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

Abstract

Failure of the functional pancreatic ␤-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 ␤-cells is important for ␤-cell proliferation in adult animals. In rat pancreatic ␤-cell lines (RIN5F), treatment with 100 nM thyroid hormone (triiodothyronine, T 3 ) enhances cell proliferation. This result suggests that T 3 is required for ␤-cell proliferation or replication. To identify the role of thyroid hormone receptor ␣ (TR␣) in the processes of ␤-cell growth and cell cycle regulation, we constructed a recombinant adenovirus vector, AdTR␣. Infection with AdTR␣ to RIN5F cells increased the expression of cyclin D1 mRNA and protein. Overexpression of the cyclin D1 protein in AdTR␣-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␣-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␣ led to the restoration of islet function and to an increase in the ␤-cell mass. These results support the hypothesis that liganded TR␣ plays a critical role in ␤-cell replication and in expansion of the ␤-cell mass during postnatal development. Thus, liganded TR␣ may be a target for therapeutic strategies that can induce the expansion and regeneration of ␤-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 homeostasis 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 cel...

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

“…We have reported that intrapancreatic injection of adenovirus vector that expresses TR␣ leads to the restoration of islet function and an increase in the ␤-cell mass in immunodeficient mice with streptozotocin (STZ)-induced diabetes (15). These results suggest that ligand-bound TR␣ plays a critical role in ␤-cell replication and expansion of the ␤-cell mass during postnatal development.…”

“…A single dose of 200 mg/kg STZ was injected intraperitoneally (15,22). Depletion of ␤-cells was confirmed by immunohistochemical staining of the pancreas as well as by detection of severe hyperglycemia.…”

“…Real-time RT-PCR-RNA was extracted from tissues or cells by using an RNeasy mini kit (Qiagen) according to the manufacturer's instructions. cDNA synthesis and real-time RT-PCR were performed as described (15). TaqMan probes for glucokinase, amylase2, insulin1, insulin2, Hnf6, Foxa2, Neurod1, Nkx2.2, Ngn3, Mafa, Pdx1, and 18S were purchased from Applied Biosystems.…”

“…Determination of the protein abundance by Western blot analysis was performed as described (15) with the following primary antibodies: anti-Ngn3 antibody (BD Biosciences); anti-MafA, anti-Akt, anti-elastase, anti-GLUT2, anti-GCK, anti-receptor-type tyrosine-protein phosphatase-like N, and anti-carboxypeptidase E antibody (Santa Cruz Biotechnology); and anti-prohormone convertase 1/3, anti-p85␣, anti-Pdx1, anti-phosphorylated Akt (Ser-473), and anti-phosphorylated Akt (Thr-308) antibody (Cell Signaling Technology).…”

“…Pancreatic exocrine cell-specific activation of these promoters has been established (17,18) and confirmed by reporter assay in AR42J cells, a rat pancreatic exocrine cell line (data not shown). The FLAG-TR␣1 plasmid (15) was used as the template for cloning human TR␣1 into pENTR-1A Dual Selection (Invitrogen) by using PCR. The PCR primers were: kozak-SalI 5Ј (GGGGTCGACCACCATGGACTACAAAGACGATGACG-ACAAG) and SpeI 3Ј (GGGCATCTCAGGATGTTAGACTTC-CTGATCCTCAAAGAC).…”