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Departments of Biochemistry-Immunology and Physiology, School of Medicine, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
Submitted 21 June 2007 ; accepted in final form 24 August 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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26–36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by 60% in both groups. The incorporation of 1.0 mM [2-14C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased 50% and 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-14C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-14C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-14C]pyruvate and [U-14C]glycerol into glucose of incubation medium (gluconeogenesis) were 140 and 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with 5%, and direct phosphorylation of glycerol by glycerokinase with 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes. phosphoenolpyruvate carboxykinase; glycerokinase; [U-14C]glucose; [2-14C]pyruvate; [U-14C]glycerol; gluconeogenesis
50% of reesterification of fatty acids in human adults after an overnight fast (21). Because glycerol-3-phosphate (G3P) is an obligatory intermediary in the process of TAG synthesis, the participation of the liver in the control of lipid metabolism requires a continuous and adequate generation of G3P by the hepatocyte. Until relatively recently, only two pathways of G3P generation in liver were known: direct phosphorylation of glycerol by glycerokinase (GyK), an enzyme that has considerable activity in liver (16), and conversion of the dihydroxyacetone produced during glycolysis to G3P by G3P-dehydrogenase. In in vivo experiments, performed in freely moving rats in the fed state, our group (3) several years ago showed, by determining simultaneously in the same animal the rate of incorporation of 3H2O and [14C]glucose into the two moieties of liver TAG, that this organ can also generate G3P from three-carbon intermediates, such as pyruvate, lactate, or glucogenic amino acids, via glyceroneogenesis. This pathway was shown many years ago (2, 9, 20, 21) to be present in adipose tissue; this pathway involves the carboxylation of pyruvate to oxaloacetate, decarboxylation of oxaloacetate to phosphoenolpyruvate (PEP) by cytosolic phosphoenolpyruvate carboxykinase (PEPCK), and subsequently the production of G3P through a partial reversal of glycolysis.
From the data of the above study (3), it could be estimated, with the use of calculations described previously (5), that, in livers from rats adapted to a high-protein, carbohydrate-free diet, rates of glyceride-glycerol synthesis via glyceroneogenesis were five times higher and via glycolysis 50% lower than in livers from control animals fed a balanced, carbohydrate-rich diet. The contribution of glyceroneogenesis to total liver TAG-glycerol synthesis could not be estimated because the technique used does not allow the measurement of TAG-glycerol formed via direct phosphorylation of glycerol, a process presumably very active in liver, considering the high levels of GyK in this organ. The contribution of glyceroneogenesis to the in vivo synthesis of hepatic glyceride-glycerol has been confirmed by Kalhan et al. (12) in humans, using a deuterium labeling of body water method to quantify the contribution of pyruvate to hepatic TAG. It was found that, after a 16-h fast, 10–60% of the plasma TAG pool was derived from pyruvate in pregnant and nonpregnant women, suggesting that glyceroneogenesis may be important for the regulation of VLDL TAG production in humans (12).
To our knowledge, no further studies on liver glyceroneogenesis have been so far reported in the literature. To obtain more information about the control of G3P supply for glyceride-glycerol synthesis in liver, in the present work we investigated the state of the pathways of G3P production in precision-cut liver slices of food-deprived and diabetic rats, two situations in which adipose tissue lipolysis and the uptake of fatty acids by the liver are increased. The generation of G3P via glycolysis, by direct phosphorylation of glycerol, and via glyceroneogenesis was evaluated by measuring the rates of incorporation of [14C]glucose, [14C]glycerol, and [14C]pyruvate, respectively, into TAG-glycerol. The activities of GyK and of cytosolic PEPCK, a key enzyme of both gluconeogenesis and glyceroneogenesis, were also determined. Parallel evaluations of gluconeogenic activity were made in experiments with [14C]glycerol and [14C]pyruvate. Gluconeogenesis has in common with glyceroneogenesis the steps of the dicarboxylic cycle and is markedly stimulated by food deprivation and diabetes, thus a good test of the metabolic effectiveness of the liver slices.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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50 yr. Rats were housed in suspended wire-bottom cages in a room kept at 25 ± 2°C with a 12:12-h light-dark cycle and fed a commercial nutritionally standard balanced diet [Nuvilab CR1, Nuvital (22% protein, 55% carbohydrate, and 4.5% lipid)] and water ad libitum. For the fasting experiments, rats were left without food but had free access to water for 48 h. For diabetes induction, streptozotocin (45 mg/kg body wt, dissolved in citrate buffer, pH 4.5) was injected under ether anesthesia into the jugular vein of rats previously fasted overnight. When used for the experiments, 3 days after streptozotocin injection, the rats had plasma glucose levels that were between 350 and 450 mg/dl. All experiments were performed between 0800 and 1000. For tissue removal, the rats were killed by cervical dislocation. The care of rats and experimental treatment of rats were approved by the Ethical Committee of the University of São Paulo.
Preparation of Precision-Cut Liver Slices
The rats were killed, and livers were quickly removed. The left lobe was separated, and precision-cut liver slices (500 µm thick) were prepared with a tissue chopper (7). Only the most uniform-shaped slices were selected for experiments. All slice manipulations were done in ice-cold Krebs-Henseleit buffer.
Incorporation of [14C]Pyruvate or [U-14C]Glycerol into TAG-Glycerol or Glucose of Medium
Liver slices (200 mg) were incubated for 1 h at 37°C with constant orbital shaking in 2 ml of Krebs-Henseleit buffer, pH 7.4, containing 1 µCi of [1-14C]pyruvate (1.0 mmol/l), [2-14C]pyruvate (0.5, 1.0, or 5.0 mmol/l), or [U-14C]glycerol (1.0 mmol/l). The procedures used for lipid extraction, isolation, and counting of TAG-glycerol were as previously described (5). The incubation medium was deproteinized by the method of Somogyi (24), and the glucose was isolated for 14C counting by the glucose pentacetate method (11).
Glycolytic Flux
Liver slices (200 mg) were incubated for 1 h at 37°C with constant orbital shaking in 2 ml of Krebs-Henseleit buffer, pH 7.4, containing D-[5-3H-glucose] (5 mmol, 1 µCi). After the reaction was stopped with 6% H2ClO4, the medium was centrifuged and neutralized and the glycolytic flux was calculated from the 3H recovered in water as described in previously (15).
Measurement of Enzyme Activity
Cytosolic PEPCK activity was assayed by the method of Chang and Lane (6) in 100,000-g supernatants after homogenization of liver in 20 mmol/l triethanolamine buffer, pH 7.5, containing 0.2 mol/l sucrose, 5 mmol/l mercaptoethanol, and l mmol/l EDTA. The incorporation of [14C]bicarbonate (2 µCi) into acid-stable product was determined in an assay mixture of composition identical to that used in a previous study (5). The protein content of homogenates was determined by the bicinchoninic acid method (23).
GyK activity was measured following the recommendations of Newsholme et al. (18) in 2,000-g supernatants obtained after homogenization of the tissue in ice-cold 1% KCl in 1 mM EDTA. The composition of the assay mixture, which contained U-[14C]glycerol, and the isolation of labeled glycerol phosphate were previously described in detail (14). The protein content of the homogenates used in GyK was determined by the method of Lowry et al. (17).
Other Methods of Chemical Analysis
Plasma glucose and liver ATP were determined with the use of commercial kits from Labtest (Lagoa Santa, Brazil) and from Bio-Orbit Oy (Turku, Finland), respectively.
Statistical Methods
Data are expressed as means ± SE, and differences between groups were analyzed by Student's t-test, with P < 0.05 as the criterion of significance.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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The concentrations of ATP in the liver slices (means ± SE: 4.3 ± 0.9 µmol/g; n = 8) did not change after 1 h of incubation (4.4 ± 0.1 µmol/g). The slices reproduced efficiently in vitro the qualitative changes in liver carbohydrate and lipid metabolism, which are well known to be induced by fasting and diabetes in intact animals. Thus lipogenic activity, assessed by the rate of incorporation of 14C from glucose into fatty acids, was reduced to 12% of normal controls in liver slices of fasted and diabetic rats (Fig. 1A), which also showed an 60% reduction of the glycolytic flux, estimated with [5-3H]glucose (Fig. 1B). Also, as expected from changes observed in vivo, gluconeogenic activity, assessed by the rates of incorporation of 14C from pyruvate and glycerol into glucose of incubation medium, increased markedly in slices of fasted and diabetic rats (
Table 3 and 4, see below for details). The above data show that, at least for 1 h (period of incubation), the slices maintain the pattern of metabolic pathways activities present in liver in different physiological conditions and are therefore useful for investigating features of these pathways not obtainable in studies in vivo.
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It should be pointed out initially that rates of incorporation of labeled substrates into final products presented in all tables and Fig. 1 were calculated directly from the specific activity of the substrate in the incubation medium, disregarding any eventual intracellular dilution.
Experiments in fasted rats. The generation of G3P via glycolysis was markedly reduced in liver slices from fasted rats, as shown by the 23% reduction in the incorporation of [U-14C]glucose (5 mM; the physiological plasma concentration of the hexose) into glyceride-glycerol, compared with rates in slices from fed rats (Table 1).
For generation of G3P via glyceroneogenesis, we confirmed the good performance of the liver slices by the rate of incorporation of [2-14C]pyruvate into glyceride-glycerol, which increased in parallel to the increase in its concentration in the incubation medium in both fed and fasted rats (Table 1). The production of G3P via glyceroneogenesis was increased in slices of fasted rats, as indicated by the increases in the rates of glycerol synthesis from 0.25 mM [2-14C]pyruvate (75%) and from 1.0 mM [2-14C]pyruvate, concentrations of pyruvate prevailing in plasma of fed and fasted rats, respectively. With 5.0 mM [2-14C]pyruvate, these rates did not differ significantly in slices from the two experimental groups, an indication that this was a saturating concentration of the substrate. Interestingly, the increased incorporation of 1.0 mM [2-14C]pyruvate in glyceride-glycerol by slices of fasted rats (42.9 ± 1.26 nmol·g–1·h–1) was not affected by the addition of 5 mM glucose to the incubation medium (44.2 ± 0.93 nmol·g–1·h–1), in contrast to the marked inhibition induced by the hexose in the synthesis of glyceride-glycerol from pyruvate by incubation with white (22) and brown (Chaves VE, unpublished observations) adipose tissue. It has been previously found (8) that high levels of glucose also failed to inhibit the increased gluconeogenesis from lactate in perfused liver from fasted rats. It would thus appear that, in ex vivo conditions, the hexose does not affect the rate-limiting steps common to liver glyceroneogenesis and gluconeogenesis below the triose-P level.
The effect of fasting on G3P generation by direct glycerol phosphorylation was less evident, with a relatively small (25%) increase in the incorporation of [U-14C]glycerol into glyceride-glycerol being observed only at the substrate concentration of 0.25 mM (Table 1).
Experiments in diabetic rats.
Table 2 shows that the results obtained were similar to those observed in liver slices of fasted rats incubated with the same concentration of substrates (Table 1). Thus the incorporation of 5 mM [U-14C]glucose into glyceride-glycerol was 36% lower and that of 1 mM [2-14C]pyruvate was 36% higher in liver slices of diabetic rats than in normal controls. The incorporation of [U-14C]glycerol did not differ significantly in the two experimental groups (Table 2).
Gluconeogenesis From Pyruvate and Glycerol
Experiments in fasted rats.
These experiments were performed in the same rats utilized for the experiments of Table 1. The incorporations of labeled glycerol and pyruvate into liver glycogen were <0.01% of those in glucose of medium (data not shown). Table 3 shows that the rates of incorporation of [2-14C]pyruvate and [U-14C]glycerol into the glucose of incubation medium also increased in parallel to the increase in the initial concentration of the two substrates in the medium for both fed and starved rats. Rates of incorporation into medium glucose were 140% higher in fasted than in fed controls in slices incubated with 0.25 and 1.0 mM [2-14C]pyruvate; with 5.0 mM [2-14C]pyruvate, the increase was 90%. The effect of fasting on the rates of incorporation of [U-14C]glycerol into glucose of the medium was relatively small, with increases of 20% being observed with the three concentrations of [U-14C]glycerol (Table 3).
Experiments in diabetic rats.
Table 4 data were obtained from the same rats of experiments shown in Table 2. Table 4 shows that rates of incorporation into medium glucose were 145% higher in diabetic than in normal controls in slices incubated with 1.0 mM [2-14C]pyruvate, an effect similar to that observed in fasted rats (Table 3). As in the experiments with fasted rats, the effect of diabetes was relatively small, with the incorporation of [U-14C]glycerol into medium glucose increasing only 14% in liver slices of diabetic rats (Table 4).
GyK and Cytosolic PEPCK Activities
In agreement with the results of the experiments of incorporation of [U-14C]glycerol into glyceride-glycerol (Tables 1 and 3), the activity of GyK in liver slices was not affected by food deprivation or diabetes (Fig. 2). In contrast, the activity of cytosolic PEPCK was markedly increased (by 84%) in slices of both fasted and diabetic rats (Fig. 2), in consonance with the observed increases in gluconeogenesis (Tables 2 and 4) and glyceroneogenesis (Tables 1 and 3) activities in these slices.
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With the assumption that there was no intracellular dilution of the label, the rates of [U-14C]glucose and of [U-14C]glycerol incorporation into glyceride-glycerol, calculated from the incubation medium-specific activity in liver slices from fasted rats (Table 1), correspond to the glyceride synthesis from these two substrates via glycolysis and by direct phosphorylation of glycerol, respectively. They can, therefore, be used to estimate the relative contribution of these two pathways to the generation of G3P for glyceride-glycerol synthesis. The same assumption, however, cannot be made for rates of incorporation of [2-14C]pyruvate calculated from the medium-specific activity. These rates, although useful for comparisons of glyceroneogenic activity in different conditions, as done above, do not correspond to the actual rate of glyceride-glycerol synthesis from pyruvate via glyceroneogenesis in a given condition. It has been known for many years (25) that, in the conversion of pyruvate to PEP, the carbons of PEP are diluted by carbons derived from acetyl-CoA and CO2. This occurs because the first intermediate in the synthesis of PEP from pyruvate is oxaloacetate, which is also an intermediate in tricarboxylic cycle, after decarboxylation of pyruvate to acetyl-CoA by pyruvate dehydrogenase (Fig. 3). Carbon-2 from pyruvate entering the mitochondria can be incorporated into oxaloacetate directly through the reaction catalyzed by pyruvate carboxylase or indirectly via the citric acid cycle, after being incorporated into acetyl-CoA produced by decarboxylation of pyruvate by pyruvate dehydrogenase. Carbon-1 from pyruvate is also incorporated into oxaloacetate by direct carboxylation of pyruvate but is removed in the CO2 and excluded from acetyl-CoA in the reaction catalyzed by pyruvate dehydrogenase, incorporating into PEP only after recycling of oxaloacetate in the citric acid cycle. Exton and Park (8) computed, from the results obtained in experiments in which they measured the incorporation of [1-14C]pyruvate and [2-14C]pyruvate into glucose, glycogen, and CO2, the ratio of pyruvate carboxylated to oxaloacetate to that decarboxylated to acetyl-CoA in perfused livers of fasted rats. Table 5 shows the results of experiments in which liver slices of fasted rats were incubated with [1-14C]pyruvate or [2-14C]pyruvate to measure the incorporation of these substrates into medium glucose (the incorporation into liver glycogen was negligible), CO2, and also into glyceride-glycerol, since glyceroneogenesis is an abbreviated form of gluconeogenesis. With the same assumptions and calculations of the experiments with perfused livers (8), it was found that in liver slices pyruvate carboxylation was practically the same as decarboxylation (ratio of carboxylation to decarboxylation = 0.94). On the basis of a model that considered all the pathways of pyruvate metabolism in the liver, Katz (13) deduced a formula to calculate the relative specific activity of liver PEP after administration of [1-14C]pyruvate that required only a knowledge of the ratio between carboxylation and decarboxylation of pyruvate (13). From the relative specific activity of PEP obtained with the above ratio (0.94), it was calculated that rates of incorporation of [1-14C]pyruvate into TAG should be multiplied by 4.2 and rates of [2-14C]pyruvate by 2.1 to obtain a value of glyceride-glycerol synthesis from pyruvate independent of intracellular dilution of pyruvate carbons. Because during fasting there is, in addition to circulating pyruvate, a large liver influx of other pyruvate-producing metabolites, such as lactate and amino acids and especially alanine, the contribution of glyceroneogenesis to G3P production in liver slices of fasted rats was estimated with the use of rates obtained with saturating amounts of substrate (5 mM) in Table 1. After correction of rates of pyruvate incorporation, it could be calculated that glyceroneogenesis contributed with 20% of G3P for glyceride-glycerol synthesis in slices of fasted rats, the glycolytic pathway with 5%, and direct phosphorylation of glycerol by GyK with 75%.
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75% contribution of glycerol to glyceride-glycerol synthesis in liver slices, Kalhan et al., who administered [13C3]glycerol tracer as prime constant rate infusion to fasted nonpregnant women, found that 6.1% of glycerol in TAG was derived from plasma glycerol, suggesting that only a small fraction of the glyceride-glycerol made by the liver during fasting is synthesized from glycerol. Species differences apart, this apparent disaccord could be partly attributed to the period during which newly formed TAGs are passing through the various stages of transport to the exterior of the hepatocyte, which precedes their appearance in plasma VLDL. Previous studies (10, 26) have shown that, after intravenous injection of radioactive FFAs, fatty acids are rapidly esterified to TAG in liver, but peak specific activities of TAG fatty acids in plasma are not reached for 1–3 h in humans. Contribution of Pyruvate and Glycerol to Gluconeogenesis in Fasted Rats
After we corrected for the rate of incorporation of pyruvate into medium glucose, we could estimate from the results obtained with saturating amounts (5 mM) of substrates in Table 3 that pyruvate contributed 54% and glycerol 46% of the gluconeogenesis in liver slices of fasted rats. Kalhan et al. (12), in their studies with [13C3]glycerol, found that a similar large fraction (50%) of the glycerol is converted to glucose in pregnant and nonpregnant fasted women.
Because in the fed state glucose is the main source of TAG-glycerol in adipose tissue, which has a low GyK activity, the glycerol released during fasting is sometimes compared with lactate, which only recycles glucose carbon, with no "new" glucose production. During fasting and diabetes, however, G3P synthesis from glucose is markedly reduced and glyceroneogenesis is very active in adipose tissue. Thus the glycerol released in large amounts from the liver in those two conditions derives mainly from three carbon substrates of glyceroneogenesis, contributing therefore to de novo synthesis of glucose.
In summary, the present study demonstrates that during fasting and diabetes, conditions in which the synthesis of triglycerides is markedly increased in the liver, the reduced generation of G3P in liver slices via glycolysis is accompanied by a significant activation of glyceroneogenesis that contributes to maintain an adequate supply of the G3P needed for triglyceride formation. Our study also shows that glycerol phosphorylation by GyK, the main generator of G3P in liver, is little affected by fasting or diabetes.
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONGRANTSREFERENCES |
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