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. 2015 Jun 2;21(6):883-90.
doi: 10.1016/j.cmet.2015.05.016.

AMPK Activation of Muscle Autophagy Prevents Fasting-Induced Hypoglycemia and Myopathy During Aging

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Free PMC article

AMPK Activation of Muscle Autophagy Prevents Fasting-Induced Hypoglycemia and Myopathy During Aging

Adam L Bujak et al. Cell Metab. .
Free PMC article

Abstract

The AMP-activated protein kinase (AMPK) activates autophagy, but its role in aging and fasting-induced muscle function has not been defined. Here we report that fasting mice lacking skeletal muscle AMPK (AMPK-MKO) results in hypoglycemia and hyperketosis. This is not due to defective fatty acid oxidation, but instead is related to a block in muscle proteolysis that leads to reduced circulating levels of alanine, an essential amino acid required for gluconeogenesis. Markers of muscle autophagy including phosphorylation of Ulk1 Ser555 and Ser757 and aggregation of RFP-LC3 puncta are impaired. Consistent with impaired autophagy, aged AMPK-MKO mice possess a significant myopathy characterized by reduced muscle function, mitochondrial disease, and accumulation of the autophagy/mitophagy proteins p62 and Parkin. These findings establish an essential requirement for skeletal muscle AMPK-mediated autophagy in preserving blood glucose levels during prolonged fasting as well as maintaining muscle integrity and mitochondrial function during aging.

Figures

Figure 1
Figure 1. During Fasting, AMPK-MKO Mice Have Hypoglycemia, Hyperketosis, and Impaired Breakdown of Muscle
(A and B) Fatty acid oxidation (A) and blood glucose (B) during fasting (n = 6–12). (C) Liver and gastrocnemius muscle glycogen (n = 5–6). (D) ATT after 24 hr fast (n = 6–10). (E and F) Serum alanine (E, n = 4–5) and serum β-hydroxybutyrate (F, n = 4–6). Data are means ± SEM. (G) Myofiber CSA change of the TA muscle presented as median ± SEM (n = 4–6). * WT versus AMPK-MKO p < 0.05, **p < 0.01, # fed versus fasted p < 0.05, ## p < 0.01, ### p < 0.001. See also Figure S1 and Table S1.
Figure 2
Figure 2. AMPK-MKO Mice Fail to Induce Autophagy Signaling in Response to Fasting
(A) Representative western blots from TA muscle. (B–I)Analysis of western blots for the following proteins: (B) AMPK Thr172 (n = 3–6), (C) ACC Ser212/ACC (n = 6–8), (D) Bnip3 (n = 5–12), (E) S6K Thr389/S6K (n = 5–12), (F) Ulk1 Total (n = 6–8), (G) Ulk1 Ser555 (n = 6–8), (H) Ulk1 Ser555/Ulk1 (n = 6–8), and (I) Ulk1 Ser757/Ulk1 (n = 5–8). Data are means ± SEM. * WT versus AMPK-MKO p < 0.05, **p < 0.01, ***p < 0.001, # fed versus fasted p < 0.05, ### p < 0.001, main effect of genotype p < 0.05, †† p < 0.01, ††† p < 0.001, § main effect of fasting p < 0.05. See also Figure S2 and Table S2.
Figure 3
Figure 3. AMPK-MKO Mice Have Impaired Autophagic Flux in Response to Fasting
(A and B) Photomicrographs (A, scale bar, 10 μm) and quantification (B, n = 3–4) of RFP-LC3 puncta in TA muscle. (C and D) LC3II/LC3I expression in mice treated with sterile water or colchicine (C, n = 9–19) and Parkin expression (D, n = 4–5). (E and F) Photomicrographs of Parkin (scale bar, 10 μm), Tom20, and merged image (scale bar, 1 μm) (n = 3 per group) (E) and quantification in TA muscle (F). Data are means ± SEM. * WT versus AMPK-MKO p < 0.05, **p < 0.01, ***p < 0.001, ### fed versus fasted p < 0.001, Φ Φ Φ fast saline versus fast colchicine p < 0.001.
Figure 4
Figure 4. Aged AMPK-MKO Mice Develop Exacerbated Myopathy and Mitochondrial Disease
(A) Percent of fibers with centrally located nuclei (n = 4–10) and (B) collagen accumulation (n = 3–10) in TA muscle. (C–K) Aged AMPK-MKO and WT mice. (C) Photomicrograph (scale bar, 100 μm) showing large rounded fibers (closed arrow), small angular fibers (open arrow), and necrotic fibers (asterisk) in TA. (D) Maximum isometric and (E) specific force production of TA muscle (n = 4–6). (F) Percent adiposity with representative CT images (n = 4–7). Mitochondrial size in the (G) IMF and (H) SS regions of the myofiber (n = 3 per group). Number of mitochondria in the (I) IMF and (J) SS myofiber regions (n = 3 per group). (K) Representative electronmicrograph showing enlarged mitochondria (closed arrow) (scale bar, 4 μm). (L and M) Number of mtDNA copies (L, n = 6–9) and mtDNA deletions (M, n = 5–10) in quadriceps muscle. (N and O) p62 (N) and Parkin (O, n = 7–11) expression in extensor digitorum longus muscle in fed state. Data are means ± SEM. * WT versus AMPK-MKO p < 0.05, **p < 0.01, ***p < 0.001, # fed versus fasted p < 0.05, ## p < 0.01, ### p < 0.001.

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