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S P Bessman

Publications and source records attributed to S P Bessman.

At least 19 recordsLinked to original sources
PubMed1997

Hexokinase binding to mitochondria: a basis for proliferative energy metabolism.

Current thought is that proliferating cells undergo a shift from oxidative to glycolytic metabolism, where the energy requirements of the rapidly dividing cell are provided by ATP from glycolysis. Drawing on the hexokinase-mitochondrial acceptor theory of insulin action, this article presents evidence suggesting that the increased binding of hexokinase to porin on mitochondria of cancer cells not only accelerates glycolysis by providing hexokinase with better access to ATP, but also stimulates the TCA cycle by providing the mitochondrion with ADP that acts as an acceptor for phosphoryl groups. Furthermore, this acceleration of the TCA cycle stimulates protein synthesis via two mechanisms: first, by increasing ATP production, and second, by provision of certain amino acids required for protein synthesis, since the amino acids glutamate, alanine, and aspartate are either reduction products or partially oxidized products of the intermediates of glycolysis and the TCA cycle. The utilization of oxygen in the course of the TCA cycle turnover is relatively diminished even though TCA cycle intermediates are being consumed. With partial oxidation of TCA cycle intermediates into amino acids, there is necessarily a reduction in formation of CO2 from pyruvate, seen as a relative diminution in utilization of oxygen in relation to carbon utilization. This has been assumed to be an inhibition of oxygen uptake and therefore a diminution of TCA cycle activity. Therefore a switch from oxidative metabolism to glycolytic metabolism has been assumed (the Crabtree effect). By stimulating both ATP production and protein synthesis for the rapidly dividing cell, the binding of hexokinase to mitochondrial porin lies at the core of proliferative energy metabolism. This article further reviews literature on the binding of the isozymes of hexokinase to porin, and on the evolution of insulin, proposing that intracellular insulin-like proteins directly bind hexokinase to mitochondrial porin.

Adenosine Triphosphate↗
PubMed1997

Insulin as a probe of mitochondrial metabolism in situ.

Our previous studies of insulin action have led us to the finding that insulin acts specifically on the mitochondrial Krebs cycle to stimulate, by 30%, the oxidation of carbons 2 and 3 of pyruvate to CO2. Insulin also stimulates the oxidation of both carbons of acetate. These carbons can be converted to CO2 only after passing through all of the reactions of the Krebs cycle more than once. Carboxyl groups, such as number 1 of pyruvate, are oxidized to CO2 without any effect of insulin, and can be converted to CO2 by extramitochondrial enzyme. We conclude that insulin must act on the complete intramitochondrial cycle and not on the four enzymes of the Krebs cycle which are present in the cytoplasm. The path taken by those carbons affected by insulin is traced through the complete Krebs cycle, and the necessity for this effect to be mitochondrial has been verified by demonstration of the same specific effect of insulin on the oxidation of the 2 and 3 carbons of succinate. The use of this phenomenon is proposed for the study not only of human diabetes, but of all mitochondrial disorders, by using 14C specifically labeled tracers in culture or biopsy material, or 13C labeled tracer material in vivo.

Animals↗
PubMed1995

Compartmentation and metabolic parameters of mitochondrial hexokinase and creatine kinase depend on the rate of oxidative phosphorylation.

Oxidative phosphorylation in rat heart mitochondria was stimulated by the presence of hexokinase, by simultaneous operation of mitochondrial hexokinase and creatine kinase, or by mitochondrial hexokinase plus exogenously added phosphofructokinase. Under these conditions, 32Pi studies were conducted to estimate the extent of ATP compartmentation in the mitochondria in the vicinity of the active sites of hexokinase and creatine kinase. In all cases studied the extent of ATP compartmentation at 500 microM ATP concentration was no more than 12%. Within the same experimental design, the extent of ATP compartmentation increased with an increase in the rate of oxidative phosphorylation. The degree of ATP compartmentation depended on the relative location of the enzyme and inner mitochondrial membrane: it was maximal in the vicinity of the creatine kinase active sites and minimal for that of phosphofructokinase. The difference in the extent of ATP compartmentation in the neighborhood of the active sites of hexokinase and creatine kinase diminished with an increase in the rate of oxidative phosphorylation. We conclude that there is an ATP concentration gradient in the mitochondrial intermembrane space during oxidative phosphorylation, the minimum concentration being at the surface of the inner membrane. It was found that stimulation of oxidative phosphorylation led to a decrease in the apparent constants, Km (MgATP) and Vmax, for the two enzymes, however, to different degrees. Possible reasons for the change in kinetic parameters of the above enzymes are discussed.

Adenosine Triphosphate↗
PubMed1995

Impaired mitochondrial protein synthesis in streptozotocin diabetic rat hepatocytes.

Diabetes is associated with decreased protein synthesis; however, there have been very few studies on the effects of insulin or diabetes on mitochondrial protein synthesis. We have recently shown that insulin has a direct stimulatory effect on mitochondrial protein synthesis in isolated rat hepatocytes. In this study we demonstrate that mitochondrial protein synthesis is severely curtailed in hepatocytes isolated from diabetic rats and is unresponsive to in vitro insulin addition. Treatment of diabetic rats with insulin for three days partially restored tracer carbon incorporation in mitochondrial proteins. These results suggest that insulin stimulates hepatic mitochondrial protein synthesis which is significantly impaired in diabetes.

Animals↗
PubMed1995

Impaired mitochondrial metabolism and reduced amphibolic Krebs cycle activity in diabetic rat hepatocytes.

Oxidation of [2,3(14)C] and [1,4(14)C] succinate carbons in the mitochondrial Krebs cycle (KC) was used as a probe to investigate the effect of insulin and diabetes on mitochondrial metabolism in isolated rat hepatocytes. The data presented show that mitochondrial oxidation of succinate carbons and their incorporation into protein and lipid was markedly lower in diabetic and insulin treated diabetic rat hepatocytes. Unlike controls, diabetic rat hepatocytes were unresponsive to in vitro insulin addition. Amphibolic channeling of [2,3(14)C] succinate carbons into amino acid fraction was reduced in hepatocytes from diabetic rats, however, more of these carbons were diverted into the gluconeogenesis pathway. These data suggest that the diminished level of anabolic activities in the diabetic rat hepatocytes may be due to impairment in the KC reactions and a subsequent reduction in amphibolic channeling of metabolic intermediates.

Animals↗
PubMed1992

Lack of hormonal stimulation of pyridoxine metabolism in isolated rat hepatocytes.

Isolated hepatocytes obtained from Sprague-Dawley rats (145-175 g) were incubated for 15 min at 30 degrees C in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 0.5 mM concentration of each of the 20 natural amino acids and either 4.5 or 23 microM [U-14C]pyridoxine. Pyridoxine, pyridoxal, pyridoxal phosphate, and pyridoxic acid separated by an anion-exchange chromatographic technique were quantified using a phosphate analyzer and a liquid scintillation counter. The conversion of [U-14C]pyridoxine to its metabolites was more than doubled by increasing the amount of pyridoxine (4.5 to 23 microM) in the incubation medium. Insulin (10 mU/ml), glucagon (1 nM), or epinephrine (10 microM) did not have any significant effect on the conversion of [14C]-pyridoxine to pyridoxal, pyridoxal phosphate, or pyridoxic acid. Our earlier observations of a large decrease in serum pyridoxal phosphate in the diabetic rat cannot be explained by any direct hormonal effects on pyridoxine metabolism.

Animals↗
PubMed1991

Amphibolic role of the Krebs cycle in the insulin-stimulated protein synthesis.

It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14CO2 formation and protein incorporation of several 1-14C-labeled or U-14C-labeled amino acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced 14CO2 formation and protein incorporation primarily of those carbons of amino acids which are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 mM), a potent inhibitor of the transamination reaction, it was shown that there exists an "insulin-sensitive" pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of 14C-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitochondrial transamination of alpha-ketoglutarate to glutamate is essential for insulin stimulation of 14C incorporation into hepatocyte protein.

Alanine↗
PubMed1991

Differential effects of insulin, epinephrine, and glucagon on rat hepatocyte mitochondrial activity.

Oxidation of [2,3-14C]succinate carbons in the mitochondrial Krebs cycle was used as a probe to investigate the effects of insulin, epinephrine, glucagon, and 2,4-dinitrophenol (2,4-DNP) on isolated rat hepatocytes. Epinephrine, glucagon, and 2,4-DNP had a far greater stimulatory effect on 14CO2 formation from [2,3-14C]succinate than insulin. Unlike insulin, epinephrine and glucagon had no significant effect on the anabolic utilization of succinate carbons for protein synthesis. Our results suggest that although epinephrine, glucagon, and 2,4-DNP enhance the movement of tracer carbons through the Krebs cycle, only insulin is capable of enhancing amphibolite utilization for protein synthesis.

2,4-Dinitrophenol↗
PubMed1991

Ammonia inhibits insulin stimulation of the Krebs cycle: further insight into mechanism of hepatic coma.

Oxidation of [2,3-14C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in 14CO2 formation from [2,3-14C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.

Ammonium Chloride↗
PubMed1991

Extracellular phosphate requirement for insulin action on isolated rat hepatocytes.

Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30 degrees in the presence of tracer [2,3-14C]succinate and a 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3-14C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of 32P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state.

Animals↗
PubMed1990

High-performance liquid chromatographic analysis of lung phospholipids and their precursors in the offspring of diabetic rats.

Pulmonary phospholipids and their precursors and metabolites were assayed in the offspring of streptozotocin-induced diabetic rats at 19 and 21 days gestation and at 2 days after birth by two unique methods that employ high performance liquid chromatography combined with automated phosphorus analysis. In general, lung phospholipids were not different between offspring of control versus diabetic mothers. Levels of phosphatidylglycerol, however, were decreased in the offspring of diabetics. Lysophosphatidylcholine appears to be increased in the lungs of offspring of diabetic mothers, suggesting that maternal diabetes is associated with alterations in the remodeling of phosphatidylcholine.

Animals↗
PubMed1989

Lack of insulin stimulation on Percoll-prepared or high-speed-centrifuged rat liver hepatocytes.

Rat liver hepatocytes isolated from a 30-31% percoll density gradient at 10,000g are refractory toward insulin stimulation of 14CO2 formation and 14C-incorporation into protein from [2,3-14C]succinate. Basal hepatocyte oxidation of succinate was not impaired by the presence of 5% percoll in the incubation medium nor was it impaired when percoll-free hepatocytes were used that had been isolated after centrifugation at 9000g; however, in both instances the stimulatory effect of insulin was lost. Hepatocyte damage may have occurred in these processes. This is in contrast to previous work which shows that insulin (10 mU/ml) will stimulate [2,3-14C]succinate oxidation and [2,3-14C]succinate carbon incorporation into protein in non-percoll-treated hepatocytes (isolated by centrifugation at 10g) by about 29%. We conclude that the latter procedure although more time consuming is the more gentle method of choice and leaves the hepatocyte in a form more closely related to an in vivo state than does treatment with a percoll density gradient at 10,000g.

Animals↗
PubMed1988

Insulin effect on [14C]-valine incorporation and its relation to hexokinase activity in developing brain.

Using minced brain cortex from fetal and postnatal rats, we studied the incorporation of [14C]-valine into protein in the presence of insulin. We also assayed the "particle bound" and soluble hexokinase in these tissues. Insulin significantly stimulated the incorporation of [14C]-valine into brain proteins from fetal stage upto 2 days of life. After this period the insulin effect was minimal, with no effect by day 5. The "particle bound" (40,000g pellet) brain hexokinase, on the other hand, remained low till about 2 days of life and then increased to almost adult level by 5 days. Our results show that there is an inverse relation between this anabolic effect of insulin and the "particle bound" hexokinase activity in the cortex of developing rat brain.

Aging↗
PubMed1988

The polysome as a terminal for the creatine phosphate energy shuttle.

The role of the creatine phosphate shuttle in the energetics of muscle protein synthesis in isolated polysomes, from rat hindlimb muscle, was studied. Triton X-100-treated polysomes, following their centrifugation through a 1 M sucrose gradient, contained 38 mU/mg RNA of bound creatine kinase. In the presence of pH 5 enzyme (obtained from rat liver), 0.5 mM ATP, and 1 microM GTP, amino acid (leucine) incorporation by polysomes in the presence of 8 mM creatine phosphate was twice that in the presence of an exogenous ATP regenerating system of 10 mM phospho(enol)pyruvate and 10 U/ml pyruvate kinase. Since added creatine kinase had no effect on incorporation supported by creatine phosphate it is clear that endogenous creatine kinase allows sufficient regeneration of ATP. These data also suggest that nucleoside diphosphokinase must have been associated with the polysome for phosphate was transferred to GTP from [33P]creatine phosphate, and the specific activities of ATP and GTP increased at equal rates, reaching the specific activity of creatine phosphate at 8 min. We conclude that skeletal muscle polysomes have bound creatine kinase activity and they act as terminals for the creatine phosphate energy shuttle. Creatine phosphate regenerates GTP, probably through an intermediate reaction catalyzed by nucleoside diphosphokinase. This provided an added support for the hypothesis of compartmentation of enzymes and substrates and that the transport form of energy between the mitochondria and energy utilizing sites in muscle is creatine phosphate rather than ATP, which extends the general role of the creatine phosphate energy shuttle.

Adenosine Triphosphate↗