Ligand-bound thyroid hormone receptor contributes to reprogramming of pancreatic acinar cells into insulin-producing cells

Abstract

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.

Keywords: Diabetes; Insulin; Pancreas; Phosphatidylinositol 3-Kinase; Thyroid Hormone.

FIGURE 1.

Ligand-dependent interaction of TRα with PI3K. a, cell lysates (500 μg) prepared from acinar cells or β-cells were immunoprecipitated (IP) with 5 μg of mouse monoclonal anti-TRα antibody, which recognizes the TRα C terminus (C3) or 5 μg of normal mouse IgG. Then, Western blot analysis of the precipitates was performed by using an antibody against an N-terminal TRα peptide. The same amount of protein extract prepared from the liver was used as a positive control. b, to analyze the specific expression of AdAmy2TRα, 10 μg of cell lysates prepared from AdAmy2TRα-infected acinar cells was immunoblotted by anti-FLAG antibody. The same amount of protein lysates from acinar cells infected with cytomegalovirus promoter-driven flag-tagged TRα-expressing adenovirus (15) was used as a positive control. c, AdAmy2TRα- or control virus-infected acinar cells were treated with or without 100 nm T3 for 24 h. Cell lysates (500 μg) were immunoprecipitated with anti-FLAG antibody or normal IgG, and then the immunoprecipitate or 10 μg of lysate was immunoblotted by anti-p85α antibody.

FIGURE 2.

Reprogramming of pancreatic acinar cells induced by AdAmy2TRα. mRNA levels for 11 genes associated with the differentiation of β-cells were assayed in AdAmy2TRα-infected acinar cells treated with 100 nm T3. From four independent samples, total RNA was isolated. The amounts of insulin1, insulin2, glucokinase, amylase2, Hnf6, Foxa2, Nkx2.2, NeuroD1, Pdx1, Ngn3, and Mafa mRNA were determined by quantitative real-time RT-PCR with 100 ng of cDNA in triplicate. Relative quantification of target cDNA was determined by arbitrarily setting the control value from untreated samples to 1. All data are expressed as the mean ± S.D. (error bars). *, p < 0.05 compared with untreated cells.

FIGURE 3.

Activation of PI3K pathway in AdAmy2TRα-infected exocrine cells. a, expression levels of phosphorylated Akt, total Akt, Pdx1, Ngn3, and MafA protein in AdAmy2TRα-infected cells exposed to 100 nm T3 for 24, 48 or 72 h with or without LY294002 treatment. b, effects of siNRA knockdown of p85α (or control siRNA) on AdAmy2TRα-infected cells incubated with 100 nm T3 for 24, 48, or 72 h and analyzed by Western blotting of cell extracts. Loading controls for γ-tubulin are shown in the bottom panel. Western blot analysis was performed with 20 μg of protein.

FIGURE 4.

Reprogramming of AdAmy2TRα-infected acinar cells. A, AdAmy2TRα-infected cells transfected with control siRNA (a–c) or p85α siRNA (d–f) and incubated with 100 nm T3 for 48 h were analyzed by immunostaining. The FLAG-tagged TRα-expressing cells were visualized by using anti-FLAG antibody and Alexa Fluor 555-conjugated secondary antibody (red). C-peptide-positive mature acinar cells were visualized with Alexa Fluor 488 (green). The 1 arrows identify the FLAG-TR TRα-expressing cells, which express insulin. The 2 arrows identify insulin-negative cells, which do not express FLAG-tagged TRα. B, the expression of prohormone convertase 1/3 (PC 1/3), carboxypeptidase E (CPE), or receptor-type tyrosine-protein phosphatase-like N (PTPRN) in AdAmy2TRα-infected cells transfected with control siRNA or p85α siRNA was analyzed by Western blotting. Loading controls for γ-tubulin are shown in the lower panel. Western blot analysis was performed with 20 μg of protein. C, electron microscopic analysis of AdAmy2TRα-infected cells transfected with control siRNA (a) or p85α siRNA (b) was performed. Scale bars, 500 nm. Insulin immunoreactivities were detected in the secretory granules in AdAmy2TRα-infected cells transfected with control siRNA (c). Scale bar, 500 nm. The arrows identify gold particles.

FIGURE 5.

A, cell lineage tracing by a lectin-associated system or Cre/loxP-based system. AdAmy2TRα-infected pancreatic acinar cells (m.o.i. 30) were stained for elastase (a marker of mature exocrine cells) and C-peptide and were analyzed by fluorescence microscopy. After 24, 48, or 72 h of incubation with 100 nm T3, Qdot 655-conjugated WGA-labeled cells (red) expressed elastase (a–c) (green) or C-peptide (d–f) (green). Pancreatic acinar cells from ROSA26-lacZ mice were labeled by infection with AdAmy2Cre. AdAmy2Cre and AdAmy2TRα-coinfected acinar cells, elastase (g–i) and C-peptide (j–l) expression was detected by using anti-elastase or anti-C-peptide antibody, respectively. Blue represents nuclear staining with DAPI. The arrows identify the insulin-producing cells, which are WGA- or β-gal-positive and express C-peptide. Scale bars, 20 μm. B, expression of FLAG-TRα and β-gal in AdAmy2TRα (30 m.o.i.)- and AdAmy2Cre (30 m.o.i.)-infected acinar cells of ROSA26-lacZ mice. After 48 h of incubation with 100 nm T3, the FLAG-tagged TRα-expressing cells were visualized by using anti-FLAG antibody and Alexa Fluor 488-conjugated secondary antibody (green). β-Gal-positive acinar cells were visualized with Alexa Fluor 555-conjugated secondary antibody (red). The arrows identify both FLAG-TRα and β-gal-positive acinar cells. C, expression of β-gal, FLAG-TRα, or elastase in AdAmy2TRα (30 m.o.i.)- and AdAmy2Cre (30 m.o.i.)-infected acinar cells of ROSA26-lacZ mice. The cells were incubated with 100 nm T3 for 24, 48, or 72 h and analyzed by Western blotting. Loading controls for γ-tubulin are shown in the bottom panel.

FIGURE 6.

Reprogramming of pancreatic acinar cells induced by AdAmy2TRα. Immunodeficient mice were injected with AdAmy2LacZ or AdAmy2TRα into the pancreatic duct. The marker of pancreatic exocrine cells, elastase, was visualized with anti-elastase antibody and Alexa Fluor 488-conjugated secondary antibody (green); transfected-TRα was visualized with an anti-FLAG antibody and Alexa Fluor 555-conjugated secondary antibody (red). Transcription factors that are essential for the differentiation of pancreatic endocrine cells were visualized with Alexa Fluor 488 (green). Insulin-producing cells were stained with anti-C-peptide antibody. Scale bars, 100 μm.

FIGURE 7.

Insulin secretory properties of pancreatic exocrine-derived cells. A and B, morning postprandial levels of blood glucose (A) and plasma insulin (B) in adenovirus-injected STZ-treated mice (n = 6). All data are mean ± S.D. (error bars). *, p < 0.05 compared with AdAmy2LacZ-treated mice. C, glucose tolerance test. Glucose tolerance improved in diabetic mice after injection with AdAmy2TRα (circles) compared with control virus (squares). n = 6 animals. *, p < 0.05. Data are means ±S .D. (error bars).