Figure 3: Effect of in vitro and in vivo inhibition of autophagy in βraKO mice.
From: Loss of mTORC1 signalling impairs β-cell homeostasis and insulin processing
(a) Immunoblotting (upper) and quantification (lower) for p62, LC3-I/II and actin in islets from βraKO and raptorf/f (30–40 days of age) treated with or without NH4Cl (20 mM) for 24 h. A representative image from four independent experiments is included and each lane shows the expression levels from one mouse. (b) Assessment of apoptosis after inhibition of autophagy in islets from raptorf/f and βraKO mice (30–40 days of age). Staining (left) and quantification (right) of TUNEL-/insulin-positive cells in dispersed islets from raptorf/f and βraKO treated or not with NH4Cl for 24 h. Scale bar, 10 μM (n=4). (c) Immunostaining for insulin (green), p62 (red) and DAPI (blue) in sections from 80-day-old raptorf/f and βraKO mice treated for 8 weeks with 3MA (15 mg kg−1 in 0.9% saline), CQ (7 mg kg−1 in 0.9% saline) or vehicle (saline). Treatment started in 18-day-old mice. Scale bar, 20 μM. (d) Random fed blood glucose levels in raptorf/f (control) and βraKO mice treated with control vehicle or CQ. (e) Random fed blood glucose levels in control (raptorf/f) and βraKO mice treated with control vehicle or 3MA. Same glucose values for control and βraKO mice are included in Fig. 2c,d. (f) Assessment of β-cell mass at 80 days of age in raptorf/f and βraKO mice treated with 3MA or control vehicle. (g,h) Assessments of β-cell proliferation and TUNEL in 80-day-old mice (n=6; Fig. 5c–h). (i) GSIS determined by static incubation of isolated islets from βraKO mice treated with 3MA or control vehicle (n=4). Data are shown as means±s.e.m., *P<0.05 versus raptorf/f (control) mice treated with saline, and #P<0.05 versus βraKO mice treated with saline; nonparametric U-test (Mann–Whitney).
