Sidney Weinhouse

Regulation of glucokinase in liver.

Publisher Summary Glucokinase is a unique glucose-adenosine triphosphate (ATP) phosphotransferase with adaptive and kinetic properties that endow it with special physiological significance in the utilization of glucose by liver. The existence of a hexokinase with unusual properties was foreshadowed by several features of liver physiology. Glucokinase is unstable, and special precautions are necessary to retain activity. The association of glucokinase with major hepatic functions suggests that this isozyme might be localized in parenchymal cells. Glucokinase activity does not drop to as low a level with glucose deprivation as with insulin deprivation. The specific role of insulin in glucokinase induction is implied by striking inhibitory effects of various glycogenolytic hormones and other agents on the glucokinase response to re-feeding of glucose. From a consideration of all the properties of glucokinase, it can be concluded that it is not involved in the physiological utilization of sugars other than glucose, it is not likely to be affected by physiological concentration variations of ATP, adenosine diphosphate (ADP), or glucose 6-phosphate; and it is free of allosteric effects. The major factors that determine its activity appear to be the concentration of glucose and the activity level of the enzyme, modulated by prior dietary history and hormonal status.

Oxidative Metabolism of Neoplastic Tissues

Publisher Summary This chapter discusses that the biochemist concerned with the cancer problem is guided and probably also motivated by the belief that the uncontrolled growth of the cancer cell has its origin in some metabolic or enzymatic peculiarity point of departure from the normal cell, which might provide a rational basis for the control or annihilation of this disease. The invasive growth of cancer cells, depending as it does on a high synthetic capacity, has directed much attention to the mechanisms by which energy is made available for anabolic processes, and since the main source of such energy is the oxidation of fats and carbohydrates, it is in this field that the bio-chemical exploration of the cancer cell has probed most deeply. Many attempts have been made to formulate differences between normal and neoplastic tissues on the basis of differences in metabolism, particularly of glucose. The chapter highlights that the concept of tumor metabolism has been raised, which maintains a fundamental thesis that the neoplastic process is somehow associated with disturbances or peculiarities of oxidative metabolism. It reviews the evidence for this concept and to examine it against our present knowledge of intermediary cell metabolism.

Automated lactic acid determination in serum and tissue extracts

Abstract Methods for assay of lactic acid in tissue extracts or blood specimens through the use of the AutoAnalyzer have been developed. One of these is a direct automation of the color development of the Barker-Summerson procedure. The other involves the use of the enzyme lactic dehydrogenase coupled through diaphorase to the oxidoreduction dye 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyltetrazolium chloride. The colored formazan produced in this reaction is held in suspension through the use of the detergent Triton X-100 (alkyl phenoxypolyethoxyethanol) and is determined colorimetrically at 500 mμ. Forty specimens per hour can be assayed by this method. These methods have been applied to lactic acid assay in blood serum and tissue extracts.

Studies of glycine oxidation in rat tissues

Abstract Using labeled substrates in vitro the oxidation of glycine α- and β-carbons was observed in rat liver and kidney but not in other organs. Glyoxylate α-carbon and formate were identified as intermediates of the oxidation of the glycine α-carbon. The interconversion of glyoxylate and glycolate occurs readily in liver and is catalyzed by DPN-specific lactic dehydrogenase. Glyoxylate was found to be oxidized by xanthine dehydrogenase to oxalate, and to formate and CO 2 by an as yet uncharacterized enzyme in rat liver. Formate is oxidized readily by a variety of rat tissues, and evidence has been presented to indicate this occurs via a peroxidative activity of catalase.

An estimation of pyruvate recycling during gluconeogenesis in the perfused rat liver

Abstract To estimate the degree of recycling of pyruvate during gluconeogenesis, an isotope tracer procedure was employed. Using the isolated, perfused rat liver with pyruvate-2- 14 C in the perfusion fluid, the 3-carbon acids lactate and pyruvate were isolated and the distribution of 14 C in each carbon was assayed. It can be shown that the degree of recycling can be approximated as twice the sum of 14 C in carbons 1 and 3. Glucose, acetoacetate, and β-hydroxybutyrate were also determined, and their 14 C distribution estimated by appropriate degradation procedures. In livers from fasted rats, recycling of pyruvate during 1 hr incubation occurred at a rate of 0.21 μmoles ± 0.02 (SE)/min/g while gluconeogenesis occurred at a rate of 0.49 ± 0.11 μmoles pyruvate-2- 14 C/min/g. In livers from carbohydrate-fed rats, the ratio was reversed, with 0.35 ± 0.06 μmoles pyruvate-2- 14 C recycled and only 0.09 ± 0.03 μmoles converted to glucose. These patterns were not affected by the simultaneous presence of octanoate in the perfusion, during which ketone body production was greatly increased. Only about 20% of the ketone bodies formed were derived from pyruvate, much less with octanoate present, and over 95% of the total radioactivity was in carbons 1 and 3 of acetoacetate as anticipated from the degree of pyruvate recycling. The glucose invariably had 3–4% of its total activity in carbons 3 and 4 and the remainder distributed approximately equally in carbons 1, 2, 5, and 6. The radioactivity in respired CO 2 indicated that about 13–25% of the total O 2 uptake was due to pyruvate oxidation to CO 2 . The data indicate that during the course of liver perfusion a part of the pyruvate substrate is recycled back to pyruvate after conversion to 4-carbon acids. A major portion is recycled directly via the dicarboxylic acid cycle while a minor portion is recycled after traversing the tricarboxylic acid cycle. The extent of recycling was increased greatly by carbohydrate feeding while conversion to glucose was greatly reduced. Recycling may occur via three mechanisms: reversal of pyruvate carboxylase, the malic enzyme, and pyruvate kinase, but it is assumed that, the major path is via pyruvate kinase; and the results point to the possibility that the activity of this enzyme may regulate the rate of glucose production.

Glucose oxidation in rat brain slices and homogenates

With the aid of C14-labeling, various glycolytic and respiratory properties of rat brain preparations were studied. Glucose oxidation as well as glycolysis occurred readily in isotonic whole brain homogenates, fortified with ATP and DPN. Under optimal conditions glucose oxidation accounted for about 60 per cent of the total respiration. Added TPN markedly enhanced the oxidation of glucose carbon-1 but had no effect on the oxidation of other glucose carbons. In short-term experiments conducted over a wide glucose concentration range, glucose oxidation in homogenates was almost maximal at 0.0002 M, whereas in slices, maximal oxidation was reached at 0.001 M. Fructose-U-C14 was readily oxidized by brain slices, but its oxidation was almost completely inhibited in the presence of glucose. Comparison of the radioactivity of lactic acid formed in brain slices and homogenates from glucose-1-C14 and glucose-U-C14 indicated the essential exclusive occurrence of the Embden-Meyerhof process. Although some glucose oxidation was observed with isolated brain mitochondria, it appeared this was due to contaminating cytoplasm, since glucose oxidation was greatly enhanced by the addition of the supernatant fraction.

Phosphorylase: A New Isozyme in Rat Hepatic Tumors and Fetal Liver

A third set of phosphorylase a and b isozymes, distinguishable kinetically and immunologically from liver and muscle forms, is present in various rat hepatomas, and is also present, together with the adult liver form, in fetal rat liver. This is one of several striking examples of suppression of isozymes of adult liver coupled with the appearance of fetal isozymes in hepatomas.

A microcolorimetric assay of inorganic pyrophosphatase

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.

Action of Insulin and Tolbutamide on Blood Glucose Entry and Removal

Although it is generally recognized that the blood sugar of the fasting animal is maintained constant by a precise balance between entry and removal rates, it is only recently, with the advent of C-labeled glucose, that reliable estimations of the rates of these processes have become possible. The availability of tracer methods has also greatly facilitated studies of the mechanism of action of agents which affect the blood sugar level. Such studies, by Searle and Chaikoff, Henderson et al., Wall et al., Berson et al., and Dunn et al. have shed considerable light on the relationships between blood glucose level and its entry and removal rates, and the effects thereon of various hormones. In our own studies, we have been struck by the marked and immediate action of insulin in suppressing the entry of new glucose into the blood of the fasting, normal dog. As a further step in the investigation of this presumably hepatic action of insulin, its effect on the normal and diabetic human has been studied, and its action on the entry and removal rates of blood glucose has been compared with that of the hypoglycemic drug, tolbutamide (Orinase).* These studies are described in the present report.

Estimation of pyruvate recycling during gluconeogenesis in perfused rat liver

Abstract An isotope tracer procedure is described for determination of pyruvate “recycling” during gluconeogenesis from pyruvate, and results are given of its application to a study of gluconeogenesis from pyruvate-2- 14 C in perfused rat liver. After one hour perfusion at 37° C, lactate, pyruvate, and glucose were isolated and radioactivity determined in each carbon atom. Recycling is estimated from the extent of incorporation of 14 C in carbons 1 and 3 of the 3-carbon acids. In livers from fasted rats approximately twice as much pyruvate was converted to glucose as was recycled, whereas in livers from fed rats only about one fourth as much was converted to glucose as was recycled. This pattern was not altered significantly with the simultaneous presence and oxidation of octanoate in the perfused livers. The results suggest a marked inhibition of pyruvate kinase in fasted rat liver as a probable factor in the conversion of pyruvate to glucose.