Effects of glucose and of lactate on phosphofructokinase flux during gluconeogenesis

Biochimica et Biophysica Acta, 676 (1981) 373-378

373

Elsevier/North-Holland Biomedical Press BBA 29705 EFFECTS OF GLUCOSE AND OF LACTATE ON PHOSPHOFRUCTOKINASE FLUX DURING GLUCONEOGENESlS ROBERT ROGNSTAD Cedars Sinai Medical Center, Los Angeles, CA 90048 (U.S.A.)

(Received February 17th, 1981)

Key words: Glucose; Lactate; Phosphofructokinase; Gluconeogenesis; (Rat hepatocyte]

We have examined the effects of glucose and lactate, the products of the gluconeogenic-glycolytic pathways, on phosphofructokinase flux during gluconeogenesis in hepatocytes from fasted rats. With dihydroxyacetone as substrate, phosphofructokinase flux is rather active. Addition of lactate, at concentrations of 5 - 1 0 raM, causes a lowering of this flux to the levels found when lactate alone is the substrate. Inhibitor studies suggest that a mitochondrially formed metabolite of lactate is the likely effector involved. Addition of glucose (10 mM or greater) to dihydroxyacetone causes an increase in phosphofructokinase flux. Only small effects are seen unless the cells are preincubated with glucose, in which case an estimated 2-3-fold increase in phosphofructokinase flux occurs.

Introduction It is now clear that many enzymes are regulated by a combination of allosteric effectors and by covalent modifications. The allosteric regulation of phosphofructokinase from glycolytic cells has long been studied [ 1 - 5 ] . The hormonal regulation of phosphofructokinase in the gluconeogenic liver cells is being actively investigated. While isotopic evidence [6-8] and intermediate analysis [9,10] both strongly suggested that hormonal control may be effected at this site (among others), it is not yet clear how this regulation occurs. This is now being actively pursued at the subcellular level [11-15]. In this paper, we present evidence that the end products or reactants of the gluconeogenic-glycolytic pathways, namely, glucose and lactate, can affect phosphofructokinase flux in rat hepatocytes, although the mechanisms of these effects are not yet known.

Methods Hepatocytes were prepared from male rats, fasted 18-24 h, as described previously [16]. The cells

(30--50 mg dry wt) were incubated in 5 ml of KrebsHenseleit butter in 25-ml Erlenmeyer flasks at 38°C with 5% CO2/95% 02 in the gas phase. When a preincubation period was required, the serum stoppers were removed for addition of the gluconeogenic substrate and [1-14C]galactose, and the flasks then were stoppered and regassed with 5% CO2/95% 02 for 1 min. The reactions were terminated by iniection of 0.5 ml of 20% HC104. The cells and medium were washed out of the flasks, made to 10.0 ml and centrifuged. A portion (9 ml) of the supernatant solution was put on a 1 × 4 cm Dowex 50 (H ÷ form, 100-200 mesh) column, on top of a 1 × 6 cm Dowex 1 (acetate form, 100-200 mesh) column, and the columns were washed with water until a 30.0 ml neutral fraction was collected. This fraction was dried in a stream of air and chromatographed on a 6 inch sheet of Whatman 3ram paper, using ethyl acetate/pyridine/ water (12/5/4, by vol.). This procedure separates [14C] glucose from any residual [14C]galactose, as well as from [14C/glycogen which remains at the origin. The radioactive bands were located with X-ray film. The purified glucose was degraded with periodate

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374 [17], after addition of carrier glucose to give a total of 200/a.mol, to give the amount of 14C on carbon-6. Glucose formation in the incubations was determined enzymically on an aliquot of the acidified medium. The assay procedure involves 0.5 ml of water, 0.5 ml of stock assay medium plus the sample in 0.1 ml or less, where the stock assay medium contains, per 100 ml total volume: 60 mmol triethanolamineHC1, 1 mmole ATP, 2 mmol MgC12, 0.1 mmol NAD ÷ at a pH of 7.5. This stock solution was kept frozen in 20-ml portions and was usable for up to 2 months. Hexokinase was from either Sigma Chemical (St. Louis, MO) or Boehringer (Indianapolis, IN) while the glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides was from Worthington (Freehold, N J). Mercaptopicolinate was a generous gift from Dr. H. Saunders, Smith, Kline and French Laboratories (Philadelphia, PA). N-Butylmalonate and cz-cyano-4hydroxycinnamate were from Aldrich Chemical (Milwaukee, WI). Aminooxyacetate was from Eastman Organic Chemicals (Rochester, NY). The c~-cyano-4hydroxy cinnamic acid was dissolved in methanol at a concentration of 0.005 M and neutralized with an equivalent amount of NaOH. The required amounts were then added to the incubation flasks and the methanol dried in a stream of air prior to addition of the other substrates. The other inhibitors were made up in water and netitralized by NaOH to pH 7.4.

Results and Discussion Most of the experiments were carried out using dihydroxyacetone as the gluconeogenic substrate. Dihydroxyacetone is not a normal physiological substrate, but has been useful in whole cell studies to clarify likely sites of hormonal action in the liver cytosol [6]. We have examined phosphofructokinase fluxes using the procedure previously described of determining ~4C incorporation on C-6. of glucose from a tracer level of [134C]galactose. The principle of this approach is as follows: If there is phosphofructokinase flux opposing the net gluconeogenic flux, [134C]fructose 6-phosphate (formed from the substrate [134C]galactose) will generate [1)4C]fruc tose-l,6-bisphosphate. Since the aldolase and triosephosphate isomerase reactions are fairly near to equilibrium, [334C]dihydroxyacetone 3-phosphate

and [3A4C]glyceraldehyde 3-phosphate will be formed, followed by formation of fructose lJ>bisphosphate labelled on both C-I and C-6. Tile phosphofructokinase flux thus leads to a degree of labelling of C-6 of glucose, although the major extent of labelling occurs at C-l, since [l)4C]glucose 6-phosphate is the initial entry point in the gluconeogenic pathway of 14C introduced from [1)4C]galactose, and since the major outflow from glucose 6-phosphate is to glucose. In this paper, the approach is used only to give a qualitative (or at best semi-quantitative) index of changes in pbosphofructokinase flux under varying conditions. Some of the errors or limitations of the method have been discussed previously [6]. It should .be pointed out that simply doubling the fractional amount of ~4C on C-6 of glucose will considerably underestimate the ratio of phosphofructokinase to net gluconeogenic flux, since (a) this neglects the fact that the hexosephosphate isomerase and aldolase reactions are not at complete isotopic equilibrium; and (b) this neglects the considerable outflow of v~C from the triosephosphate pool to lactate, which is pronounced with dihydroxyacetone as the substrate. Thus, a factor of 5 or 6, rather than 2, would provide a more suitable conversion of fractional ~4C on C-6 to relative phosphofructokinase flux under these conditions. With a substrate such as lactate, where the net flux is strongly from pyruvate to triosephosphate, the conversion factor should be considerably lower, but still greater than 2. However, in this case (and in general, with the case involving dihydroxyacetone being anomalous in that the rate of triosephosphate isomerase is not a factor [6]) another factor is required for lack of complete isotopic equilibration in the triosephosphate isomerase reaction, which is well documented for the liver [18,19]. Table I shows that there is considerable phosphofructokinase flux, opposing the net gluconeogenic flux, when dihydroxyacetone is the sole substrate in hepatocytes from fasted rats. The values of about 3-5% of t4C on C-6 of glucose from incubations with [134C]galactose are similar to those found previously [6], from which we previously estimated that the rates of phosphofructokinase flux ranged from about 15% to nearly 40% of the net gluconeogenic flux. Addition of high concentrations ( 5 - 2 0 raM) of lactate to the dihydroxyacetone medium caused a

375 TABLE 1 EFFECT OF ADDED LACTATE ON PHOSPHOFRUCTOKINASE FLUX DURING GLUCONEOGENESIS FROM DIHYDROXYACETONE Hepatocytes from fasted rats were incubated with substrates as shown in 5 ml of Krebs-Henseleit buffer with [1-14C]galactose (approx. 1 gCi) added as a tracer (<0.1/~mol). DHA, dihydroxyacetone. Experiment

Glucogenic substrates

Glucose formation 01mol/g per h)

From [ 1-14C]galactose 14C on C-6 of glucose (% of total)

1

DHA (10 mM) DHA (10 mM) + lactate (10 mM) Lactate (10 mM)

92 130 54

4.5 2.4 2.2

2

DHA (10 mM) DHA (10 mM) + lactate (20 mM) Lactate (20 mM)

94 126 53

5.0 1.9 1.6

3

DHA (5 mM) DHA (5 mM) + lactate (5 mM) DHA (5 mM) + pyruvate (5 mM)

83 117 126

2.9 1.2 0.9

4

DHA (10 mM) DHA (10 mM) + lactate (I0 mM) DHA (10 mM) + pyruvate (10 mM) Lactate (10 mM) Pyruvate (10 mM)

120 155 175 52 49

4.6 1.8 1.4 1.5 1.0

TABLE I1 EFFECT OF INHIBITORS ON LACTATE CONTROL OF PHOSPHOFRUCTOKINASE FLUX Hepatocytes from fasted rats were incubated with the substrates and inhibitors shown, with a tracer of [1-14C]galactose. DHA, dihydroxyacetone. Experiment

Glucogenic substrates

Inhibitor

5

DHA (10 mM

None Aminooxyacetate (0.2 mM) Mercaptopicolinate (1 mM) a-Cyano-4-hydroxycinnamate (1 mM)

DHA (10 mM) + lactate (10 mM)

DHA (5 mM)

6

DHA (5 mM) + lactate (5 mM)

Glucose formation 0zmol/g per h)

From [1-14C] galactose 14C on C-6 of glucose (% of total)

88 90 81 76

4.4 4.2 4.0 6.9

None Aminooxyacetate (0.2 mM) Mercaptopicolinate (1 mM) c~-Cyano-4-hydroxycinnamate (1 mM)

124 102 103

2.6 2.6 2.4

86

6.7

None Aminoxyacetate (0.2 mM) Mercaptopicolinate (1 mM) ~-Cyano-4-hydroxycinnamate (1 mM)

79 84 80

3.2 2.7 3.1

68

4.3

115 107 104

1.2 1.5 1.0

80

4.9

None Aminooxyacetate (0.2 mM) Mercaptopicolinate (1 mM) c~-Cyano-4-hydroxycinnamate (1 mM)

376 marked decrease in the phosphofructokinase flux (Table I) such that the values for percent 14C on C-6 at glucose approach the lower values obtained when lactate alone is used as the gluconeogenic substrate in hepatocytes from fasted rats. In a fewer number of experiments, pyruvate at high concentrations also decreased phosphofructokinase backflow. (While we are focusing on phosphofructokinase flux in this paper, the probable major cause of the increase in gluconeogenesis caused by addition of lactate or pyruvate to dihydroxyacetone is the reversal of the direction of net flux between triose phosphate and

We have used a nmnber of inhibitors in order to suggest possible mechanisms for the effect of lactate o13 phosphofructokinase flux (Table ll). The effect of lactate is completely blocked by c~-cyano-4hydroxycinnamate, an inhibitor of pyruvate transport into the mitochondria [20]. This indicates that lactate or pyruvate itself is not an allosteric regulator, but that metabolism of lactate on pyruvate is required to produce a regulator. Mercaptopicolinate, which at 1 mM largely inhibits gluconeogenesis from lactate by blocking conversion of oxalacetate to phosphoenolpyruvate [21,22], does not diminish tile lactate-induced decrease in phosphofructokinase flux.

pyruvate.)

TABLE III EFFECT OF PREINCUBATION WITH GLUCOSE ON PHOSPHOFRUCTOKINASE FLUX Hepatocytes from fasted rats were preincubated (where noted) with glucose, and then further incubated with 10 mM dihydroxyacetone (or 5 mM xylitol, Experiment 13 only) together with the substrate present during the preincubation period. The cells were not washed free of any glucose present during preincubation, and only slight changes in glucose concentration occurred in the preincubation period. DHA, dihydroxyacetone. Experiment

Glucose concn, during preincubation (raM)

Preincubation time (rain)

Glucose concn, for final incubation (raM)

final incubation time (rain)

From [ 1.14 C] galactose 14 C on C-6 of Glucose (% of total)

7

0 0

0 0

0 10

30 30

3.9 4.1

8

0 0

0 0

0 10

40 40

4.3 4.7

9

0 10 10 10

60 15 30 60

10 10 10 10

20 20 20 20

3.9 5.2 6.8 7.1

10

0 27

60 60

0 27

30 30

2.0 6.7

11

0 10

20 20

10 10

40 40

4.7 6.3

12

0 0

30 0

0 l0

10

. 5

10

I0 10 10 10 10

10 15 20 25 30

10 10 10 10 10

30 30 30 30 30 30 30 30

1.6 2.2 2.7 3.0 3.1 3.6 3.9 3.9

0 l0 20 30

40 40 40 40

0 10 20 30

30 30 30 30

3.3 6.4 6.5 6.9

13

Xylitol Xylitol Xylitol Xylitol

377 TABLE IV EFFECT OF GLUCOKINASE INHIBITORS ON CONTROL OF PHOSPHOFRUCTOKINASE FLUX Hepatocytes from fasted rats were preincubated with the substrates noted, then further incubated with 10 mM dihydroxyacetone (DHA) plus [ 1-14C ]galactose. Experiment

Glucose concn, (mM)

Glucosamine concn, (nM)

14

0 10 10 0

0 0 10 10

t5

0 0 10 10 0 10 0

0 0 0 10 10 0 0

Time (min)

From [ 1 - 1 4 C ] galactose 14C on C-6 of glucose (% of total)

0 0 0 0

45 45 45 45

4.7 6.1 1,9 1.9

0 0 0 0 0 20 20

30 30 30 30 30 30 30

3.5 4.4 6.3 2.4 2.1 2.5 2.3

Final incubation period

Preincubation period N-Acetyl glucosamine concn. (nM)

Time (min)

Glucose concn, (raM)

Glucosamine concn, (raM)

0 0 0 0

15 15 15 15

0 10 10 0

0 0 10 10

0 0 0 0 0 20 20

30 30 30 30 30 30 30

0 10 10 10 0 10 0

0 0 0 10 10 0 0

This result suggests that the effector produced by lactate metabolism is not a phosphorylated intermediate in the pathway of gluconeogenesis, but rather a compound produced in the mitochondria. Also, the lack of effect o f aminooxyacetate, a general transaminase inhibitor, suggests that accumulation o f an amino acid such as glutamate, aspartate or alanine is probably not involved. A likely candidate for control of phosphofructokinase flux would, of course, be citrate, as this compound has been shown to inhibit phosphofructokinase from a number of tissues, including liver [23,24]. However, at present we have insufficient data to establish whether lactate may control phosphofructokinase flux by increasing cytosolic citrate. Similar effects are seen when pyrurate is used instead of lactate, together with the inhibitors used in Table II (results not shown). It can be seen (Table II) that not only did a-cyano4-hydroxycinnamate block the inhibitory effect of lactate on phosphofructokinase flux, but also that this compound, by itself, increased phosphofructokinase flux (whether or not lactate was present). One can only speculate at present on the mechanism of this effect. One possibility is that maintenance of

N-Acetyl glucosamine concn. (raM)

the levels o f the intermediates of the Krebs cycle in the mitochondria depends upon a balance between toss to the cytosol and reformation via pyruvate carboxylation. Thus, outflow of malate from the mitochondria to the cytosol, followed by the sequence of malate -* oxalacetate-~ phosphoenolpyruvate ~ p y r u v a t e may occur continuously to some degree, and blocking pyruvate reentry into the mitochondria could lead to a lowering of tricarboxylic acid cycle intermediates. While lactate, the major physiological substrate for gluconeogenesis, produces an inhibition of phosphofructokinase flux, glucose, the product of gluconeogenesis, causes an increase in phosphofructokinase flux under the conditions used (Table Ill). Here, however, the effect of glucose is rather small when it is added at the same time as dihydroxyacetone (plus [1-14C]galactose). But if the cells are preincubated with glucose (concentrations of 10 mM or higher were used in these experiments), a 2 - 3 - f o l d increase in phosphofructokinase can be produced. Similar results of glucose preincubation were seen when xylitol replaced dihydroxyacetone as the gluconeogenic substrate. However, when lactate was used as the glu-

378 coneogenic substrate during the final incubation period, we could demonstrate no significant effect o f glucose preincubation on phosphofructokinase flux (results not shown). This suggests that, whatever the mechanisms, the 'lactate effect' may override the 'glucose effect'. This has as of yet only been tested at high (10 mM or greater) lactate concentrations. Again we have used inhibitors in an attempt (here unsuccessful) to clarify whether the glucose effect might involve the glucose molecule itself or whether metabolism of glucose is required. In these experiments we employed glucosamine or N-acetylglucosamine as inhibitors of glucokinase [25]. As seen in Table IV, if these inhibitors were present together with glucose during the preincubation period, no activation of phosphofructokinase flux during the subsequent incubation was apparent. Rather, a depression of this flux occurred. However, glucosamine or N-acetylglucosamine decrease phosphofructokinase also in the absence of glucose. These experiments, thus, do not clarify whether or not glucose metabolism is required to produce a stimulation of phosphofructokinase. The apparent time dependence of the glucose effect (Table III) is analogous to the effect of glucose in the control o f glycogen synthesis or degradation reported by Hers and coworkers [26]. The relative importance, however, o f glucose or glucose 6-phosphate in the regulation of glycogen metabolism in the liver is highly controversial [27,28]. Brand and Soling [29] have reported previously an effect of glucose on activation of phosphofructokinase measured in a cell-free homogenate. Van Schaftingen et al. [30] have reported recently that intravenous injection of glucose (200 rag/100 g body weight to fasted rats caused a marked activation o f phosphofructokinase flux, estimated by the [1-14C] galactose procedure. Our results indicate that both glucose and lactate may exert partial control of gluconeogenesis (or glycolysis) by action at the site of phosphofructokinase. Hormonal control also seems now to be exerted partially at this locus, superimposed on these substrate effects. References 1 Mansour, T.E. and Mansour, J.M. (1962) J. Biol. Chem.

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