Fig 1: Amino acids promotes the stability and nuclear localization of Pdx1 in a Rab1A- and mTORC1-dependent manner(A) Pdx1 is required for aas to stimulate insulin mRNA expression. MIN6 cells transfected with Pdx1 or control siRNA were starved for 24 h and stimulated with aas for 2 h. Ins2 mRNA expression was assayed by qRT-PCR. Results are presented as mean ± SEM of three independent experiments (n = 3). *p < 0.05, **p < 0.01, unpaired Student’s t test for two groups.(B) Amino acids are required for maintaining Pdx1 protein level and nuclear localization. MIN6 cells were cultured in normal or aa starvation medium for 24 h. Pdx1 protein (green) was analyzed by IF staining. Scale bars, 10 µm.(C) Amino acids promote Pdx1 protein level in an mTORC1-dependent manner. MIN6 cells were starved from aas for 24 h and re-stimulated for 2 h in the absence or presence of 100 nM rapamycin. Samples were analyzed by immunoblot.(D) Amino acids promote Pdx1 protein level and nuclear localization in an mTORC1-dependent manner. MIN6 cells were starved from aas for 24 h and were then re-stimulated with aas for different times in the absence or presence of 100 nM rapamycin. Pdx1 (green) was analyzed by IF staining. Scale bars, 10 µm.(E) Branched chain amino acids (BCAA) promote Pdx1 nuclear accumulation. MIN6 cells was starved from full aas and restimulated with full aas, BCAA, leucine, isoleucine, valine, glutamine or arginine for 2 h, and Pdx1 was analyzed by immunofluorescence staining. Scale bars, 10 µm.(F) Amino acids promote Pdx1 protein level and nuclear localization in n Rab1A-dependent manner. MIN6 cells were transfected with control or Rab1A short hairpin RNA (shRNA) and starved of aas for 24 h before re-stimulated with aas for 2 h. Pdx1 was analyzed by IF. Scale bars, 10 µm.(G) Amino acids regulate Pdx1 protein stability. MIN6 cells were cultured in normal medium (+aa) or aa starvation medium (-aa) for 24 h and then treated with cycloheximide (CHX) for different times. PDX1 protein was analyzed by immunoblot. Actin was used as a loading control.(H) Rab1A is required for maintaining Pdx1 protein stability. MIN6 cells were transfected with control or Rab1A shRNA, and then treated with CHX for different times. Pdx1 protein was analyzed by immunoblot.(I) aa-Rab1A signaling regulates proteasome-dependent degradation of Pdx1. MIN6 cells starved from aas and then re-stimulated with aas without or with Rab1A knockdown in the absence or presence of MG-132. Pdx1 protein was analyzed by immunoblot.(J) Amino acids and glucose cooperate in the regulation of Pdx1 stability. MIN6 cells starved from aas and glucose and then re-stimulated with aas and/or glucose without or with Rab1A knockdown. Pdx1 protein was analyzed by immunoblot.(K) Rab1A knockout leads to downregulation of Pdx1 protein expression and mTORC1 signaling. Pdx1, p-S6K, S6K, p-Akt, and Akt were analyzed by immunoblot analysis of the pancreas of Rab1A+/+ and Rab1A-/- mice (1 week post TAM). Shown are two different animals in each group.(L) Same as (K) except Pdx1 was analyzed by IHC. Arrowheads indicate loss of Pdx1 in the nucleus. Scale bar, 50 µm.
Fig 2: Construction for AAV vector of GCG promoter drive geneA schematic showing the construction of the AAV vector and GCG promoter driving GFP and Cre. For AAV-GCG-Pdx1-MafA, the GCG promoter drives Pdx1 and MafA open reading frames (ORFs), and the GFP tag was connected to the 2A peptide sequence, pAAV-GCG-Pdx1-P2A-MafA-T2A-GFP (A). The Pdx1 and MafA expression of plasmids was determined by western blot. Lanes 1, 2, and 3 are three individual vector-transduced samples (C) and GFP cell transduction efficiency in alpha-TC1 clone 9 cells (B). Min6, Min-6 cell line, a positive control. Bright field and fluorescence images, ×100.
Fig 3: AAV8 GCG-driven Pdx1 and MafA normalize blood glucose in ALX-induced diabetic miceBlood glucose levels were normalized 3 weeks post AAV8-GCG-PM viral infusion (A). IPGTT showed that glucose tolerance was better than that of the pancreas infused with the AAV-GCG-GFP virus (B). Compared with AAV8-GCG-GFP viral-infused islets (C, e–h) in ALX-induced diabetic mice, ALX islets were normalized by reprogrammed alpha cells into insulin-producing cells in ALX-induced C57/6 mice pancreas (C, i–l). Beta-like cell mass significantly increased compared with the cell mass of AAV8-GCG-GFP viral-infused pancreas (p < 0.01) and up to 65% of WT mice 4 weeks after AAV8-GCG-PM viral infusion (D). Immunofluorescence staining, ×200, scale bar, 25 μM.
Fig 4: The GCG promoter drives Pdx1 and MafA in reprogrammed insulin-producing, beta-like cells and reduces immune cell homing to the islets of the pancreas in NOD/ShiLtJ miceCD45-positive immune cells infiltrated the islets of NOD/ShiLtJ mice, but did not infiltrate the AAV8-GCG-PM viral-infused pancreatic islets of NOD/ShiLtJ mice. Immunofluorescence staining, ×200, scale bar, 25 μM.
Fig 5: AA8 GCG-driven Pdx1 and MafA normalize blood glucose in NOD/ShiLtJ mice with autoimmune diabetesNOD/ShiLtJ mouse blood glucose levels 4 weeks after AAV8-GCG-PM viral infusion (A). Immunohistochemistry of glucagon and insulin in pancreatic tissue of AAV8-GCG-GFP viral-infused NOD/ShiLtJ mice (B, a–d). Pancreatic islets were normalized by reprogramming alpha cells into insulin-producing cells in AAV8-GCG-PM viral-infused NOD/ShiLtJ mice (B, e to l, shows two pancreatic tissue samples). Immunofluorescence staining, ×200, scale bar, 25 μM. IPGTT showed that glucose tolerance was better than that of the pancreas infused with AAV-GCG-GFP (C). Beta-like cell mass was increased at 4 weeks (∗p < 0.01) (D) and maintained until the end of the 30-week experiment. None of the AAV8-GCG-GFP-infused NOD/ShiLtJ mice survived after 6 weeks from viral infusion (D). Concentrations of triglycerides, free fatty acids, and β-hydroxybutyrate in the serum of AAV8-GCG-PM virus-infused mice had normal insulinemia (E). HG, hyperglycemic NOD/ShiLtJ mice; NG, NOD/ShiLtJ normoglycemic; NOD + GCG-PM, AAV8-GCG-PM-infused NOD/ShiLtJ mice. ∗p < 0.01, #p < 0.05.
Supplier Page from Abcam for Anti-PDX1 antibody [EPR22002]