Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes.

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of atractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40-50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.

Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids.

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].

Swelling of rat hepatocytes stimulates glycogen synthesis.

In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following observations. 1) The extent of stimulation of glycogen synthesis by both metabolizable and nonmetabolizable amino acids was directly proportional to their ability to increase cell volume, except for proline, which stimulated glycogen synthesis more than could be accounted for by the increase in cell volume. 2) Both cell swelling and stimulation of glycogen synthesis by amino acids were prevented when hepatocytes were incubated in hyperosmotic media containing sucrose or raffinose. 3) Increasing the cell volume by incubating hepatocytes in Na(+)-depleted media in the absence of amino acids also stimulated glycogen synthesis. 4) Stimulation of glycogen synthesis by Na+ depletion was prevented by restoring the normal osmolarity with sucrose, but not with choline chloride which, by itself, stimulated glycogen synthesis and increased the cell volume. It is concluded that stimulation of glycogen synthesis by amino acids is due, at least in part, to an increase in hepatocyte volume resulting from amino acid uptake, and that hepatocyte swelling per se stimulates glycogen synthesis.

A combination of intracellular leucine with either glutamate or aspartate inhibits autophagic proteolysis in isolated rat hepatocytes.

It has been shown previously that the inhibition of autophagic proteolysis in liver by a physiological mixture of amino acids can be mimicked completely by addition of leucine in combination with alanine [Leverve, X. M., Caro, L. H. P., Plomp, P. J. A. M. and Meijer, A. J. (1987) FEBS Lett. 219, 455-458]. We have now further defined conditions which lead to this inhibition. Isolated rat hepatocytes were incubated in the perifusion system in which the cells can be maintained at a steady state in the presence of low amino acid concentrations. Combinations of leucine (0.5 mM) with either alanine, glutamine, asparagine or proline (2 mM) inhibited proteolysis by 40-50%. Under these conditions, both in the absence and presence of the transaminase inhibitor, aminooxyacetate, a correlation was found between the extent of inhibition of proteolysis and the sum of the total intracellular amounts of aspartate and glutamate. Inhibition of proteolysis by leucine and leucine analogues did not correlate with their ability to activate glutamate dehydrogenase.

Energy dependence of different steps in the autophagic-lysosomal pathway.

The energy dependence of the autophagic-lysosomal pathway was investigated in isolated rat hepatocytes, using electroinjected [14C]lactose as an autophagy probe and atractyloside to alter intracellular ATP levels. Since autophagocytosed lactose is hydrolyzed in lysosomes, several steps in the pathway could be analyzed. The following observations were made. 1) The overall autophagic degradation of electroinjected [14C]lactose was strongly energy-dependent. More than 85% inhibition was obtained when the ATP content decreased from the control value of 10 mumol/g dry weight to 4 mumol/g dry weight. 2) The initial step, i.e. the autophagic sequestration of [14C]lactose, measured in the presence of vinblastine to prevent transfer of lactose to lysosomes, was as sensitive to small changes in ATP as was the overall lactose degradation. 3) The steady state level of sequestered [14C]lactose remained constant as ATP decreased from 10 to 4 mumol/g dry weight, indicating that the sequestration step and some postsequestrational process were inhibited to a similar extent by ATP depletion. 4) The final step in the pathway, intralysosomal hydrolysis, was measured by allowing [14C]lactose to preaccumulate intralysosomally in the presence of the reversible lysosome inhibitor propylamine. Following propylamine removal and inhibition of further sequestration by 3-methyladenine, ATP-dependent hydrolysis of the intralysosomal [14C]lactose could be demonstrated. However, this hydrolysis step was not as sensitive to small changes in ATP as was the sequestration step or the overall autophagic lactose degradation. Control of the autophagic-lysosomal pathway in response to energy deprivation would therefore not seem to occur at the lysosomal level, but may be exerted both at the sequestration step and at a postsequestrational, prelysosomal step.

3-Methyladenine, an inhibitor of autophagy, has multiple effects on metabolism.

3-Methyladenine is generally used as an inhibitor of autophagy [P. O. Seglen & P. B. Gordon (1982) Proc. Natl Acad. Sci. USA 79, 1889-1892]. Using isolated hepatocytes, we observed that 3-methyladenine has other effects as well. 1. 3-Methyladenine promoted glycogen breakdown and inhibited flux through phosphofructokinase and pyruvate kinase. These effects proved to be unrelated to inhibition of autophagic proteolysis and were caused by cAMP, which slightly increased in the presence of 3-methyladenine. 2. Addition of 3-methyladenine to intact hepatocytes increased the intralysosomal pH and caused a lower density of the lysosomal population upon centrifugation in a Percoll density gradient. No increase in the intralysosomal pH was effected by 3-methyladenine in isolated lysosomes.

Hepatic autophagy and intracellular ATP. A morphometric study.

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.

Thermodynamics of the control of metabolism.

A theory is presented, describing the control analysis of metabolic systems in terms of Gibbs free energies, extending earlier work of Kacser and Burns (25), and Heinrich and Rapoport (29). It is shown that relationships exist between flux control coefficients (the degree to which enzymes control steady-state fluxes) and free-energy elasticity coefficients, defined as the fractional change in the rate of a reaction induced by a standard change in one free-energy difference, while all the other free-energy differences are kept constant. Application of this extended control analysis to some biochemical reactions, including proton translocation, demonstrates that 1. Problems arising in the control analysis because of conservation (sum concentration of substrate and product constant) can be circumvented. 2. Although free-energy elasticity coefficients are maximal when the reaction is close to equilibrium, they can also be significant when the reaction is not close to equilibrium. 3. Problems in the control analysis caused by compartmentation can be resolved by defining control parameters that refer to the organelle as a whole. 4. These latter control parameters obey the above-mentioned relationships.

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator.

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.

Control of proteolysis in perifused rat hepatocytes.

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and 1-2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.

Energy dependence of autophagic protein degradation in isolated rat hepatocytes.

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. 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. There was a linear relationship between the autophagic sequestration of cytosolic [14C]sucrose and intracellular ATP, the sequestration rate decreasing with decreasing ATP concentrations. ATP depletion did not cause release of [14C]sucrose previously sequestered in autophagosomes and lysosomes at high ATP levels. Intracellular accumulation of chloroquine, used as an indicator of the pH inside lysosomes and other acidic cell compartments, diminished with decreasing cellular ATP content. 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.

Mechanism of the stimulation of respiration by fatty acids in rat liver.

The mechanism of stimulation of hepatic respiration by fatty acids was studied in isolated rat hepatocytes. Stimulation of respiration by fatty acids varied from about 35% to about 105% depending on chain length. The stimulatory effect of octanoate (1 mM) or oleate (0.5 mM) was prevented by oligomycin (2 micrograms/ml). With carboxyatractyloside (100 microM) and ouabain (2 mM) the stimulation of respiration was partially inhibited (by 50-70 and 50-60%, respectively). From these results it can be concluded that the increased rate of respiration after addition of fatty acids is coupled to ATP synthesis. A large part (50-60%) of this ATP is utilized by the (Na+ + K+)-ATPase.

Influence of metabolic end-products on the growth efficiency of Klebsiella aerogenes in anaerobic chemostat culture.

Progressively increasing the input concentration of growth-limiting nutrient (glucose, ammonia, K+) to anaerobic chemostat cultures of Klebsiella aerogenes (D = 0.38 h-1; 35 degrees C; pH 6.8) led to a non-linear increase in bacterial cell concentration. At modest population densities, residual growth-limiting substrate levels increased substantially, with increasing input concentration, and the culture bacterial dry weight tended to a constant value. With the glucose-limited culture, increasing the glucose input concentration above 20 g X 1(-1) led to accumulation of unused glucose and a change in the fermentation pattern. There was a concomitant lowering of the yield value with respect to glucose consumption, and the calculated YATP value similarly declined. Addition of extra essential (non-limiting) nutrients to the culture was without effect. Similarly, addition of individual fermentation products (acetate, ethanol, D-lactate, 2,3-butanediol, succinate) to the feed medium, in varying concentrations and in different combinations, failed to influence the fermentation pattern or the energetics of cell synthesis. However, a clear correlation was observed between the yield values (of both glucose- and K+-limited cultures) and the steady state concentration of CO2 in the effluent gas. Increasing the concentration CO2 either by increasing the population density or lowering the sparging rate of nitrogen gas through the culture, effected a lowering of the yield values. It is suggested that dissolved CO2 exerts an effect on both metabolism and the energetics of cell synthesis. A possible mechanism of energy dissipation (i.e., a futile cycle) involving carboxylation and decarboxylation reactions is proposed.