Review
doi: 10.2337/db11-1199.
Mapping autophagy on to your metabolic radar
Affiliations
- PMID: 22275084
- PMCID: PMC3266408
- DOI: 10.2337/db11-1199
Item in Clipboard
Review
Mapping autophagy on to your metabolic radar
Diabetes.
2012 Feb.
No abstract available
Figures
Molecular constituents of autophagy. Autophagy requires more than 30 Atg proteins that orchestrate the formation of a de novo limiting membrane, which sequesters cytosolic cargo and then seals upon itself to form an autophagosome. The fusion of autophagosomes to lysosomes leads to cargo degradation and release of nutrients into the cytosol. JNK, Jun NH2-terminal kinase 1; PE, phosphatidylethanolamine; P, phosphorylation of JNK.
Autophagy is regulated by mTOR and AMPK signaling. Nutrient availability and growth factors activate mTOR that phosphorylates ULK1 to inhibit autophagy by trapping the ULK1-FIP200-Atg13 complex in an inactive state. Starvation reduces mTOR activity, which releases its inhibition on autophagy and on DAP1, the activation of which prevents uncontrolled activation of autophagy during starvation. Energy depletion activates AMPK that phosphorylates ULK1 at distinct residues to activate autophagy. Cells may bypass chronic mTOR activation by upregulating sestrins that upregulate autophagy by increasing AMPK activity.
Autophagic degradation of lipid droplets. Autophagy degrades hepatocellular lipid droplets under basal conditions or following an acute exposure to lipids by delivering droplets to lysosomes. A: Breakdown of lipid droplets releases free fatty acids that undergo β-oxidation in the mitochondria. B: Chronic lipid stimulus impairs delivery of lipids to lysosomes and promotes hepatic steatosis.
Hypothetical links between autophagy and insulin resistance. A: Inhibition of autophagy leads to lipid accumulation that promotes hepatic insulin resistance by activating inflammatory signaling pathways and endoplasmic reticulum stress. Excessive lipids, activated nuclear factor-κB, and the hyperactivation of mTOR during obesity inhibit autophagy and further lead to hepatic steatosis and insulin resistance. B: Obesity activates autophagy in the adipose tissue to promote fat accumulation and inflammation, which increases circulating lipids that accumulate in ectopic sites, such as liver and muscle. C: Blocking adipose-selective autophagy switches adipose differentiation into brown adipose-like tissue that increases fat oxidation and improves insulin sensitivity. D: Induction of β-cell autophagy in response to chronic lipid stress promotes β-cell expansion and insulin secretion. Blocking β-cell–selective autophagy results in β-cell injury and reduced insulin secretion. E: Dysregulated skeletal muscle autophagy may occur from excessive lipid accumulation or disturbed Akt and mTOR signaling, which may affect muscle control of glucose homeostasis.
Representative immunoblots for LC3. Steady state LC3 levels in NIH3T3 cells and mouse embryonic fibroblasts (MEF) cultured in serum-supplemented Dulbecco's modified Eagle's medium (Fed) or in response to serum removal for 2 h (Stv). The effect of serum starvation is increased levels of LC3-II (lanes 2 and 4), reflecting increased autophagosome content.
Representative indirect immunofluorescence for LC3. Indirect immunofluorescence for endogenous LC3 in hypothalamic GT1–7 cells cultured in serum-supplemented medium (Fed) or following serum removal for 2 h (Stv). Distinct LC3 puncta (white arrows) are observed in response to serum removal and are in green (fluorescein isothiocyanate). Nuclei are in blue (diaminido phenylindol). (A high-quality digital representation of this figure is available in the online issue.)
Schematic representation of the LC3 flux assay. A: Experimental plan for the LC3 flux assay: Cells cultured in serum-supplemented (Fed) or serum-starved medium (Stv) treated in presence or absence of inhibitors of lysosomal degradation (Inh) for 2 h, following which cell lysates are subjected to immunoblotting for LC3. B: Cartoon depicting immunoblots and densitometry for LC3-II from cells harvested according to plan in A. C: Calculations for determination of net LC3 flux. Densitometric values of samples are subtracted from corresponding inhibitor-treated value, and these represent residual amounts of LC3-II within lysosomes. Higher values correspond to increased autophagic flux.
Methods to modulate autophagy. Autophagosome formation can be blocked by pharmacological agents that inhibit class III PI3K (3MA, wortmannin) or through deletion of autophagy genes (atg5 or atg7). Autophagosome-lysosome fusion can be inhibited by agents that affect microtubule function (vinblastine, nocodazole) or that interfere with lysosomal pH (bafilomycin). Lysosomal degradation is blocked by dissipating lysosomal pH (ammonium chloride, bafilomycin) or by inhibiting lysosomal proteases (leupeptin, pepstatin, E64d). Autophagy may be activated by inhibiting mTOR (rapamycin, torin 1) or through mechanisms independent of mTOR (lithium, trehalose). PE, phosphatidylethanolamine; P-mTOR, phosphorylated mTOR.
References
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- Kiffin R, Bandyopadhyay U, Cuervo AM. Oxidative stress and autophagy. Antioxid Redox Signal 2006;8:152–162 - PubMed
