PMC

Starvation induces autophagy in beta cells in vivo. A, immunostaining for LC3 (green), insulin (red), and DAPI (blue) in islets from fed or 24-h fasted mice. A representative area from an islet was magnified (High mag) in gray from each inset. Starvation increased the number of LC3-positive punctae/beta cell in fasted compared with fed mice. B, LC3 punctae were increased from 2.36 ± 0.21 punctae in fed mice to 6.18 ± 1.24 punctae in starved mice (*, p < 0.01) (n = 3).

Comparison of Pdx1^+/− and Pdx1^+/−Becn1^+/− mice after 1 week on a high fat diet. A, blood glucose concentrations during intraperitoneal glucose tolerance tests after 1 week on a high fat diet in 4-week-old Pdx1^+/− and Pdx1^+/− Becn1^+/− mice (n = 4) (top panel) and Becn1^+/− (n = 4) and WT mice (n = 3) (lower panel). B, total areas under the glucose curves (AUC) from A. The AUC was reduced in Pdx1^+/− Becn1^+/− mice compared with Pdx1^+/− mice (*, p < 0.05) and was similar to the AUC in the WT and Becn1^+/− mice. C, fasting and 30-min serum insulin levels measured during intraperitoneal glucose tolerance tests in Pdx1^+/− Becn1^+/− mice (n = 4) were significantly greater than in Pdx1^+/− mice (n = 4) (*, p < 0.05; **, p < 0.01) and similar to the levels obtained in WT (n = 4) and Becn1^+/− mice (n = 4).

Amino acid deprivation-induced cell death in MIN6 and NIH 3T3 cells. A, PI-positive cells were measured in control, Atg5, or Becn1 KD MIN6 cells incubated for 72 h in amino acid (aa)-deprived medium. 48 ± 5% of cells incubated in amino acid-starved medium stained positive for PI, and this was decreased to 30 ± 2 and 26 ± 2% in Atg5 KD and Becn1 KD MIN6 cells, respectively (*, p < 0.01) (n = 3). B, representative Western blots of cleaved caspase-3 from the cells at 72 h described for A. Cleaved caspase-3 levels were increased in MIN6 cells maintained in amino acid-deprived medium compared with normal medium. Note that Atg5 KD and Becn1 KD did not affect the level of cleaved caspase-3 in amino acid-starved MIN6 cells (n = 3). C, in NIH 3T3 cells, PI-positive cells were increased to 28 ± 3% when cells were incubated in amino acid-deprived medium for 72 h, and this was further increased to 40 ± 2 and 42 ± 2% in Atg5 KD and Becn1 KD NIH 3T3 cells, respectively (*, p < 0.05) (n = 3). D, amino acid deprivation increased cleaved caspase-3 levels in NIH 3T3 cells, and this was further elevated in Atg5 KD and Becn1 KD NIH 3T3 cells at 72 h (n = 3).

Nutrient deprivation induces autophagy in MIN6 cells. A, MIN6 cells were cultured for 48 h in normal medium, amino acid-starved (aa starv) medium plus amino acids, HBSS medium that does not contain either amino acids or serum, or amino acid-starved medium. LC3-II levels were increased in HBSS medium and amino acid-starved medium, and the addition of amino acids in amino acid-starved medium prevented the increase in LC3-II levels as assessed by Western blot (n = 3). B, MIN6 cells were cultured in normal medium, HBSS medium, or amino acid-starved medium with or without 1 mm 3-MA for 24 h. Six days after infection with a lentivirus containing shBecn1, MIN6 cells were cultured without amino acid for 24 h. C, LC3 punctae were increased from 4.31 ± 1.37 punctae/cell in normal medium to 28.0 ± 3.51 punctae/cell in HBSS medium and 26.4 ± 3.77 punctae/cell in normal medium without amino acids. The number of punctae/cell was significantly decreased to 9.87 ± 0.27 in HBSS medium with 3-MA, 8.74 ± 2.17 in amino acid-starved medium with 3-MA, and 3.21 ± 1.47 in amino acid-starved Becn1 KD MIN6 cells. D, human islets were incubated for 48 h with or without amino acids in the presence or absence of 1 mm 3-MA. Amino acid starvation increased LC3-II levels, and this was prevented by3-MA (n = 3).

Beta cell autophagy, apoptosis, proliferation and mass in pancreatic islets from mice after 1 week on a high fat diet. These analyses were conducted in the mouse groups depicted in . A, the number of LC3 punctae was decreased from 10.19 ± 3.36 punctae in Pdx1^+/− beta cells to 4.33 ± 2.34 punctae in Pdx1^+/− Becn1^+/− beta cells (*, p < 0.001). The number of LC3 punctae in beta cells from WT and Becn1^+/− mice were 2.54 ± 0.21 and 2.30 ± 0.22 punctae (p = ns), respectively. B, the number of cells staining positive for cleaved caspase-3 was significantly reduced in islets from Pdx1^+/− Becn1^+/− mice compared with Pdx1^+/− mice (*, p < 0.01). The percentage of cells staining positive for cleaved caspase-3 was similar in islets from Becn1^+/− and WT mice (n = 3). C, the number of Ki-67-positive beta cells per islet was increased in Pdx1^+/− Becn1^+/− and Becn1^+/− mice compared with Pdx1^+/− and WT mice, respectively (*, Pdx1^+/− Becn1^+/− versus Pdx1^+/−, p < 0.05; **, Becn1^+/− versus WT, p < 0.01) (n = 3). D, beta cell area at 1 week after high fat diet was estimated using insulin immunoreactivity as described under “Experimental Procedures” and normalized to the total pancreas area. Five or six sections were analyzed from each animal (n = 4). Pdx1^+/− Becn1^+/− beta cell area/pancreas area (0.76 ± 0.09%) was preserved compared with that of Pdx1^+/− (0.29 ± 0.05%) (*, p < 0.01) and comparable with that seen in WT (0.76 ± 0.03%) and Becn1^+/− mice (0.78 ± 0.09%).

Autophagy is increased in Pdx1^+/− beta cells. A, immunostaining for LC3 (green), insulin (red), and DAPI (blue) in islets from 11-week-old male Pdx1^+/− and Pdx1^+/+ mice. A representative area from an islet was magnified (High mag) in gray from each inset. Pdx1^+/− beta cells revealed more frequent LC3 punctae formation compared with Pdx1^+/+ beta cells. B, the number of LC3 punctae/beta cell was increased from 1.49 ± 0.11 in Pdx1^+/+ beta cells to 9.38 ± 1.64 in Pdx1^+/− beta cells (*, p < 0.0001). C, Western blots of LC3 in islets from 3-week-old male Pdx1^+/− mice showed an increased LC3-II and LC3-I/LC3-II ratio compared with Pdx1^+/+ islets (n = 3). D, pancreatic islets isolated from 3-week-old male Pdx1^+/+ (panel a) and Pdx1^+/− (panels b–f) mice analyzed by transmission electron microscopy. Pdx1^+/+ islets showed numerous granules surrounded by halos in beta cells (panel a) in the absence of autophagosomes, autolysosomes, and apoptotic bodies. Beta cells from Pdx1^+/− mouse islets contained autophagosomes and autolysosomes (arrows), which are shown at several magnifications (panel b–f). A representative area from the beta cell was magnified from each inset. Note that rough endoplasmic reticulum is visible in autophagosomes in panel d from a Pdx1^+/− beta cell. Panels e and f show several apoptotic bodies (asterisks) with autophagosomes and autolysosomes (arrows) from Pdx1^+/− mouse islets. The scale bars represent 2 μm. E, the effect of 0.3 mg/kg BafA1 on autophagic flux was studied by LC3 staining. BafA1 treatment increased in LC3-positive punctae in Pdx1^+/− beta cells from 8.85 ± 3.56 to 17.31 ± 2.26, indicating increased autophagic flux in Pdx1^+/− compared with Pdx1^+/+ beta cells (*, p < 0.01).

Effects of Becn1 and Atg5 KD in Pdx1-reduced MIN6 cells. A, Becn1 and cleaved caspase-3 levels were measured by Western blot 11 days after infection with the control or shBecn1 vector. Nearly complete suppression of Becn1 to inhibit basal autophagy induced cleaved caspase-3. MIN6 cells exposed to 1 μm thapsigargin for 24 h were used as a positive control for cleaved caspase-3 (n = 2). PI-positive cells were increased from 3 ± 1 to 67 ± 4% in Becn1 KD MIN6 cells (*, p < 0.0001). B, Becn1 protein levels 7 days after coinfection with shPdx1 and shBecn1Δ30, shBecn1Δ50, or shBecn1Δ90 are shown in the inset (n = 3). Becn1Δ30 and Becn1Δ50 reduced the proportion of Pdx1 KD cells staining positive for PI from 41 ± 2 to 18 ± 2% (*, p < 0.01) and 7 ± 1% (**, p < 0.001), respectively. Becn1 KD by 90% (Becn1Δ90) increased the proportion of Pdx1 KD MIN6 cells staining with PI to 55 ± 4% (***, p < 0.001) (n = 3). C, following infection with a Pdx1 KD vector, MIN6 cells were exposed to lentivirus containing control, shBecn1, or shAtg5 vectors. On day 7, 39 ± 3% of Pdx1 KD cells stained positive for PI, whereas only 12 ± 2 and 7 ± 1% of Pdx1/Becn1 KD cells and Pdx1/Atg5 KD cells, respectively, stained with PI. On day 9, the proportion of PI-positive Pdx1/Becn1 KD cells and Pdx1/Atg5 KD cells increased to 44 ± 4 and 33 ± 6%, respectively (n = 3). Pdx1 KD versus Pdx1/Becn1 KD (*, p < 0.001) and Pdx1/Atg5 KD (*, p < 0.001) on day 7. The right panels show Western blots of Pdx1, Becn1, and Atg5 from the aforementioned experiments on day 7 (n = 3).

Inhibition of autophagy in Pdx1-reduced MIN6 cells. A and B, 5 days after infection with the shPdx1 or control vector in the absence or presence of 1 mm 3-MA, MIN6 cells were fixed and stained for LC3. A representative area (box) from Pdx1 KD MIN6 cells was magnified (High mag). The number of LC3 punctae per cell increased from 8.61 ± 1.54 in control cells to 27.0 ± 3.14 in Pdx1 KD cells (*, p < 0.001), and this increase was prevented by treatment with 1 mm 3-MA (7.86 ± 3.87) (*, p < 0.001). C, representative electron micrographs of Pdx1 KD or control MIN6 cells on day 5. Pdx1 KD cells showed the presence of autophagosomes and autolysosomes (arrows) with cytoplasmic contents ranging from granular cytoplasm with occasional organelles to well formed autophagosomes containing degenerating organelles. Autophagosomes and autolysosomes were rarely seen in control MIN6 cells. The scale bars represent 2 μm. D, following infection with a control or Pdx1 KD vector, MIN6 cells were exposed to 1 mm 3-MA, 10 μm DEVD-CHO, or no treatment. On day 9, 63 ± 11% of Pdx1 KD cells stained positive for PI, whereas only 19 ± 12% of DEVD-CHO-treated Pdx1 KD cells and 53 ± 16% of 3-MA-treated Pdx1 KD cells stained for PI (n = 3). Pdx1 KD versus Pdx1 KD with DEVD-CHO on day 9 (*, p < 0.001). E, cleaved caspase-3 levels in 1 mm 3-MA or 10 μm DEVD-CHO treated Pdx1 KD MIN6 cells. Increased cleaved caspase-3 levels were observed in 3-MA-treated Pdx1 KD cells on day 9 compared with day 5 and day 7, whereas cleaved caspase-3 was not detected in DEVD-CHO-treated Pdx1 KD cells (n = 4).

Pdx1 deficiency induces MIN6 cell death accompanied by increased autophagy. A, MIN6 cells were infected with a lentiviral vector that drives expression of an shRNA targeting Pdx1 transcript (Pdx1 KD) or control lentiviral vector. Five days after infection, Pdx1 mRNA levels assayed by real time reverse transcription-PCR were reduced to 52 ± 3% of control values (n = 3) (*, p < 0.0001). B, six days after infection, Pdx1 protein levels assayed by Western blot were reduced in the Pdx1 KD MIN6 cells compared with control (n = 3). C, following infection with the control or Pdx1 KD vector, MIN6 cells received no treatment (panels a and b), 1 mm 3-MA (panels c and d), 0.3 μm chloroquine (CQ) (panels e and f), or 30 nm wortmannin (Wm) (panels g and h). PI (red)/DAPI (blue) staining revealed that all three treatments prevented the Pdx1 KD-induced cell death on day 7. 40 ± 3% of Pdx1 KD stained with PI, and this was reduced (n = 3). Bottom panel, control versus Pdx1 KD (*, p < 0.001), Pdx1 KD versus Pdx1 KD with 3-MA (**, p < 0.001), Pdx1 KD versus Pdx1 KD with chloroquine (**, p < 0.001), and Pdx1 KD versus Pdx1 KD with wortmannin (**, p < 0.001). D, in the upper panel, LC3-II levels in MIN6 cells infected with a control or Pdx1 KD lentiviral construct were measured on days 5 and 7. LC3-II levels were increased in Pdx1 KD MIN6 cells on day 5 before cell death became evident and on day 7 when death of these cells was evident (n = 3). The lower panel shows LC3-II levels in control or Pdx1 KD MIN6 cells cultured in the absence or presence of 1 nm BafA1 on day 7. LC3-II levels were increased in the presence of BafA1 in Pdx1 KD MIN6 cells, indicating increased autophagic flux in Pdx1 KD MIN6 (n = 3). The intensity of LC3-I and LC3-II bans was quantified, and the calculated LC3-II/LC3-I ratio is indicated.