Stimulation of rat-kidney hexose monophosphate shunt dehydrogenase activity by chronic metabolic acidosis

The effect of chronic metabolic acidosis on the kinetic behaviour of renal glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been investigated. Acidosis induced a significant increase in both enzyme activities at all substrate concentrations used. Saturation curves of both dehydrogenases were hyperbolic with no evidence of sigmoidicity. Maximum activities were found after 7 days of acidosis with no significant change in the Km values. The results suggest that stimulated renal hexose monophosphate dehydrogenases activities are probably due to an increased intracellular concentration of these enzymes. The relationship between these changes and those generated in the metabolic acidosis are also discussed.     

Roles of cAMP and free fatty acids on the activity of the hexose monophosphate shunt

Summary Basal glucose utilization by isolated rat adipocytes have been found to be increased ten times in the presence of certain preparations of albumin. In these conditions the effects of several adrenergic agonists and related compounds on glucose oxidation, lipolysis and triacylglycerol synthesis in isolated fat cells have been studied. Oxidation of D(1-¹⁴C) glucose in rat adipocytes was almost completely inhibited by norepinephrine and isoproterenol when added to incubated fat cells. Agents able to modify intracellular AMP cyclic levels by different mechanisms display a similar ability to imitate the effect of lipolytic agents. The inhibition of glucose oxidation due to norepinephrine and isoproterenol is partially reverted by propanolol. Under the same conditions in which norepinephrine and isoproterenol markedly reduced glucose conversion to ¹⁴CO₂, they stimulated lipolysis and triacylglycerol synthesis and in this case propanolol also reverted those actions. However, in these experimental conditions, norepinephrine and isoproterenol did not raise CAMP levels 10 min after hormone addition.It is concluded from these data that glucose oxidation through hexose monophosphate shunt, activation of lipolysis and triacylglycerol synthesis in isolated rat fat cells by lipolytic agents occurs by a mechanism(s) that depend(s) on intracellular free fatty acids levels.     

Dynamic assessment of hexose monophosphate shunt activity in the intact rabbit lens by proton NMR spectroscopy

A proton nuclear magnetic resonance technique is demonstrated for ascertaining the real-time contribution of the hexose monophosphate shunt to glucose metabolism in the intact incubated rabbit lens. This measurement requires incubation of the tissue in medium supplemented with [1-13C]glucose, and depends on the presence of the 13C label in the methyl position of lactate which creates satellite resonances by way of 13C - 1H spin-spin scalar coupling. The assumptions required to make the measurement are presented. For lenses maintained under control conditions, a basal level corresponding to 5% hexose monophosphate shunt activity was determined. An eight-fold increase in activity was observed under conditions known to stimulate the shunt.     

Interrelations between glycolysis and the hexose monophosphate shunt in erythrocytes as studied on the basis of a mathematical model

A mathematical model is presented which comprises the reactions of glycolysis, the hexose monophosphate shunt (HMS) and the glutathione system in erythrocytes. The model is used to calculate stationary and time-dependent metabolic states of the cell in vitro and in vivo. The model properly accounts for the following metabolic features observed in vitro: (a) stimulation of the oxidative pentose pathway after addition of pyruvate due to a NADP-dependent lactate dehydrogenase as coupling enzyme between glycolysis and the oxidative pentose pathway, (b) relative share of the oxidative pentose pathway in the total consumption of glucose amounting to approximately 10% in the normal case and to approximately 90% under conditions of oxidative stress excreted by methylene blue. From the application of the model to in vivo conditions it is predicted that (c) under normal conditions glycolysis and the HMS are independently regulated by the energetic and oxidative load, respectively, (d) under conditions of enhanced energetic or oxidative load both glycolysis and the HMS are mainly controlled by the hexokinase; in this situation the highest possible values of the energetic and oxidative load which are compatible with cell integrity are strongly coupled and considerably restricted in comparison with the normal case, (e) the stationary states possess bifurcation points at high and low values of the energetic load.     

Regulation and rate of the hexose monophosphate shunt in Rana ridibunda erythrocytes

1. Resting rates of Rana ridibunda erythrocyte glucose consumption and 14CO2 production from 1-14C-glucose were found to be significantly lower than the respective values in human erythrocytes. 2. In the presence of 1-14C-glucose Methylene Blue stimulated 14CO2 production 7-fold, while in the presence of 6-14C-glucose Methylene Blue stimulated 14CO2 production 1.2-fold. 3. The Km of G-6-PD for G-6-P and NADP were 29 and 12 microM, respectively while the Km of 6-PGD for 6-PG and NADP were 83 and 32 microM, respectively. The Ki of G-6-PD and 6-PGD for NADPH were 80 and 12 microM, respectively. 4. Excess amounts of NADP resulted in a significant decrease of 14CO2 production from 1-14C-glucose in total haemolysates. 5. ATP, ADP and fructose diphosphate inhibited both G-6-PD and 6-PGD, the latter being more sensitive than G-6-PD to their inhibitory effect, 2,3-DPG and reduced and oxidized glutathione showed a marked inhibitory effect on 6-PGD, while the phosphorylated trioses inhibited only G-6-PD. 6. Physiological concentrations of oxidized glutathione decreased the inhibition exercised by NADPH on G-6-PD. 7. The possible role of the two dehydrogenases in the regulation of the HMS is discussed.     

Quinone induced stimulation of hexose monophosphate shunt activity in the guinea pig lens: role of zeta-crystallin.

The response of the hexose monophosphate shunt (HMS) in organ-cultured guinea pig lens to 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone (juglone) has been investigated. Both these compounds, which are substrates of guinea pig lens zeta-crystallin (NADPH:quinone oxidoreductase), were found to cause increases in the rate of 14CO2 production from 1-14C-labelled glucose. Exposure of lenses to 15 microM 1,2-naphthoquinone or 20 microM juglone yielded 5.9- and 7-fold stimulation of HMS activity, respectively. Unlike hydrogen peroxide-induced stimulation of HMS activity, these effects were not abolished by preincubation with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1 nitrosourea (BCNU). While hydrogen peroxide produced substantial decrements in lens glutathione (GSH) levels, incubation with quinones was not associated with a similar reduction in GSH concentration. Protein-bound NADPH content in quinone-exposed guinea pig lenses was decreased, with a concomitant increase in the amounts of free NADP+. This finding supported the involvement of zeta-crystallin bound NADPH in the in vivo enzymic reduction of quinones. Hydrogen peroxide, on the other hand, caused decreases in the level of free NADPH alone, serving to confirm our earlier inference that quinone stimulated increases in the guinea pig lens HMS could be mediated through zeta-crystallin NADPH:quinone oxidoreductase activity.     

Glucose metabolism and the hexose monophosphate shunt in clones of hamster cells.

A Chinese hamster ovary cell line has been isolated in cell culture which exhibited resistance to the cytotoxic effects of fluorocitrate. Unlike previously reported fluorocitrate-resistant lines this variant does not contain an altered aconitase activity. Instead it was found that the variant cells contained a more active than wild type hexose monophosphate shunt. It is postulated that this higher than normal activity in the variant cells is important in overcoming the partial fluorocitrate block of the tricarboxylic acid cycle that occurs when the resistant cells are grown in the presence of the drug.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

The effect of hexose monophosphate shunt inhibitors on adenohypophyseal and ceruloplasmin reactions to oestrogen.

Oestradiol benzoate, as an aqueous microcrystal suspension, was administered i.m. to rats in doses of 1 mg twice a week; it induced adenohypophyseal hyperplasia and an increase of the thyroxine-binding capacity of the adenohypophyseal proteins in vitro and raised the blood ceruloplasmin level. The simultaneous administration of a hexose monophosphate shunt inhibitor--6-aminonicotinamide (200 microgram/rat/day in food) or oxythiamine (8 mg/rat/day in food)--did not modify the reaction of the adenohypophysis; the hexose monophosphate shunt thus probably does not play a significant role in the adenohypophyseal reaction to oestrogens. By themselves, both inhibitors raised the blood ceruloplasmin level and their effect summated with that of oestradiol. The mechanism of action of the inhibitors is not known, but a nonspecific stress effect leading to an increase in the ceruloplasmin level as an "acute phase protein" is considered to be the most likely.     

Long-term adaptive response to dietary protein of hexose monophosphate shunt dehydrogenases in rat kidney tubules

We have studied the effects of several different macronutrients on the kinetic behaviour of rat renal glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Rats were meal-fed with high-carbohydrate/low-protein, high-protein/low-carbohydrate and high-fat diets. High-protein increased renal G6PDH and 6PDGH activities by 66 per cent and 70 per cent respectively, without significantly changing the Km values of either and each Hexose monophosphate dehydrogenase activity increased steadily, reaching a significant difference on day 4. A rise in carbohydrate or fat in the diets, produced no significant change in either the activity or the kinetic parameters, Vmax and Km of the two dehydrogenases. In addition, the administration of a high-protein diet for 8 days significantly increased both the pentose phosphate pathway flux (92.6 per cent) and the kidney weigth (35 per cent), whereas no significant changes in these parameters were found when the animals were treated with the other diets. Our results suggest that an increase in the levels of dietary protein induces a rise in the intracellular levels of these enzymes. The possible role of this metabolic pathway in the kidneys under these nutritional conditions is also discussed.     

An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion.

When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.     

Establishment of a human preadipose cell line,HPB-AML-I: refractory to PPARgamma-mediated adipogenic stimulation

In this study, we established a unique cell line, HPB-AML-I (AML-I), from peripheral blood mononuclear cells collected from a patient with acute myeloid leukemia (AML: M1). Morphological and phenotypical analyses of the established AML-I cells demonstrated that they belong to a preadipocyte cell line as indicated by their storage of lipid droplets and expression of surface antigens similar to those found on bone marrow stromal cells (MSC). Through cell culture under adipogenic conditions, effective differentiation of AML-I cells into adipocytes was induced by an adipogenesis inducing cocktail (INC) made up of a mixture of methylisobutylxanthine, hydrocortisone, and indomethacin. By contrast, activation of peroxisome proliferator-activated receptor (PPARgamma), which plays a key role in lipids metabolism and is highly expressed in AML-I cells, decreased the number of lipid droplets in AML-I cells. Here we report the establishment of a unique human derived-preadipocyte cell line, AML-I, and its bi-directional adipogenic response to different type stimulation, i.e., one is a refractory response to troglitazone, a well-known adipogenic stimulator, and a positive response to INC, an adipogenesis induction cocktail. These results suggest that, based on the adipogenic response, there might be some distinct lineages in human adipocytes and that the unique differentiation of AML-I cells should be useful for analyzing both the differentiation and regulation of human preadipocytes.