(A) Western blot analysis of protein lysate obtained from mice with a conditional deletion of Atg7 within skeletal muscle (Atg7^F/F:MCK-Cre) or control animals (Atg7^F/+:MCK-Cre). Relative levels of Atg7, p62 and actin (loading control) were assessed. (B) Representative stained histological sections from soleus muscle. (C) Electron micrographs of skeletal muscle from 8 week old Atg7^F/+:MCK-Cre or Atg7^F/F:MCK-Cre mice. Electron micrographs demonstrate the accumulation of dysmorphic mitochondria within the Atg7^F/F:MCK-Cre muscle. (D) Western blot analysis of purified mitochondria obtained from Atg7^F/F:MCK-Cre or control animals Atg7^+/+:MCK-Cre demonstrating an apparent similar level of electron transfer subunit composition. Mitochondrial protein lysates were probed for the Complex I component protein NDFS3 (NADH-ubiquinone oxidoreductase 30 kDa subunit), the Complex II component FP (flavoprotein subunit of complex II ), the Complex III protein Core 1 (Ubiquinol-cytochrome-c reductase complex core protein 1) and the mitochondrial membrane protein VDAC. (E) Blue Native gel electrophoresis using mitochondrial extracts isolated from control and Atg7^-/- tissues demonstrating similar stoichiometry and assembly of electron transfer complex components. (F) Representative oxygen consumption tracings of mitochondria isolated from the skeletal muscle of 12 month old Atg7^+/+:MCK-Cre or Atg7^F/F:MCK-Cre mice. Atg7-deficient skeletal muscle demonstrated a pronounced reduction in respiration when assessed in the presence of the Complex II dependent substrate succinate. Rotenone was routinely added to prevent reverse electron transport. Measurements were made in the absence (State IV) and presence (State III) of ADP and following the Complex III inhibitor antimycin A (AA). (G) Composite determinations of mitochondrial respiration in the presence of succinate and rotenone. Graph represents the mean +/- SEM from Atg7^+/+:MCK-Cre (n=3) or Atg7^F/F:MCK-Cre mice (n=3). * p≤0.05; ** p≤0.01.

(A) Intracellular ROS levels as assessed by DCFDA fluorescence intensity (arbitrary units) in WT and Atg7^-/- MEFs. ROS measurements were made from three independent WT or Atg7^-/- MEF primary cell isolates and the fluorescent intensity of more than 250 cells of each genotype were assessed. (B) NAC treatment reduces the levels of ROS in MEFs lacking Atg7. Levels of ROS were assessed by DCFDA fluorescence in Atg7^-/- MEFs untreated or treated with NAC (500 μM) for 4 days prior to imaging. Values represent the normalized fluorescent intensity (arbitrary units) of approximately 300 cells per condition. Graphs represent the mean +/- SEM.

(A) Western blot analysis of purified pancreatic islets obtained from Atg7^F/+:Rip2-CRE or Atg7^F/F:Rip2-CRE mice demonstrating the relative expression of Atg7, p62 and actin (loading control). (B) Intracellular insulin levels (mean +/- SEM) in pancreatic tissue of 8-9 week old Atg7^F/+:Rip2-Cre (n=4 mice) or Atg7^F/F:Rip2-Cre mice (n=5 mice). The slight reduction in insulin levels in the Atg7^F/F:Rip2-Cre mice was not significant when compared to the control. (C) Pancreatic sections of control Atg7^F/+:Rip2-Cre or Atg7^F/F:Rip2-Cre mice were stained for non-β cell components within the islets with the simultaneous use of anti-glucagon, anti-somatostatin, and anti-polypeptide antibodies. (D) Serial sections were used to visualize β cells with an anti-insulin antibody. Eight week old mice lacking autophagy in β cells have qualitatively similar levels of α, δ, and polypeptide producing cells within their islets, as well as similar levels of β cells when compared to control mice. (E) Electron micrographs demonstrating the accumulation of swollen, dysmorphic mitochondria within the Atg7-deficient β cells. (F) Isolated islets from control and Atg7^-/- mice were assessed for fold +/- SEM changes in basal respiration (Atg7^F/+:Rip2-Cre isolated islets=1), and for oxygen consumption in the presence of oligomycin (0.5 μM) or FCCP (0.5 μM). Results are normalized to islet protein concentration and are from n=4 mice per genotype. (G) Impaired glucose tolerance in Atg7^F/F:Rip2-Cre mice. Blood glucose measurements were made in 8-10 week-old control mice Atg7^F/+:Rip2-Cre (n=10 mice) or Atg7^F/F:Rip2-Cre mice (n=8 mice) following the IP injection of D-glucose (1 g/kg). Data represent the mean +/- SEM. *p≤0.05; **p≤0.01.

(A) Male Atg7^F/+:Rip2-Cre (n=6 mice), Atg7^F/+:Rip2-Cre (+NAC; n=6 mice), Atg7^F/F:Rip2-Cre (n=11 mice) or Atg7^F/F:Rip2-Cre (+NAC; n=11 mice) were fasted overnight and subsequently injected with 1 g/kg D-glucose. Serum glucose levels were measured and the untreated β cell Atg7 deficient mice were found to be statistically different at the indicated time points, while the other three groups of mice were statistically indistinguishable over the 2 hr timecourse. (B) Insulin levels were determined by tail vein blood sampling at time 0 and 15 min following glucose administration: Atg7^F/+:Rip2-Cre (n=6 mice), Atg7^F/+:Rip2-Cre (+NAC; n=4 mice), Atg7^F/F:Rip2-Cre (n=8 mice) or Atg7^F/F:Rip2-Cre (+NAC; n=5 mice). Data are represented as the mean +/- SEM. * p≤0.05; ** p≤0.001.

(A) Western blot analysis of wild type (+/+) or Atg7^-/- MEFs for the expression of Atg7, p62 and actin (loading control). (B) Measurement of oxygen consumption for WT and Atg7^-/- MEFs under basal conditions, following the addition of the mitochondrial electron chain inhibitor oligomycin (0.5 μM), or in the presence of the mitochondrial uncoupler FCCP (1 μM), to determine maximal oxidative capacity. Shown is the average fold change +/- SEM in oxygen consumption (WT MEFs basal respiration=1) obtained from 5 experiments each performed in triplicate. (C) Assessment of mitochondrial number in WT or Atg7^-/- MEFs. DNA was isolated from WT (n=3 independent WT MEF cell isolates) and Atg7^-/- MEFs (n=3 independent Atg7^-/- MEF cell isolates) and quantitative PCR analysis performed for the mitochondrial-encoded gene ND1 and the nuclear-encoded gene H19. (D) Relative extracellular acidification rates indicating lactic acid production and hence glyolytic rates in WT or Atg7^-/- MEFs. Shown is the average +/- SEM fold change in lactic acid production from 8 experiments each performed in triplicate. * p≤0.05; ** p≤0.01.

(A) Western blot analysis of wild type (+/+) or Atg7^-/- MEFs for the expression of Atg7, p62 and actin (loading control) cultured in the presence or absence on the antioxidant NAC (500 μM) for ten days. (B) Primary wild-type and Atg7^-/- MEFs that were cultured in the absence or presence of 500 μM NAC for 10 days prior to cellular respiration measurement. Shown is a representative tracing of oxygen consumption performed in triplicate under basal conditions, following the addition of oligomycin (0.5 μM), the pharmacological uncoupler FCCP (1 μM) or the Complex III inhibitor antimycin A (0.25 μM). (C) Averaged metabolic profile from 4 separate experiments employing 3 independent primary isolates of WT and Atg7^-/- MEFs. Shown is the fold change +/- SEM in oxygen consumption (WT MEF basal respiration =1) for WT MEFs and for Atg7^-/- MEFs that were cultured in the absence or presence of 500 μM NAC for 10 days prior to metabolic assessment.* p≤0.05; ** p≤0.01; NS= not significant.

(A) Atg7^F/+:Rip2-Cre or Atg7^F/F:Rip2-Cre mice that were untreated or treated with the antioxidant NAC for 12 weeks. At 16 weeks of age, mice were sacrificed and serial sections of pancreatic tissue were analyzed for p62 and insulin or (B) nitrotyrosine and insulin. (C) Quantification of nitrotyrosine staining in islets of control mice, Atg7-deficient animals or Atg7-deficient mice treated for 12 weeks with NAC (n=3 animals per group). Graph represents the mean+/- SEM. **; p≤0.01.