Pdx1^+/− mice develop diabetes in response to HFD-induced insulin resistance. (A) Baseline glucose tolerance test and body weights of 3.5-week-old male Pdx1^+/− mice (n = 16) and their wild-type Pdx1^+/+ littermates (n = 15). GTT was performed after a 6 h fast; P < 0.001 by ANOVA. (B) Weight gain profile of Pdx1^+/− mice and their littermate controls after randomization into HFD or normal chow (NC) groups. *, P < 0.05, **, P < 0.005 Pdx1^+/+ HFD vs. NC; ^‡, P < 0.05, ^‡‡, P < 0.005 Pdx1^+/− HFD vs. NC. (C) Insulin tolerance test after 5 months on HFD or NC (by ANOVA, P < 0.005 Pdx1^+/+ HFD vs. NC; P = 0.06 Pdx1^+/− HFD vs. NC; P = 0.985 Pdx1^+/+ HFD vs. Pdx1^+/− HFD). (D) Six-hour fasting blood glucose levels of mice 5 months in HFD or NC group. *, P < 0.001, Pdx1^+/− HFD vs. NC; ^‡, P < 0.001, ^‡‡, P < 0.00001 relative to diet-matched Pdx1^+/+ littermates. (E and F) IP glucose tolerance tests on mice fasted overnight after 1 month (E) and 4 months (F) on HFD or NC. In (E), P < 0.005 for Pdx1^+/− HFD vs. NC and P < 0.001 for all other pair-wise comparison groups by ANOVA. In (F), P < 0.0001 for all comparison groups by ANOVA. Glucose values of 600 mg/dL represent the upper limit of detection. (G) Acute insulin release in mice 3 months on HFD or NC. Plasma insulin was measured by ELISA after IP glucose injection. *, P < 0.05, **, P < 0.005 relative to time 0; ^‡, P < 0.05, ^‡‡, P < 0.01 relative to diet-matched Pdx1^+/+ littermates; #, P < 0.05, ##, P < 0.01 relative to corresponding NC controls. For (B–G), n = 8 mice per group, and data represent the mean ± standard error.

Pdx1 haploinsufficiency limits β cell mass expansion by decreasing β cell survival and blocking compensatory hypertrophy without impairing proliferation. (A) Representative images of insulin immunohistochemistry (brown) performed on pancreas sections from mice 5 months on either HFD or NC. (Scale bars, 50 μm.) (B) Quantification of β cell mass (n = 5–8 mice per group; *, P < 0.05 relative to corresponding NC controls). (C) Quantification of β cell proliferation using BrdU/insulin double immunofluorescence. n = 4–6 mice per group and >1,000 insulin-positive cells were counted per animal; *, P < 0.05, **, P < 0.005 relative to NC controls. (D) Rates of β cell apoptosis as assessed by TUNEL. For sections containing TUNEL/insulin double positive cells, cell death was quantified by normalizing to total number of insulin positive cells (≈3,000–9,000 per slide). n = 4–6 mice per group; **, P < 0.01 relative to NC control. (E) Mean β cell size was approximated by dividing total insulin-positive area per islet by number of insulin-positive nuclei per islet. n = 4–6 mice per group and 7–17 islets were analyzed per animal (**, P < 0.001 relative to NC control).

β cells of HF-fed Pdx1^+/− mice exhibit evidence of increased ER stress. (A–D) Electron micrographs of pancreata from Pdx1^+/+ and Pdx1^+/− mice fed either HFD or NC for 4 months. The β cells of both HF-fed Pdx1^+/+ (B) and NC-fed Pdx1^+/− mice (C) show mild ER dilation compared with NC-fed Pdx1^+/+ controls (A). However, the HFD induces striking distention of the ER in β cells of Pdx1^+/− mice (D). Magnification of left panels, 5,000×; magnification of middle panels, 30,000×. The arrow in (D) indicates normal ER appearance in an islet alpha cell. (E) Quantitative RT-PCR analysis of Bip mRNA levels in islets isolated from individual Pdx1^+/+ or Pdx1^+/− mice fed HFD or NC for 8 weeks. (F) Bip immunohistochemistry on pancreas sections from mice 5 months on either diet showing increased Bip staining in Pdx1^+/− islets. (Scale bars, 50 μm.) (G and H) qPCR measurement of spliced Xbp1 transcript (G) and total Xbp1 (H) in isolated islets. For (E, G, and H) raw data are normalized to HPRT (n = 4–6 mice per group; *, P < 0.05, **, P < 0.01 relative to NC-fed Pdx1^+/+; #, P < 0.05 relative to corresponding NC control). (I and J) Induction of spliced Xbp1 (I) and Bip (J) transcript in AdshPdx1- or AdshLuc-infected Min6 cells after treatment with thapsigargin, tunicamycin, or DMSO (vehicle). (n = 3 replicates per condition; *, P < 0.05; **, P < 0.001 relative to treatment-matched AdshLuc-infected cells).

Pdx1 deficiency enhances β cell susceptibility to ER stress-related apoptosis. (A) Cleaved caspase 12 protein levels in Min6 cells following silencing of Pdx1 with a pool of four siRNA duplexes (siPdx1). A pool of nontargeting siRNA duplexes (siNT) was used as a control. (B) Annexin V flow cytometry analysis of Min6 cell apoptosis after nucleofection with pooled siPdx1 duplexes and subsequent ER stress induction with 1 μM thapsigargin (n = 6, ***, P < 0.0001 relative to siNT; ^‡, P < 0.001 relative to DMSO). (C) β cell and acinar cell apoptosis in Pdx1^+/− mice and their Pdx1^+/+ littermates 96 h after IP injection of tunicamycin (TM). Cell death was quantified using TUNEL immunofluorescence. n = 3–4 mice per group; *, P < 0.05 (one-tailed t test). (D) Glucose levels in the tunicamycin-injected mice just before sacrifice.

Pdx1 regulates multiple genes involved in maintaining ER homeostasis. (A) Quantitative RT-PCR confirmation of ER-related genes downregulated in Mouse PancChip 6.1 cDNA microarray analyses of AdshPdx1-infected Min6 cells compared with either uninfected or AdshLuc-infected controls. (n = 3; *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001 relative to AdshLuc-infected cells). (B) Upper, Schematic of the genomic sequence 1.5 kb upstream of the mouse Atf4 promoter highlighting two regions of evolutionary conservation with the human sequence and the Pdx1 consensus sites contained within (adapted from rVista 2.0). Lower, Pdx1 directly binds the Atf4 promoter as assessed by chromatin immunoprecipitation (ChIP) from Min6 cells. (C) Pdx1 also occupies the Wfs1 promoter in Min6 cells as assessed by quantitative ChIP, consistent with the high-throughput ChIP on chip data. (D) ChIPs performed on primary islets freshly isolated from 8-week-old mice demonstrate Pdx1 occupancy at the two conserved regions of the Atf4 promoter and the Wfs1 promoter in vivo. Primers designed to amplify a known Pdx1-binding region of the insulin promoter and a piece of the albumin promoter were used as positive and negative controls, respectively. n = 4 individual experiments, data are mean ± s.e.m, *, P < 0.05 relative to IgG control pulldown. (E and F) Pdx1 and Atf4 mRNA (E) and protein (F) levels in Min6 cells transfected with either a pool of four siPdx1 duplexes (siPdx1) or a nontargeting control pool (siNT). n = 3 replicates per condition; **, P < 0.01. In (F), nuclear extracts were analyzed by Western blot, using histone H3 as a loading control. (G) qPCR analysis of Atf4, known Atf4 targets and Wfs1 expression in islets isolated from individual 12-week-old Pdx1^+/+ or Pdx1^+/− mice. n = 4–6 mice per group; *, P < 0.05; **, P < 0.01.