AdLacZ- (lanes 1 and 2) or AdTRα (lanes 3–6)-infected RIN5F cells cotransfected with si-control (lanes 1–4) or si-cyclin D1 (lanes 5 and 6). The loading controls using γ2-tubulin are shown in the lower panel. Western blot analysis was carried out using 20 μg of nuclear protein. B, BrdUrd incorporation by AdLacZ- and AdTRα-infected cells. Cyclin D1 expression was knocked down in AdTRα-infected cells by si-cyclin D1. The ratio of cells with BrdUrd uptake to DAPI-stained cells is shown. Data are expressed as the mean ± S.D. (n = 6 to 10). *, p < 0.05; **, p < 0.01 compared with AdTRα-infected cells incubated with T3.
FIGURE 4.
Influence of liganded TRα on cell cycle progression and apoptosis. A, the levels of TRα protein expression in AdLacZ- (lanes 1, 2, 5, and 6) or AdTRα (lanes 3 and 4)-infected RIN5F cells cotransfected with si-control (lanes 1-4) or si-TRα (lanes 5 and 6). The loading controls using γ2-tubulin are shown in the lower panel. Western blot analysis was carried out using 20 μg of nuclear protein. B, the cells were synchronized in the G0/G1 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 T3. Cells harvested at various times after release were subjected to FACS analysis to define the phase of the cell cycle. *, p < 0.05 compared with T3 depletion. C, induction of apoptosis in RIN5F cells by STZ treatment. After transfection with adenovirus and siRNA, the cells were incubated with 15 m
m
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 ± S.D. *, p < 0.05.
FIGURE 5.
Effects of TRα on β-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, β-cell mass was calculated using the following formula: islet β-cell mass (mg) = the area stained by insulin antibody/the area of the whole pancreas × pancreas weight. All data are mean ± S.D. (n = 6–10). Bars represent the mean ± S.D. *, p < 0.05. compared with control.
FIGURE 6.
Intraperitoneal glucose tolerance test in AdTRα- or AdLacZ-infected mice with STZ-induced diabetes. Each group of mice (n = 6) was injected with glucose and blood glucose levels (A) and plasma insulin levels (B) were measured after 0, 5, 15, 30, 60, and 120 min. Bars represent the mean ± S.D. *, p < 0.05.
FIGURE 7.
Islet architecture in STZ-treated mice following AdTRα or AdLacZ infection. A, islet architecture 7 days after STZ treatment of non-infected control mice (a-c). Fourteen days following STZ treatment, AdTRα (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 ± S.D. of 30 islets in AdLacZ- and AdTRα-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 ± S.D. *, p < 0.05. C, the mean diameters ± S.D. of β-cells or nuclei in 30 islets from AdLacZ- and AdTRα-infected mice are shown. There are no differences between AdLacZ- and AdTRα-infected mice.
FIGURE 8.
TRα stimulates islet β-cell replication in STZ-treated mice. A, histochemical staining of islet sections of the pancreas in AdLacZ-infected (a and b) or AdTRα-infected (c and d) mice. Islet sections were stained with antibodies that detect BrdUrd incorporation (a and c) or BrdUrd (green nuclear staining) and insulin (red cytosolic staining) (b and d). B, immunofluorescence analysis of BrdUrd incorporation in AdTRα-treated islets. BrdUrd (a) and TRα (b) were stained with anti-FLAG (M2) (green staining) and anti-BrdUrd (red staining) antibodies and visualized with labeled secondary antibodies. Panel c shows nuclear staining with DAPI. Panel d shows the overlay of all three signals. The yellow arrows identify the BrdUrd-labeled nuclei, which in all cases co-stain with TRα and DAPI. TRα-positive and BrdUrd-negative nuclei are marked as circles.
FIGURE 9.
Islet apoptosis in AdTRα-infected mice following vehicle or STZ administration. STZ was administered to AdLacZ and AdTRα 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 ± S.D. *, p < 0.05.
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