Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells

Drosophila melanogaster has become a prominent and convenient model for analysis of insulin action. However, to date very little is known regarding the effect of insulin on glucose uptake and metabolism in Drosophila. Here we show that, in contrast to effects seen in mammals, insulin did not alter [(3)H]2-deoxyglucose uptake and in fact decreased glycogen synthesis ( approximately 30%) in embryonic Drosophila Kc cells. Insulin significantly increased ( approximately 1.5-fold) the production of (14)CO(2) from D-[1-(14)C]glucose while the production of (14)CO(2) from D-[6-(14)C]glucose was not altered. Thus, insulin-stimulated glucose oxidation did not occur via increasing Krebs cycle activity but rather by stimulating the pentose phosphate pathway. Indeed, inhibition of the oxidative pentose phosphate pathway by 6-aminonicotinamide abolished the effect of insulin on (14)CO(2) from D-[U-(14)C]glucose. A corresponding increase in lactate production but no change in incorporation of D-[U-(14)C]glucose into total lipids was observed in response to insulin. Glucose metabolism via the pentose phosphate pathway may provide an important source of 5'-phosphate for DNA synthesis and cell replication. This novel observation correlates well with the fact that control of growth and development is the major role of insulin-like peptides in Drosophila. Thus, although intracellular signaling is well conserved, the metabolic effects of insulin are dramatically different between Drosophila and mammals.     

Pentitols and insulin release by isolated rat islets of Langerhans

1. Insulin secretion was studied in isolated islets of Langerhans obtained by collagenase digestion of rat pancreas. In addition to responding to glucose and mannose as do whole pancreas and pancreas slices in vitro, isolated rat islets also secrete insulin in response to xylitol, ribitol and ribose, but not to sorbitol, mannitol, arabitol, xylose or arabinose. 2. Xylitol and ribitol readily reduce NAD(+) when added to a preparation of ultrasonically treated islets. 3. Adrenaline (1mum) inhibits the effects of glucose and xylitol on insulin release. Mannoheptulose and 2-deoxy-glucose, however, inhibit the response to glucose but not that to xylitol. 4. The intracellular concentration of glucose 6-phosphate is increased when islets are incubated with glucose but not with xylitol, suggesting that xylitol does not promote insulin release by conversion into glucose 6-phosphate. 5. Theophylline (5mm) potentiates the effect of 20mm-glucose on insulin release from isolated rat islets of Langerhans, but has no effect on xylitol-mediated release. These results indicate that xylitol does not stimulate insulin release by alterations in the intracellular concentrations of cyclic AMP. 6. A possible role for the metabolism of hexoses via the pentose phosphate pathway in the stimulation of insulin release is discussed.     

The pentose cycle and insulin release in isolated mouse pancreatic islets during starvation.

When islets from mice were incubated with 16.7 mM-glucose, previous starvation for 48 h decreased the rate of insulin release by approx. 50% and glucose utilization was decreased by approx. 35%. The maximally extractable activity of glucose 6-phosphate dehydrogenase was diminished by 28% after starvation. The formation of 14CO2 from both [1-14C]glucose was, however, higher than the rate of oxidation of [6-14C]-glucose in islets from both fed and starved mice. The fraction of glucose utilized that was oxidized (specific 14CO2 yield) ranged from one-fifth to one-third and was higher in islets from starved mice with both [1-14C]glucose and [6-14C]glucose as substrate. The contribution of pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose-cycle oxidation to total glucose metabolism was small (3% in the fed state and 4% in the starved state). The absolute rates of glucose carbon metabolism via the pentose cycle and the turnover of NADPH in this pathway were identical in islets from fed and starved animals. After incubation at 16.7 mM-glucose for 30 min the contents of glucose (6-phosphate and 6-phosphogluconate were both unchanged by starvation. It is concluded that there is no correlation between the decreased sensitivity of the insulin secretory mechanism during starvation and the metabolism of glucose via the pentose cycle, the islet content of glucose 6-phosphate or 6-phosphogluconate.     

The effect of 2,4-dinitrophenol on adipose-tissue metabolism

1. The effect of dinitrophenol on the metabolism of glucose labelled with (14)C and tritium by epididymal fat-pad segments from fed rats was studied. Dinitrophenol at concentrations of 0.1-0.3mm: (a) had little effect on glucose utilization; (b) depressed synthesis of fatty acids and greatly increased that of lactate; (c) increased the T/(14)C ratio in fatty acids synthesized from [U-(14)C,3-T]glucose and decreased that in fatty acids synthesized from [U-(14)C,4-T]glucose; (d) abolished randomization of (14)C from [6-(14)C]glucose in lactate. 2. Dinitrophenol stimulated oxidation of pyruvate and greatly inhibited the oxidation of lactate. It inhibited lipogenesis from pyruvate and lactate. 3. From the isotope data it was calculated that: (a) dinitrophenol stimulates oxidation via the tricarboxylic acid cycle three- to six-fold; (b) dinitrophenol depresses markedly the operation of the pentose cycle; (c) in the presence of dinitrophenol, NADPH formed in the pentose cycle provides all the hydrogen equivalents for fatty acid reduction, whereas, in its absence, NADPH provides 50-70% of the hydrogen equivalents; (d) in the presence of dinitrophenol, there is an excess of ATP produced in the cytoplasm, which flows into the mitochondria. A reverse flow operates in the absence of dinitrophenol. 4. A balance of formation and utilization of reduced nicotinamide nucleotides in the cytoplasm was established. With dinitrophenol there is some excess of NADH. There are indications that this excess may be transferred into mitochondria in the form of malate. 5. Our results are interpreted to indicate the absence from adipose tissue of the alpha-glycerophosphate shuttle for transferring reducing equivalents from the cytoplasm to mitochondria. 6. The effects of dinitrophenol are accounted for in terms of decreased ATP concentrations in the cells, leading to marked decrease in pyruvate carboxylation in the mitochondria and depression of fatty acid synthesis in the cytoplasm.     

Oxidation of glucose, ribose, alanine, and glutamate by Leishmania braziliensis panamensis

The metabolism of [1-14C]- and [6-14C]glucose, [1-14C]ribose, [1-14C]- and [U-14C]alanine, and [1-14C]- and [5-14C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of 14CO2 produced from [1-14C]glucose to that from [6-14C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of 14CO2 from [1-14C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of 14CO2 production from [1-14C]glucose was almost linear with time of incubation, whereas that of [6-14C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26 degrees C to 34 degrees C had no appreciable effect on the rates or time courses of oxidation of either [1-14C]- or [6-14C]glucose or of [1-14C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of 14CO2 produced from [1-14C]- to [U-14C]alanine and from [1-14C]- to [5-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of [13C]glycerol carbon via the pentose cycle. Implications for gluconeogenesis measurement by mass isotoper distribution analysis

Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods.     

Effect of cytochalasin B on glucose uptake, utilization, oxidation and insulinotropic action in tumoral insulin-producing cells

Cytochalasin B (17-3 microM) virtually abolished 3-O-methyl-D-[U-14C]glucose uptake and D-[5-3H]glucose utilization in tumoral insulin-producing cells of the RINm5F line. This coincided with a marked decrease in D-[U-14C]glucose oxidation and suppression of the stimulant action of D-glucose upon insulin release. Cytochalasin B, however, augmented basal insulin release by the tumoral cells. The RINm5F cells appeared much more sensitive than normal islet cells to cytochalasin B, as judged by the relative magnitude of inhibition in either hexose uptake or utilization. In both cell types, the inhibitory action of cytochalasin B upon glucose metabolism seemed to be competitive, being more marked at low than high glucose concentration. These results are interpreted in support of the view that a decreased efficiency of hexose transport across the plasma membrane represents an essential deficiency of the RINm5F cells.     

Glucose metabolism in mouse pancreatic islets

1. Rates of glucose oxidation, lactate output and the intracellular concentration of glucose 6-phosphate were measured in mouse pancreatic islets incubated in vitro. 2. Glucose oxidation rate, measured as the formation of (14)CO(2) from [U-(14)C]glucose, was markedly dependent on extracellular glucose concentration. It was especially sensitive to glucose concentrations between 1 and 2mg/ml. Glucose oxidation was inhibited by mannoheptulose and glucosamine but not by phlorrhizin, 2-deoxyglucose or N-acetylglucosamine. Glucose oxidation was slightly stimulated by tolbutamide but was not significantly affected by adrenaline, diazoxide or absence of Ca(2+) (all of which may inhibit glucose-stimulated insulin release), by arginine or glucagon (which may stimulate insulin release) or by cycloheximide (which may inhibit insulin synthesis). 3. Rates of lactate formation were dependent on the extracellular glucose concentration and were decreased by glucosamine though not by mannoheptulose; tolbutamide increased the rate of lactate output. 4. Islet glucose 6-phosphate concentration was also markedly dependent on extracellular glucose concentration and was diminished by mannoheptulose or glucosamine; tolbutamide and glucagon were without significant effect. Mannose increased islet fructose 6-phosphate concentration but had little effect on islet glucose 6-phosphate concentration. Fructose increased islet glucose 6-phosphate concentration but to a much smaller extent than did glucose. 5. [1-(14)C]Mannose and [U-(14)C]fructose were also oxidized by islets but less rapidly than glucose. Conversion of [1-(14)C]mannose into [1-(14)C]glucose 6-phosphate or [1-(14)C]glucose could not be detected. It is concluded that metabolism of mannose is associated with poor equilibration between fructose 6-phosphate and glucose 6-phosphate. 6. These results are consistent with the idea that glucose utilization in mouse islets may be limited by the rate of glucose phosphorylation, that mannoheptulose and glucosamine may inhibit glucose phosphorylation and that effects of glucose on insulin release may be mediated through metabolism of the sugar.     

Anomeric specificity of D-glucose metabolism in rat adipocytes

The anomeric specificity of D-glucose metabolism was investigated in rat adipocytes exposed for 60 min at 8 degrees C to pure alpha- or beta-D-glucose or to equilibrated D-glucose. The rate of D-[5-3H]glucose utilization was higher with alpha- than beta-D-glucose. However, as judged from the oxidation of D-[1-14C]glucose and D-[6-14C]glucose anomers, the fraction of D-glucose catabolism occurring via the pentose cycle was higher with beta- than alpha-D-glucose. In the presence of equilibrated D-glucose, the utilization of alpha-D-[5-3H]glucose and the oxidation of both alpha-D-[1-14C]glucose and alpha-D-[6-14C]glucose were higher, relative to the anomer concentration, than the corresponding values for beta-D-glucose. It is concluded that the anomeric specificity of D-glucose metabolism is operative in adipocytes, even when they are exposed to equilibrated D-glucose.     

Anomeric specificity of D-glucose phosphorylation and oxidation in human erythrocytes

1. In human erythrocytes, alpha-D-[U-14C]glucose is more efficiently oxidized than beta-D-[U-14C]glucose at a low concentration of the hexose (0.1 mM), but not so at higher glucose concentrations. 2. This unexpected situation may be attributable in part to the lower Km of hexokinase for alpha- than beta-D-glucose, this difference in affinity compensating for the higher maximal velocity found with the beta- rather than alpha-anomer. 3. A contributive role for aldose reductase in the anomeric control of D-glucose 6-phosphate circulation in the pentose phosphate pathway should not be ruled out, since aldose reductase inhibitors decrease the production of 14CO2 by erythrocytes exposed to D-[U-14C]glucose. 4. Nevertheless, the essential role of hexokinase in such an anomeric control is supported by the finding that, in the presence of menadione, which augments considerably D-[U-14C]glucose oxidation but fails to affect D-[5-3H]glucose utilization, the anomeric alpha/beta ratio in 14CO2 production from D-[U-14C]glucose follows, at increasing concentrations of the hexose, the same pattern as that found for its phosphorylation.     

Interrelationship and control of glucose metabolism and lipogenesis in isolated fat-cells. Effect of the amount of glucose uptake on the rates of the pentose phosphate cycle and of fatty acid synthesis

In order to study the quantitative relationship between fatty acid synthesis and pentose phosphate-cycle activity under different hormonal and dietary conditions affecting the extent of glucose uptake, cells isolated from rat epididymal adipose tissue were incubated in bicarbonate buffer containing [U-(14)C]-, [1-(14)C]- or [6-(14)C]-glucose. From the amount of glucose taken up, the production of lactate and pyruvate, and the incorporation of (14)C from differently labelled [(14)C]glucose into CO(2), fatty acids and glyceride glycerol, the rates of glucose metabolism via different pathways and the extent of lipogenesis under various experimental conditions were determined. The contribution of the pentose phosphate-cycle to glucose metabolism under normal conditions was calculated to be 8%. Starvation and re-feeding, and the presence of insulin, caused an enhancement of glucose uptake, pentose phosphate-cycle activity and fatty acid synthesis. Plots of both pentose phosphate-cycle activity and fatty acid synthesis versus glucose uptake revealed that the extent of glucose uptake, over a wide range, determines the rates of fatty acid synthesis and glucose metabolism via the pentose phosphate cycle. A balance of formation and production of nicotinamide nucleotides in the cytoplasm was established. The total amount of cytoplasmic NADH and NADPH formed was only in slight excess over the hydrogen equivalents required for the synthesis of fatty acids, glyceride glycerol and lactate. Except in cells from starved animals, the pentose phosphate cycle was found to provide only about 60% of the NADPH required for fatty acid synthesis. The results are discussed with respect to an overall control of the different metabolic and biosynthetic reactions in the fat-cells by the amount of glucose transported into the cell.     

Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.     

Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species.

1. The patterns of 14CO2 evolution from specifically labeled glucose substrates by washed bull, ram, boar, rabbit, dog, rooster and turkey spermatozoa were similar and indicated the Embden-Meyerhof and Kreb's cycle pathways as the major route of energy metabolism. 2. Honey bee spermatozoa metabolized glucose-3,4-[14C], glucose-[U-14C] or fructose-[U-14C], but not glucose-1-[14C], glucose-2-[14C]or glucose-6-[14C], indicating the presence of the glycolytic pathway, but the absence of respiration via the Kreb's cycle. 3. The rate of glycolysis exceeded the rate of respiration in the spermatozoa of all the species studied. 4. A preferential utilization of glucose-1-[14C] over glucose-6-[14C] was evident in some sperm samples, but no consistent indication of pentose cycle metabolism was observed, due to considerable variability between samples within each group. 5. Fructose metabolism was greater than glucose metabolism in the rooster, less in the dog, boar and turkey, and similar in the spermatozoa from the other species examined. 6. Only ram and bull spermatozoa metabolized acetate-1-[14C] to any extent.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

Early effects of phytohaemagglutinin on glucose metabolism of normal human lymphocytes

1. Phytohaemagglutinin induced early changes in the catabolism of glucose by normal human lymphocytes suspended in a bicarbonate buffer. During 4hr. incubation glucose utilization was almost doubled. 2. The rates of several reactions in the metabolism of glucose were estimated. Total pyruvate formation, lactate production and fatty acid synthesis were stimulated to the same degree as was glucose utilization. The pentose cycle and the glycogen synthesis were also stimulated but less than was glucose utilization. The pentose cycle was found to account for 1.4% and 0.9% of the total glucose utilization without and with phytohaemagglutinin respectively. In these cells rates of triose phosphate iso-merization were at least six to seven times the rate of glucose phosphorylation. On an average 55-60% of the total carbon dioxide evolved was derived from decarboxylation of pyruvate, 25-30% from the tricarboxylic acid cycle and about 15% from the pentose cycle. Observed ratios of (14)C specific yields in glycogen from [1-(14)C]- and [6-(14)C]-glucose could possibly be explained by assuming the existence of two separate glucose 6-phosphate pools. 3. During 4hr. incubation in bicarbonate buffer (14)C from [U-(14)C]serine was incorporated into perchloric acid-insoluble material. This incorporation was stimulated by phytohaemagglutinin but was almost completely inhibited by puromycin. Puromycin also abolished phytohaemagglutinin-induced stimulation of glycolysis.     

Channeling of alpha-D-glucose 6-phosphate in tumoral islet cells exposed to D-galactose

In human erythrocytes, in which the fractional turnover rate of glucose 6-phosphate is rather low, menadione increases to almost the same relative extent the oxidation of D-[U-14C]glucose and D-[U-14C]galactose. However, in pancreatic tumoral islet cells (RINm5F line), in which the fractional turnover rate of glucose 6-phosphate is considerably higher, menadione increases the oxidation of D-[1-14C]glucose but not that of D-[1-14C]galactose. These results suggest that alpha-D-glucose 6-phosphate generated from exogenous D-galactose is channeled preferentially into the glycolytic rather than pentose phosphate pathway. Such was no more the case, however, when the RINm5F cells were exposed simultaneously to both D-glucose and D-galactose.     

Further evidence for the classical pentose phosphate cycle in the liver

Isolated rat hepatocytes were incubated with [3-(14)C]xylitol or d-[3-(14)C]xylulose plus xylitol or glucose at substrate concentrations. The glucose formed was isolated and degraded to give the relative specific radioactivities in each carbon atom. C-4 of glucose had the highest specific radioactivity, followed by C-3, with half to one-fifth that of C-4. Only about 1% of the total radioactivity was in C-1. The data are compared with the predictions of the classical pentose phosphate cycle [Horecker, Gibbs, Klenow & Smyrniotis (1954) J. Biol. Chem.207, 393-403], and the proposed new version of the pentose phosphate cycle in liver [Longenecker & Williams (1980) Biochem. J.188, 847-857], which they denoted as the ;L-type pentose cycle'. The Williams pathway predicts that the specific radioactivity of C-1 of glucose should be half that of C-4 (after correction for approximately equal labelling on C-3 and C-4 of hexose phosphate in the pathway involving fructose 1,6-bisphosphatase). The actual labelling in C-1 is 20-350-fold less than this. When the hepatocytes are incubated with phenazine methosulphate, to stimulate the oxidative branch of the pentose phosphate cycle, the predicted relationship between (C-2/C-3) and (C-1/C-3) ratios of specific radio-activities is nearly exactly in accord with the classical pentose phosphate cycle. Glucose and glucose 6-phosphate were isolated and degraded from an incubation of hepatocytes from starved/re-fed rats with [3-(14)C]xylitol. Although the patterns were of the classical type, there was more randomization of (14)C into C-2 and C-1 in the glucose 6-phosphate isolated at the end of the incubation than in the glucose which was continuously produced.     

Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

Utilization of pyruvate and pyruvate precursors by normal and carcinogen-altered rat tracheal epithelial cells in culture

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen-altered cells derived from dimethylbenz(a)anthracene-exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate-supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three- to four-fold higher incorporation of radioactivity from [2-14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U-14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two-fold greater glucose consumption and two-fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two-fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U-14C]glutamine or [U-14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7-fold higher utilization of [1-14C]glucose along the oxidative arm of the pentose cycle in the normal cells.     

Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue

Glucose utilization was studied in isolated fat cells prepared from rat adipose tissue which had been cultured for 18 hr in TC 199 medium. When 1% bovine serum albumin (BSA) was in the culture medium, basal rates of (14)CO(2) and [(14)C]triglyceride production from [1-(14)C]glucose were markedly depressed and there was no effect of insulin. With 4% BSA, basal (14)CO(2) production was the same as in cells prepared from fresh tissue and basal triglyceride production was greatly increased. Insulin effect on these cells was minimal. One-minute uptake of [(14)C]2-deoxyglucose was stimulated by 800-1000% in fresh cells and 300-500% in cells cultured with either 1% or 4% BSA. Oxidation of [U-(14)C]glucose showed a much smaller impairment in cultured cells than for [1-(14)C]glucose, suggesting that the pentose phosphate shunt was more severely impaired than glycolysis. Glyceride-glycerol production was increased in cultured cells relative to preculture (fresh) cells. There was no effect of insulin in the culture medium in any of these systems. Rates of free fatty acid and glycerol release were markedly increased in cultured cells, especially when insulin was present in the culture medium. The acute antilipolytic effect of insulin was retained, so that insulin in the test incubation decreased lipolysis by 40-80%. Nevertheless, cell-associated fatty acids were increased in cultured cells and FFA/albumin ratios in the medium often reached potentially toxic levels. The reduction in pentose phosphate shunt activity, lipogenesis, and insulin effect resembles other models of insulin insensitivity. The impaired metabolism is probably due to an intracellular defect. A possible toxic role of either intracellular or extracellular fatty acids cannot be excluded. This system should be a useful model in which to study the cellular mechanisms of insulin insensitivity in adipocytes.-Bernstein, R. S. Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue.