High Km glucose-phosphorylating (glucokinase) activities in a range of tumor cell lines and inhibition of rates of tumor growth by the specific enzyme inhibitor mannoheptulose.

Differences in modes of control of glycolysis in tumor cells, compared with normal cells, have suggested that phosphofructokinase may not catalyse the rate-controlling step. Instead, hexokinase activity may assume a more important regulatory role. Hexokinase activities are consistently lower than those of phosphofructokinase in tumor cells, and the former enzyme may be saturated with its substrate (M. Board et al., Biochem. J. 265: 503-509, 1990). The present work has focused on the glucose-phosphorylation step in tumor cell glycolysis. A range of eight human tumor cell-lines, one human tumor tissue, and four rat tumor cell lines were found to have an additional glucose-phosphorylating activity, with properties similar to hepatic glucokinase. Maximal activities range from 1.1-20 nmol/min/mg cell protein, and the activity is consistently absent from any untransformed cell line or tissue tested, except rat liver tissue (18 nmol/min/mg cell protein). Tumor cell glucokinase activity has been characterized by its high Km for glucose (8-11.8 mM); inhibition by the specific glucokinase inhibitor, mannoheptulose (I50, 12.5 mM); and lack of inhibition by 10 mM glucose-6-phosphate. Mannoheptulose also causes inhibition of glucose uptake by tumor cells (25-75% at 30 mM mannoheptulose) and inhibition of rates of growth of cultured tumor cell lines (I50, 21.4 mM). Rates of growth of human tumors in experimental animals are dramatically reduced (by 65-79%) by a dose of 1.7 mg/g mannoheptulose daily for 5 days. The potential of the naturally occurring sugar, mannoheptulose (which is purified from avocados and is assumed to be of low toxicity), as a cancer treatment is discussed.

The transport of glutamine into rat mesenteric lymphocytes.

The transport of glutamine into isolated rat mesenteric lymphocytes was studied. This transport appears to be dependent upon the Na+ gradient. The Km for glutamine transport was about 1.0 mM. A large number of amino acids were shown to inhibit the rate of transport of both serine and glutamine into lymphocytes. The transport of glutamine was competitively inhibited by serine and that for serine was similarly inhibited by glutamine. In contrast, histidine and 2-(methylamino)isobutyrate inhibited the transport of both serine and glutamine noncompetitively. It is concluded that glutamine is transported into rat mesenteric lymphocytes by a process similar to System ASC described for other cells.

Distribution of adenosine metabolising enzymes between adipocyte and stromal-vascular cells of adipose tissue.

Rat adipose tissue was digested with collagenase and separated into adipocytes and stromal-vascular cells. The adipocytes accounted for 40% of the total adipose tissue adenosine deaminase activity, 32% of 5'-nucleotidase activity and 87% of adenosine kinase activity. This distribution suggests that adipocyte are the major cell type involved in adenosine utilization in adipose tissue. Furthermore, it suggests that the high sensitivity of isolated adipocytes to adenosine is representative of their sensitivity of isolated adipocytes to adenosine is representative of their sensitivity in vivo.

The effect of fructose 2,6-bisphosphate on muscle fructose-1,6-bisphosphatase activity.

Rat and rabbit muscle fructose 1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) are inhibited by fructose 2,6-bisphosphate. In contrast with the liver isozyme, the inhibition of muscle fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is not synergistic with that of AMP. Activation of fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate has been observed at high concentrations of substrate. An attempt is made to correlate changes in concentrations of hexose monophosphate, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate with changes in fluxes through 6-phosphofructokinase and fructose-1,6-bisphosphatase in isolated epitrochlearis muscle challenged with insulin and adrenaline.

Influence of cell culture conditions on diet-induced changes in lymphocyte fatty acid composition.

The effect of a range of dietary lipids on the fatty acid composition and membrane fluidity of lymphocytes was investigated. The effects of subsequent culture of these lymphocytes in medium containing autologous serum, foetal calf serum or a serum-free supplement were assessed; this was considered important, since many studies investigating the effects of dietary lipid manipulation on immune function have used protocols involving a variety of cell culture conditions when performing tests of immune function. Weanling Lewis rats were fed for 10 weeks on a low-fat (LF; 20 g/kg) diet or on high fat diets containing 200 g/kg of either hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO), evening primrose oil (EPO) or menhaden oil (MO). The fatty acid composition of the phospholipid fractions of lymphocytes from the spleen was altered by dietary lipid manipulation, whereas the fatty acid composition of thymic lymphocytes was not modified significantly. In general, the changes in the fatty acid composition of spleen lymphocytes reflected the fatty acid composition of the diets themselves. Despite the considerable changes in the fatty acid composition of lymphocytes from spleen, dietary lipid manipulation had no effect on the plasma membrane fluidity of these cells. Culturing lymphocytes in autologous serum allowed some, but not all, of the diet-induced changes in fatty acid composition to be maintained. The effects of dietary lipid manipulation were totally reversed when lymphocytes were cultured in FCS. Culturing lymphocytes in serum-free medium not only reversed any effects of dietary manipulation, but also markedly increased the appearance of palmitoleic and oleic acids, at the expense of palmitic and stearic acids, suggesting activation of the delta 9 desaturase when these cells were cultured in the absence of exogenous lipid. This study suggests that cell culture conditions have significant influence on the changes in lymphocyte fatty acid composition brought about by dietary lipid manipulation and may therefore influence the outcome of functional tests.

Effects of varying the type of saturated fatty acid in the rat diet upon serum lipid levels and spleen lymphocyte functions.

To obtain further information about the effects of specific dietary saturated fatty acids, weanling male rats were fed for 6 weeks on low fat (7.7% by weight) or high fat (17.8% by weight) diets which differed according to the principal fatty acids present. The diets were rich in caprylic and capric acids (medium chain triacylglycerols; MCT), lauric acid, palmitic acid at the sn-1(3) position, palmitic acid at the sn-2 position or stearic acid. The total proportions of saturated (42-46%), monounsaturated (36%), n-6 polyunsaturated (15%) and n-3 polyunsaturated (2.2%) fatty acids were the same in all diets. Serum cholesterol concentrations were not different among rats fed the different diets, except that the concentration in the serum of rats fed the high fat diet with palmitic acid in the sn-2 position was high. This was reflected in higher HDL and LDL cholesterol concentrations in the serum of animals fed this diet. Triacylglycerol (TAG) concentrations tended to be higher in the serum of rats fed the low fat diets compared with those fed the high fat diets. They were lowest in the serum of MCT-fed rats irrespective of the level of fat in the diet and were highest in the serum of rats fed the low fat diet rich in stearic acid. These differences were due to lower chylomicron and VLDL TAG concentrations in the serum of MCT-fed rats and higher chylomicron and VLDL TAG concentrations in the serum of low fat, stearic acid-fed animals. The fatty acid compositions of the serum and of spleen lymphocytes were influenced by that of the diet fed. The ex vivo proliferation of lymphocytes from the spleens of rats fed the high fat diet rich in palmitic acid at the sn-2 position was greater than that of lymphocytes from animals fed the other diets. Natural killer (NK) cell activity tended to be lower for spleen lymphocytes from rats fed high fat diets than for those fed low fat diets irrespective of the principal saturated fatty acid present. NK cell activity was highest for spleen lymphocytes from animals fed the diets rich in palmitic acid and was lowest for those from animals fed the high fat diet rich in stearic acid. Spleen lymphocytes from the latter animals had the lowest proportion of CD16+ cells, a marker for NK cells. Thus, this study shows that the type of saturated fatty acid present in the diet not only has subtle effects upon blood lipid and lipoprotein levels but can significantly affect lymphocyte functions. Spleen lymphocyte NK cell activity is decreased as the fat content of the diet increases. NK cell number and activity are reduced by a high fat diet rich in stearic acid. Spleen lymphocyte proliferation is enhanced by palmitic acid-rich diets, particularly if palmitic acid is in the sn-2 position of dietary TAG.

Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria).

A procedure is described for the partial purification of pyruvate carboxylase (pyruvate:CO2 ligase (ADP-forming), EC 6.4.1.1) from the flight muscle of the locust (Schistocerca gregaria). Characterisation of the kinetic properties of this enzyme indicates that it is activated by acetyl-CoA, is insensitive to inhibition by di- and tricarboxylic acids and exhibits an apparent Km for HCO3-(16 mM) which differs by an order of magnitude from that observed for other pyruvate carboxylases. It is suggested that activation of this locust flight muscle pyruvate carboxylase during the rest leads to flight transition may result from increases in the concentrations of pyruvate and HCO3- under these conditions.

Recent developments in metabolism that impinge on research into the nature and treatment of diabetes mellitus.

It has been established that adenosine, its agonists, or antagonists can cause dramatic changes in insulin sensitivity in isolated soleus muscle and, moreover, can modify changes in sensitivity caused by pathophysiological conditions. Addition of adenosine deaminase to the incubation medium, which is known to lower the concentration of adenosine, increases the sensitivity of glycolysis to insulin. Addition of an adenosine-receptor agonist decreases sensitivity by about 10-fold, whereas addition of an adenosine-receptor antagonist increases sensitivity by about 10-fold. The latter totally removes the resistance of glucose utilization to insulin in the isolated soleus muscle obtained from either the genetically obese rat or from the rat fed a high sucrose diet. These findings strongly support the view that changes in insulin sensitivity in muscle can be brought about either by acute changes in the local concentration of adenosine or in the affinity or number of receptors for adenosine in muscle. However, in many of the conditions investigated, in which insulin sensitivity in muscle is changed, there was no correlation between the change in the adenosine content of the muscle and altered insulin sensitivity. It has also been shown that prostaglandin E1 can increase dramatically the sensitivity of glycolysis to insulin and that this is a specific effect of prostaglandins of the E series. It is not produced by prostacyclins, thromboxanes, or leukotrienes. It is unclear if there is a relationship between the effects of adenosine and prostaglandins. Chronic elevation of catecholamines may increase the sensitivity of glucose utilization to insulin and also increase the rate of thermogenesis by substrate cycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of a range of dietary lipids upon serum and tissue lipid composition in the rat.

Since the type of fat consumed in the diet may play a role in the development of several disorders, it is important to ascertain the effects of different dietary fats upon parameters such as serum lipid levels and adipose deposition. The aim of this study was to determine the effects of feeding rats a range of fats with differing fatty acid compositions. Weanling male rats were fed for 10 weeks on a low fat (LF) diet or on one of five high fat diets, which contained 20% by weight of either hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO), evening primrose oil (EPO) or menhaden (fish) oil (MO). Food intake, animal growth, tissue weights at sacrifice, serum and liver lipid concentrations and serum, heart, brain and adipose tissue fatty acid compositions were studied. The food intake of the LF-fed animals was greater than that of animals fed on the high fat diets; there were no differences in food intake between animals fed the high fat diets. The total energy intake was lower for animals fed on the HCO diet than for those fed on the LF, OO, EPO or MO diets; there were no other differences in energy intake between the groups. Animals fed the different diets had almost identical rates of weight gain up to 5 weeks; after this period of rapid growth, the increase in weight was slower in all groups but especially in the LF-fed animals. The LF-fed rats had a lower total weight gain and smaller final weights than rats fed on the high fat diets. Animals fed on the MO diet had a greater weight gain than those fed on the OO or EPO diets and their final weights were greater. The MO diet resulted in greatly increased liver weight compared with each of the other diets. The HCO, OO and EPO diets also increased liver weight compared with the LF diet. The total lipid content of the livers from rats fed the high fat diets was greater than that of those from rats fed the LF diet; the livers from animals fed the MO diet contained more lipid than those from animals fed each of the other diets. MO feeding increased the free cholesterol, cholesterol ester and triacylglycerol contents of the liver.(ABSTRACT TRUNCATED AT 400 WORDS)

A method for testing the stability of a steady-state system during the calculation of a response to large changes in regulator concentrations.

A modification of the 'finite decomposition' method (Crabtree and Newsholme (1985) Curr. Top. Cell. Regul. 25, 21-76) for calculating physiological responses from sensitivities is described, to enable the system to be tested for stability at each step of the procedure. Instability is indicated by a change of sign of the determinant of the square matrix (N) in the governing equation for the system. The method cannot be used to predict a change of sign of the determinant of the square matrix (N) in the governing equation for the system. The method cannot be used to predict responses beyond any step at which instability occurs.

The maximum capacity of glycolysis in brown adipose tissue and its relationship to control of the blood glucose concentration.

The maximum activity of the key glycolytic enzymes, hexokinase and 6-phosphofructokinase, was measured in tissues of control and cold-acclimated rats. The only significant change in activity was seen in brown adipose tissue where the activity of these enzymes was increased 2-fold. This increase in glycolytic capacity along with the hypertrophy of BAT observed in cold acclimation suggests that this tissue could play an important role in glucose utilisation by the rat.

Maximal activities of glutaminase, citrate synthase, hexokinase, phosphofructokinase and lactate dehydrogenase in skin of rats and mice at different ages.

The activities of key glycolytic enzymes are, in general, similar at the three different ages of the animals used in this work (very young, adult and old). Glutaminase is present in skin of both mice and rats, but the activity was much lower in adult animals compared to the very young or the old. It is suggested that this activity is important for the provision of nitrogen for the de novo synthesis of purine and pyrimidine nucleotides during the growth of skin in the young animal and for DNA repair in the old animals; it might be important in the adult skin in response to wound healing.

Effect of adenosine deaminase and an adenosine analogue on insulin sensitivity in soleus muscle of the rat.

The concentration of insulin that produces half-maximal stimulation of glycolysis in stripped soleus muscle preparations is decreased from approximately 100-10 muunits/ml by the presence of adenosine deaminase in the incubation medium. This suggests that adenosine decreases insulin sensitivity. The effect of the deaminase is abolished by addition of the adenosine analogue, N6-phenylisopropyladenosine which is not metabolised by the deaminase. The effect of the deaminase in isolated soleus muscle is similar to that of a period of physical training of the rat.

Maximal activities of enzymes involved in adenosine metabolism in muscle and adipose tissue of rats under conditions of variations in insulin sensitivity.

The maximal activities of 5'-nucleotidase, adenosine deaminase and adenosine kinase were measured in quadriceps or soleus muscle from animals in which the sensitivity to insulin was changed. Most conditions caused no effect on the activities but exercise-training increased the activity of adenosine deaminase and cold exposure increased the activity of 5'-nucleotidase in soleus muscle: in addition, ageing decreased markedly the activities of all three enzymes in both muscles. When the activities are based on mg protein they are much higher in both white and brown adipose tissue than in muscle, suggesting that changes in adenosine concentration may be important in changing insulin sensitivity in adipose tissue whereas changes in adenosine receptor number may be more important in muscle.

Maximum activities of some key enzymes of glycolysis, glutaminolysis, Krebs cycle and fatty acid utilization in bovine pulmonary endothelial cells.

Despite the importance of endothelial cells little is known about their metabolic fuel requirements. To provide some information in this area, the maximum catalytic activities of key enzymes of important metabolic pathways have been measured in bovine pulmonary endothelial cells. The results suggest that both glucose and glutamine are important fuels for these cells: in addition, the oxidation of fatty acids may also be of quantitative significance. The activity of glutaminase in these cells was about 20-fold higher than that in lymphocyte, a cell which exhibits high rates of glutaminolysis. It is suggested that a high rate of glutamine metabolism by endothelial cells is important not only for energy provision but also for provision of nitrogen for biosynthetic purposes including production of local messengers.

The effect of insulin and noradrenaline on the uptake of 2-[1-14C]deoxyglucose in vivo by brown adipose tissue and other glucose-utilising tissues of the mouse.

The uptake of 2-[1-14C]deoxyglucose in vivo by brown adipose tissue was greater than that of brain or heart in control mice on a whole tissue basis. When mice were treated with noradrenaline the rate of uptake of 2-[1-14C]deoxyglucose by brown adipose tissue was increased 6-fold with the only other change occurring in heart where a 5-fold increase was observed. After administration of insulin the uptake of 2-[1-14C]deoxyglucose in vivo was increased in heart, brown adipose tissue, white adipose tissue and muscle. The amount of 2-[1-14C]deoxyglucose taken up by brown adipose tissue compared to other tissues and the changes in this uptake after administration of noradrenaline or insulin suggest that brown adipose tissue is capable of playing a quantitatively important role in glucose removal from the blood.

Effects of analogues of adenosine and methyl xanthines on insulin sensitivity in soleus muscle of the rat.

The concentration of insulin that produces half-maximal stimulation of glycolysis by stripped soleus muscle preparations is markedly increased by the adenosine analogues, 2-chloroadenosine and N6-phenylisopropyladenosine, but is markedly decreased by the methyl xanthine analogue, 8-phenyltheophylline. 2-Chloroadenosine increases the concentration of insulin required to stimulate glycolysis half maximally, from about 100 to 2000 mu units/ml. 8-Phenyltheophylline decreases this concentration of insulin from about 100 to 10 mu units/ml, an effect which is similar to that produced either by addition of adenosine deaminase to the medium or to exercise-training of the donor animals for 4 weeks.

Increased insulin sensitivity in soleus muscle from cold-exposed rats: reversal by an adenosine-receptor agonist.

The effect of 0.5, 2, 7 and 14 days cold exposure at 4 degrees C on insulin sensitivity was investigated in the stripped soleus muscle preparation incubated in vitro. Cold-exposure for 2 or 7 days increased the sensitivity of glycolysis, but did not affect the sensitivity of glycogen synthesis to insulin. Cold-exposure for 0.5 or 14 days had no effect on the sensitivity of either process to insulin. The increased sensitivity to insulin after exposure of animals to the cold for 2 days was completely reversed by addition of the adenosine receptor agonist, 2-chloroadenosine, to the incubation medium. This suggests that cold exposure may increase insulin sensitivity in the muscle, either by a decrease in the concentration of adenosine in the muscle, or by a decrease in the number or affinity of the adenosine receptors.

Substrate cycles: their metabolic, energetic and thermic consequences in man.

The role of substrate cycles in metabolic control was first indicated over ten years ago, but the recent interest in such cycles has developed from the direct demonstration by isotopic techniques of their existence in various tissues. I propose that substrate cycles form part of a logical series of biochemical mechanisms that exist to increase the sensitivity of non-equilibrium reactions to changes in concentrations of metabolic regulators. The possible importance of such cycles for provision of precise metabolic regulation in the tissues of the normal subject and the trained athlete is proposed. Furthermore, cycling may provide a mechanism by which hormones can change the magnitude of response in a tissue to a given metabolic signal, without interfering in the biochemistry of the basic control mechanism. It is, however, possible to extend the role of cycling to heat generation and thus to controlled energy loss by an organism. Heat generation by substrate cycles may be important as an acute mechanism for maintaining the body temperature in man in response to a sudden decrease in the environmental temperature; alcoholic hypothermia would be explained by inhibition of substrate cycling in the liver, and accidental hypothermia in the elderly could be explained by decreased capacity of substrate cycles with age. If heat generated by the cycles is rapidly lost to the environment, the expenditure of energy to maintain this heat loss could explain, in part, the physiological phenomena of the thermic response to food and the oxygen debt which is always observed after exercise. Finally, the energy expended in these ways could be part of a general biochemical mechanism for maintenance of the correct body weight; a decrease in the capacity of substrate cycles might be one factor involved in the development of obesity.