Marc E. Tischler

Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats

Abstract Hepatocytes isolated from fed or starved rats were rapidly lysed using the recently described technique of turbulent flow (M. E. Tischler, P. Hecht, and J. R. Williamson, 1977, Arch. Biochem. Biophys. , 181 , 278–292) . Pyridine nucleotide and metabolite contents were measured in the particulate fraction of both whole and disrupted cells after centrifugation through silicone oil. Lactate/pyruvate, β-hydroxybutyrate/acetoacetate, isocitrate/α-ketoglutarate, and malate/pyruvate ratios were determined for calculation of the free NADH NAD + and NADPH NADP + ratios in the cytosol and mitochondria. Lactate/pyruvate ratios measured in the extracellular and cytosolic compartments were in good agreement. Ratios of β-hydroxybutyrate/acetoacetate measured in the extracellular, cytosolic, and mitochondrial compartments also agreed well. Addition of ammonia to fed or starved rat liver cells incubated with lactate, pyruvate, β-hydroxybutyrate, and acetoacetate caused an oxidation of both the NAD and NADP redox states in the mitochondria and cytosol, although the NADP system was oxidized to a greater extent. Calculation of the free NADH and NAD concentrations in the cytosol provided values of about 1 and 400 to 500 μ m , respectively, under control conditions. The concentrations of free NADH and NAD in the mitochondria were considerably higher, being 300 to 400 μ m and 4 to 6 m m , respectively. The free andm bound NAD systems in both the cytosol and mitochondria were more oxidized in the presence of ammonia. NAD and NADP redox potential differences across the mitochondrial membrane (Δ E h ) were not significantly affected by ammonia addition and were generally similar in cells from both fed and starved rats: −52 and −56 mV for the NAD system and −19 to −29 mV for the NADP system. For the NAD system the cytosolic potential was −260 mV in the absence of ammonia and −250 mV in its presence, the mitochondrial values being −315 and −303 mV, respectively. The average cytosolic NADP potential, on the other hand, was −400 mV in the absence and −384 mV in the presence of ammonia. The mitochondrial fractions yielded NADP potentials of −420 mV in the absence of ammonia with both fed and starved rats. Ammonia decreased the mitochondrial NADP potential to −404 mV in fed rats and to −415 mV in starved rats. The calculated free NADH NAD + and NADPH NADP + ratios as well as metabolite concentrations were used to evaluate the mass action ratios of both cytosolic and mitochondrial enzymes. Cytosolic alanine aminotransferase remained near equilibrium in the absence and presence of ammonia, while cytosolic and mitochondrial aspartate aminotransferase reactions deviated up to fivefold. The glutamate dehydrogenase reaction was in near equilibrium with the NAD system, but deviated by three to four orders of magnitude from equilibrium with the NADP system in the direction favoring glutamate synthesis rather than deamination. Cytosolic malate dehydrogenase deviated from equilibrium by about one order of magnitude, while mitochondrial malate dehydrogenase and citrate synthase deviated by two to six orders of magnitude. These data emphasize the importance of regulation of the citric acid cycle at the citrate synthase step.

Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption

Abstract A new technique is described for determining the distribution of metabolites between the cytosol and mitochondria. Rapid lysis of the cell plasma membrane is obtained by forcing isolated liver cells under high pressure through a small diameter needle. The cells, after disruption by the shearing forces generated during the turbulent flow through the needle, are exposed to mitochondrial anion transport inhibitors to prevent efflux of mitochondrial metabolites. Maximal release of cytosolic metabolites was obtained when release of lactate dehydrogenase was greater than 70%, which corresponded with minimal release of mitochondrial enzymes (5–9%). A measured Reynolds number between 7600 and 8000 was indicative of optimal disruption. Mitochondria in the disrupted cells were still functional, as shown by the ability of ADP to stimulate respiration when glutamate plus malate were provided as substrates. Measurement of the subcellular volumes yielded values of 2.0 and 0.2 ml/g dry wt, respectively, for the cytosol and mitochondria. Calculation of the mitochondrial ΔpH (pHin-pHout) in the isolated liver cell based on 22 individual measurements of mitochondria/cytosol gradients for citrate, isocitrate, α-ketoglutarate, malate, glutamate, and pyruvate yielded a value of 0.41 ±0.03. The excellent relationship of these gradients to a common ΔpH lends credence to the technique. Cytosolic and mitochondrial ATP ADP ratios were similar in liver cells isolated from starved and fed rats. Fed rat liver cells, however, had a higher cytosolic adenine nucleotide content (16.8 μmol/g dry wt) than those from starved rats (14.5 μmol/ g dry wt) whereas the mitochondrial content was the same (16 nmol/mg of mitochondrial protein). Data obtained by the disruption technique are compared with other previously published data obtained using either digitonin treatment of isolated hepatocytes or nonaqueous solvent extraction of lyophilized freeze-clamped perfused livers.

Different mechanisms of increased proteolysis in atrophy induced by denervation or unweighting of rat soleus muscle

Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents (eg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which antagonizes sarcoplasmic reticulum release of Ca2+, reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to denervation. Calcium ionophore (A23187) increased proteolysis more so in unweighted than control muscles whether or not Ca2+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (1) atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome, and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2(+)-dependent proteolysis and/or thiol proteases.

Response to trauma of protein, amino acid, and carbohydrate metabolism in injured and uninjured rat skeletal muscles☆

Soft tissue injury to one hindlimb produced trauma in rats without affecting their food intake or weight gain. Histologic examination showed damage to the soleus and gastrocnemius muscles but not to the extensor digitorum longus muscle. The protein content of the injured soleus muscle was lower than that of the contralateral soleus at one day after injury, and was reflected in vitro by a faster rate of protein degradation. The injured soleus also showed greater rates of protein synthesis, glucose uptake, glycolysis, oxidation of glucose, pyruvate, and leucine, and de novo synthesis of alanine. During three days after the injury, urinary nitrogen excretion increased progressively and was paralleled by a faster rate of protein degradation in uninjured muscles incubated with glucose, insulin, and amino acids. In these muscles, the inhibition of protein degradation by insulin diminished, while its stimulation of protein synthesis was unaffected. This insensitivity of proteolysis to insulin in trauma can explain the increased rate of this process. The oxidation of glucose and pyruvate were lower in the diaphragms of traumatized than of normal rats incubated with leucine, while glycolysis and uptake of 2-deoxyglucose did not differ. The degradation of leucine and isoleucine was greater in the diaphragms of traumatized animals and was associated with a faster de novo synthesis of alanine. For the uninjured soleus muscles of the traumatized rats, the slower rates of oxidation of glucose, glycolysis, and uptake of 2-deoxyglucose in the presence of insulin showed an insensitivity of glucose metabolism to this hormone. In contrast, no differences were seen in these various metabolic processes between the extensor digitorum longus muscles of traumatized and normal rats. These data suggest that the response of skeletal muscles to trauma may depend on their physiologic and biochemical characteristics.

Hormonal regulation of protein degradation in skeletal and cardiac muscle.

Abstract A number of hormones produce either anabolic or catabolic effects on protein degradation in muscle. These effects can account for the changes in muscle proteolysis associated with a variety of physiological and pathological states. Thus the balance of hormones within the organism seems to play an important role in the overall regulation of this process. In the fed state, insulin may be the single most important factor maintaining low rates of proteolysis, whereas the catabolic effects of the glucocorticoid hormones in fasting seem to predominate. The proportions of these hormones may be important not only during starvation, but also in trauma and in diseases associated with their altered production and secretion (e.g., diabetes, Cushings syndrome). Hyperthyroidism too causes catabolic effects on muscle proteolysis.

Mechanism of glutamate-aspartate translocation across the mitochondrial inner membrane.

Abstract In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl- p -phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (p K = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline p K , aspartate efflux is inhibited with increased matrix [H + ] due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H + ]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Δ p ) unfavorable to entry of ions with net negative charge as well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H + ] and by addition of an energy source. The glutamate-glutamate exchange, however, showed a slight stimulation by increased [H + ] and was unaffected by the energy state. The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 m m , however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (1–35 °C and 5–25 °C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (1–2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed.

Time Course of the Response of Myofibrillar and Sarcoplasmic Protein Metabolism to Unweighting of the Soleus Muscle

Contributions of altered in vivo protein synthesis and degradation to unweighting atrophy of the soleus muscle in tail-suspended young female rats were analyzed daily for up to 6 days. Specific changes in myofibrillar and sarcoplasmic proteins were also evaluated to assess their contributions to the loss of total protein. Synthesis of myofibrillar and sarcoplasmic proteins was estimated by intramuscular (IM) injection and total protein by intraperitoneal (IP) injection of flooding doses of 3H-phenylalanine. Total protein loss was greatest during the first 3 days following suspension and was a consequence of the loss of myofibrillar rather than sarcoplasmic proteins. However, synthesis of total myofibrillar and sarcoplasmic proteins diminished in parallel beginning in the first 24 hours. Therefore sarcoplasmic proteins must be spared due to a decrease in their degradation. In contrast, myofibrillar protein degradation increased, thus explaining the elevated degradation of the total pool. Following 72 hours of suspension, protein synthesis remained low, but the rate of myofibrillar protein loss diminished, suggesting a slowing of degradation. These various results show (1) acute loss of protein during unweighting atrophy is a consequence of decreased synthesis and increased degradation of myofibrillar proteins, and (2) sarcoplasmic proteins are spared due to slower degradation, likely explaining the sparing of plasma membrane receptors. Based on other published data, we propose that the slowing of atrophy after the initial response may be attributed to an increased effect of insulin.

Relationship of the reduction-oxidation state to protein degradation in skeletal and atrial muscle.

Abstract Changes in proteolysis were correlated with the cell reduction-oxidation state in rat diaphragm and atrium. Protein degradation was measured in the presence of cycloheximide as the linear release of tyrosine into the medium. Intracellular ratios of lactate/pyruvate, total NADH NAD , and malate/pyruvate were used as indicators of the muscle reduction-oxidation state. Incubation of diaphragms with leucine (0.5–2.0 m m ) or its transamination product, sodium α-ketoisocaproate (0.5 m m ), resulted in a lower rate of proteolysis and a higher ratio of lactate/pyruvate and NADH NAD . These effects of leucine could be abolished by inhibiting its transamination with l -cycloserine. Unlike leucine, neither isoleucine nor valine alone produced any change in these parameters. Incubation of diaphragms with glucose (20 m m ) or atria with sodium lactate (2 m m ) produced a diminution of tyrosine release from the muscles and a rise in the ratio of total NADH NAD . Similarly, in incubated diaphragms of fasted rats, the anabolic effects of insulin, epinephrine and isoproterenol on protein degradation were associated with a higher malate/pyruvate ratio. In catabolic states, such as fasting, cortisol treatment of fasted, adrenalectomized rats or traumatization, enhanced muscle proteolysis was observed. Fresh-frozen diaphragms from these rats had both lower lactate/pyruvate and malate/pyruvate ratios than did muscles from control animals. These data show that diminution of proteolysis in diaphragm is accompanied by an increase of the NAD(P)H NAD(P) ratios. In contrast to these findings, chymostatin and leupeptin, which inhibit directly muscle proteinases, caused a decrease of the lactate/pyruvate and malate/pyruvate ratios. These results suggest that protein degradation in diaphragm and atrium is linked to the cellular redox state.

Time course of the response of carbohydrate metabolism to unloading of the soleus

The time course of the response of carbohydrate metabolism to unloading was studied in the soleus muscle of rats subjected to tail-cast suspension. In the fresh soleus, just 12 hours of unloading led to higher concentrations of glycogen and lower activity ratios of both glycogen synthase and glycogen phosphorylase. These changes were still evident on day 3. This initial accumulation of glycogen was likely due to its decreased degradation in response to muscle disuse. Thereafter, the increased glycogen concentration apparently diminished the activity ratio of glycogen synthase, leading to a subsequent fall in the total glycogen content after day 1. After 24 hours of unloading, when no significant atrophy was detectable, there was no differential response to insulin for in vitro glucose metabolism. As reported for day 6 (reference 6), on day 3 the soleus atrophied significantly and displayed a greater sensitivity to insulin for most of these parameters compared to the weight-bearing control muscle. However, insulin sensitivity for glycogen synthesis was unchanged. These results showed that the increased sensitivity to insulin of the unloaded soleus is associated with the degree of muscle atrophy, likely due to an increased insulin binding capacity relative to muscle mass. This study also showed that insulin regulation of glucose uptake and of glycogen synthesis is affected differentially in the unloaded soleus muscle.

Metabolism of amino acids by the atrophied soleus of tail-casted, suspended rats

Amino acid metabolism was investigated in atrophied soleus muscle from rats subjected to six days of tail-cast, hindlimb suspension. The fresh-frozen unloaded muscle showed higher concentrations of tyrosine and glutamate but lower amounts of aspartate, glutamine, ammonia, and a lower ratio of glutamine to glutamate than normal muscle. The atrophied muscle also showed faster in vitro production of alanine and tyrosine, and slower utilization of glutamate and aspartate. Despite a greater activity of glutamine synthetase, synthesis of glutamine was slower in the soleus muscle of suspended rats than in control muscle. Provision of ammonium chloride and/or glutamate showed that this slower synthesis of glutamine in the atrophied soleus probably was due to limiting amounts of free ammonia and not of glutamate. Flux through AMP deaminase was probably slower as demonstrated by the maintenance of a greater pool of total adenine nucleotides and by the slower release of nucleosides by the incubated soleus muscle of suspended v control rats. The extensor digitorum longus muscles of suspended animals showed greater glutamine production, glutamine synthetase activity, and aspartate utilization than control muscles. Data from muscles of intact, adrenalectomized and adrenalectomized, cortisol-treated rats suggested that the greater glutamine synthetase activity was mediated possibly by higher circulating glucocorticoid hormones and a greater response of the soleus muscle to these hormones. Glutamine synthesis in skeletal muscle may be regulated primarily by the availability of ammonia, which is associated with the degradation of adenine nucleotides, and secondarily by the amount of glutamine synthetase and glutamate in the tissue.