Energy dependence of autophagic protein degradation in isolated rat hepatocytes

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 ['4C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations.3. ATP depletion did not cause release of ['4C]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.Autophagic degradation of endogenous liver cell protein is an energy-dependent process. This conclusion can be drawn on the basis of experiments with rat liver slices or with isolated rat hepatocytes, showing that proteolysis via autophagy is greatly diminished under conditions where mitochondria1 ATP production is strongly, if not completely, suppressed by high concentrations of inhibitors [l -31 or by replacing oxygen with nitrogen [l, 4, 51. Although these experiments do, indeed, suggest that ATP is required for proteolysis, they do not give any information about the steps in the autophagic pathway that require ATP; nor do they give information on the sensitivity of these steps towards small, more physiological changes in intracellular ATP concentration.The experiments described in this paper were designed to answer these questions. As an experimental system we used isolated rat hepatocytes, incubated under amino-acid-free conditions to ensure maximal rates of autophagic protein degradation [3, 61. MATERIALS AND METHODS Isolation of hepatocytesHepatocytes were isolated from 20-h-starved male Wistar rats (200-280 g) either according to Berry and Friend [7] with the modifications described by Groen et al. [8] or, in the experiments in which autophagic sequestration of intracellular sucrose was measured, by the method of Seglen [9]. Gordon and Seglen [lo, 111: hepatocytes were electropermeabilized with five high-voltage pulses and incubated with [14C]sucrose (5-10 pCi/ml) for 1 h at 0"C, followed by a resealing period of 30 min at 37°C and washing at 0 "C to remove extracellular radioactivity.Autophagic sequestration of the intracellular ['4C]sucrose was measured exactly as described by Gordon et al. [1...

Cell Swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes

“…3F). We have previously shown that proteolysis in hepatocytes is quite sensitive to inhibition by relatively small decreases in intracellular ATP concentration, a 25% fall in intracellular ATP resulting in 20% inhibition of proteolysis [26]. It can be concluded, therefore, that a small part of the inhibition of proteolysis by high concentrations of added amino acids under our experimental conditions was due to the fall in ATP concentration.…”

“…Since autophagic proteolysis is dependent on ATP and sensitive to relatively small changes in intracellular ATP concentration [26], this parameter was also measured as a function of medium osmolarity. The intracellular level of ATP slightly increased with decreasing osmolarity and was unaffected by the presence of amino acids (Fig.…”

Cell swelling inhibits proteolysis in perfused rat liver

“…On the other hand, exposure of perfused liver to hyperosmotic media (from 305 to 385 mosmol kg-') resulted in a 22 +2 % (n = 6) stimulation of branched-chain amino acid (leucine plus valine plus isoleucine) release from 168 + 5 to 203 + 7 nmol/min per g (n = 6), owing to a 15-26% increase in the release of the individual amino acids. Because branched-chain amino acids are neither synthesized nor catabolized in rat liver [16], their release into effluent perfusate of isolated perfused rat liver can be used as an estimate of the proteolytic rate [2][3][4]6,7]. The possibility that the decreased leucine release is explained by an inhibition of leucine transport out of the cell during hypo-osmotic cell swelling is very unlikely, because steady-state release rates are reached, and restoring normo-osmotic perfusion conditions is not followed by an overshoot in leucine release, as one might expect when leucine would accumulate intracellularly during hypo-osmotic cell swelling.…”

“…Amino acids such as glutamine are known to be potent inhibitors of proteolysis in rat liver; however, the underlying mechanism is not understood [1][2][3][4][5][6][7]. Recent studies have indicated that amino acids, even at physiological concentrations [8], elicit liver cell swelling and volume-regulatory ion-flux responses [8-1 1].…”

NAD and axon degeneration: From the Wlds gene to neurochemistry

“…19,20 Defects in ATP synthesis, which might be due to failure in energy homeostasis, occurs at an early stage of necrosis, while a certain level of ATP synthesis can still be maintained until the late stages of apoptosis and autophagy. [21][22][23][24] Results from our lab revealed that axonal ATP levels start to decrease while the AMP level increases at early stages of Wallerian degeneration. 25 These observations further support the idea that mechanisms of axon degeneration might share more similarities with the mechanisms of necrosis than with those of apoptosis or autophagic cell death.…”