Energy dependence of autophagic protein degradation in isolated rat hepatocytes

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Abstract
The effect of small changes in intracellular ATP on autophagic flux was studied in isolated rat hepatocytes by using inhibitors of ATP production or by varying the metabolic conditions. The following observations were made. 1. There was a linear relationship between endogenous protein degradation and intracellular ATP, the rate of proteolysis declining with decreasing ATP concentrations. 15% of the maximal proteolysis is either independent of ATP or has a very high affinity for this metabolite. 2. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. 3. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. 4. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. 5. Amino acids inhibited proteolysis without affecting ATP levels or chloroquine accumulation. We conclude from the high sensitivity of autophagy towards relatively small changes in the concentration of intracellular ATP that, besides amino acids, ATP is a very important factor in controlling the rate of autophagy in rat hepatocytes.