Reduced autophagy in livers of fasted, fat-depleted, ghrelin-deficient mice: Reversal by growth hormone
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Contributed by Joseph L. Goldstein, December 12, 2014 (sent for review November 19, 2014)
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Fig. 1.Levels of blood glucose (A), plasma GH (B), and hepatic LC3-II (C–E) before and after feeding in calorie-restricted WT and Goat−/− mice. Sixteen male WT and 16 Goat−/− littermates (8 wk old) were subjected to 60% calorie restriction for 8–9 d as described in Materials and Methods. Four mice from each group were killed at the indicated time. (A and B) At 1 h before sacrifice, mice were injected i.p. with leupeptin (15 mg/kg). Immediately before sacrifice, blood was obtained from the retro-orbital sinus for measurement of blood glucose and plasma GH. Each value in A and B represents mean ± SEM of data from four mice. Asterisks denote the level of statistical significance (Student t test) between WT and Goat−/− mice: **P < 0.01; ***P < 0.001. (C–E) Liver supernatants were processed as described in Materials and Methods, after which individual (C) or pooled (D) supernatants were subjected to immunoblotting with the indicated antibody. For immunoblotting of LC3 and GAPDH, 20 μg of liver supernatant was loaded in each lane; for p-STAT5 and t-STAT, 60 μg was loaded. Each pooled sample in D was derived from equal amounts of the four individual samples in C. (E) Relationship between the amounts of LC3II and p-STAT5. The relative amounts of LC3II (normalized to GAPDH) and p-STAT5 (normalized to total STAT5) were quantified by scanning the autoradiograms in D. The data were analyzed with ImageJ.
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Fig. 2.LC3 levels in muscle (A), heart (B), and kidney (C) before and after feeding in calorie-restricted WT and Goat−/− mice. The mice used in this experiment are the same as those used in the experiment shown in Fig. 1. Tissues were processed as described in Materials and Methods, after which 20 µg of each supernatant was subjected to immunoblot analysis with the indicated antibody.
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Fig. 3.Electron micrographs of liver from calorie-restricted WT (A–D) and Goat−/− (F–I) mice at 9:30 AM and 5:30 PM on day 8. WT and Goat−/− littermates (8 wk old) were subjected to 60% calorie restriction for 8 d. Mice were perfused at 9:30 AM or 5:30 PM on day 8 of calorie restriction, after which the liver was fixed and processed as described in Materials and Methods. Pictures from 20 different unit areas (36 × 26 μm per unit area) from two WT and two Goat−/− livers at each time point were taken at 5,000 amplitude; representative pictures are shown. White arrows denote typical autolysosomes. (Scale bar: 5 μm; magnification, 5,000×.) (Insets) Enlarged images of each arrow-denoted autolysosome (magnified 5×). (E and J) The number of autolysosomes per unit area was determined by three independent examiners as described in Materials and Methods. Each value represents mean ± SEM of data from 20 images. Asterisks denote level of statistical significance (Student t test) between autolysosome numbers at 9:30 AM and 5:30 PM of WT (E) and Goat−/− (J) mice: ***P < 0.001.
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Fig. 4.Preservation of autophagy in calorie-restricted Goat−/− mice by infusion of GH. Alzet osmotic minipumps containing either vehicle or GH (15 μg/24 h) were implanted at 3 d before calorie restriction as described in Materials and Methods. Mice were then subjected to 60% calorie restriction for 8 d. Mice were dissected at 5:30 PM on day 8, at 1 h after leupeptin was injected i.p. (A and B) Immunoblot analysis with the indicated antibody was carried out on liver supernatants from individual mice (A) or pooled supernatants (B) from the five individual mice as described in Fig. 1. (C) Blood glucose levels were measured at 5:30 PM on day 8. Each value represents mean ± SEM of data from five mice. Asterisks denote level of statistical significance (Student t test) between WT and Goat−/− mice infused with vehicle or GH: ***P < 0.001.
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Fig. 5.(A–J) Electron micrographs of livers from GH-injected, calorie-restricted WT (A–D) and Goat−/− (F–I) mice. Male WT and Goat−/− mice littermates (8 wk old) were subjected to 60% calorie restriction for 8 d. On day 7, each mouse received three s.c. injections of either vehicle or GH (30 µg per mouse) at 2:30 PM, 3:30 PM, and 4:30 PM. At 5:30 PM, blood was obtained for glucose measurements, after which mouse livers were fixed, perfused, and processed as described in Materials and Methods. Pictures from 20 different unit areas (36 × 26 µm per unit area) from two WT and two Goat−/− mice were taken at 5,000 amplitude; representative pictures are shown. White arrows denote typical autolysosomes. (Scale bar: 5 µm; magnification, 5,000×.) (E and J) The number of autolysosomes per unit area was determined by three independent examiners as described in Materials and Methods. Each value represents mean ± SEM of data from 20 images. Asterisks denote level of statistical significance (Student t test) between autolysosome numbers of vehicle or GH-injected WT (E) and Goat−/− (J) mice: ***P < 0.001. (K and L) Failure of acute injections of GH to restore blood glucose in Goat−/− mice. WT and Goat−/− mice (five in each group) were calorie-restricted and treated with three s.c. injections of GH as described above. On day 7, mice were killed at 5:30 PM, at 1 h after i.p. injection of leupeptin (15 mg/kg). Blood was obtained from the retro-orbital sinus for measurement of glucose (K). Supernatants from pooled livers of five mice were immunoblotted for LC3, GAPDH, p-STAT5, and t-STAT5 (L) as described in Fig. 1.
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Fig. 6.Failure to restore liver content of ATP in calorie-restricted Goat−/− mice by acute injection of GH. Male WT and Goat−/− mice littermates (8 wk old) were subjected to 60% calorie restriction for 8 d. On day 8, each mouse received three s.c. injections of vehicle (−GH) or 30 µg of GH (+GH) at 2:30 PM, 3:30 PM, and 4:30 PM. Mice were killed at 5:30 PM, liver samples were freeze-clamped, and blood glucose level (A) and hepatic content of ATP (B) were measured as described in Materials and Methods. In A, individual values from two independent experiments (n = 13 mice) were combined (mean ± SEM; ***P < 0.001). In B, each individual value from the two experiments is shown. Results from one experiment are denoted by open symbols; results from the other experiment, by closed symbols.
