Studies on the Mechanism of Action of Glucagon on Gluconeogenesis*

SUMMARY The mechanism by which glucagon stimulates gluconeogenesis was studied in livers perfused with 1 mM lactate, 0.1 mM pyruvate, and several different 14C-labeled substrates. In livers perfused for 235 min with [14C]lactate plus [‘“Cl-pyruvate, glucagon increased [14C]glucose synthesis 3-fold, increased the labeling of tissue P-pyruvate, aspartate, alanine, and glycogen, and decreased the labeling of pyruvate, oxalacetate, malate, citrate, cr-ketoglutarate, and glutamate. The hormone also decreased the tissue levels of pyruvate, malate, citrate, cr-ketoglutarate, and glutamate and increased those of aspartate and P-pyruvate. These changes are in-terpreted as indicating that glucagon stimulated the conversion of oxalacetate to aspartate and P-pyruvate. Tryptophan produced changes in livers perfused with [14C]lactate plus [Wlpyruvate which were consistent with inhibition


SUMMARY
The mechanism by which glucagon stimulates gluconeogenesis was studied in livers perfused with 1 mM lactate, 0.1 mM pyruvate, and several different 14C-labeled substrates.
In livers perfused for 235 min with [14C]lactate plus ['"Clpyruvate, glucagon increased [14C]glucose synthesis 3-fold, increased the labeling of tissue P-pyruvate, aspartate, alanine, and glycogen, and decreased the labeling of pyruvate, oxalacetate, malate, citrate, cr-ketoglutarate, and glutamate. The hormone also decreased the tissue levels of pyruvate, malate, citrate, cr-ketoglutarate, and glutamate and increased those of aspartate and P-pyruvate.
These changes are interpreted as indicating that glucagon stimulated the conversion of oxalacetate to aspartate and P-pyruvate.
Tryptophan produced changes in livers perfused with [14C]lactate plus [Wlpyruvate which were consistent with inhibition of gluconeogenesis at P-enolpyruvate carboxykinase. It blocked completely the effects of glucagon on the labeling of glucose and P-pyruvate at 244 or 20 min, and caused the hormone to induce accumulation of malate and aspartate. The extra malate and aspartate which accumulated under these circumstances were derived from unlabeled sources (e.g. endogenous protein) and not from pyruvate as indicated by their decreased or unchanged radioactivity. In livers perfused with H14C03 for 1 min, glucagon decreased the radioactivities of oxalacetate, malate, citrate, and a-ketoglutarate and increased those of aspartate, Ppyruvate, succinate, and fumarate. The specific radioactivities of intermediates were consistent with fixation of CO2 predominately into malate, aspartate, and citrate. The changes induced by glucagon indicated stimulation of aspartate and P-pyruvate formation from oxalacetate and of succinate formation from or-ketoglutarate.
Further evidence for an effect of glucagon at a site between oxalacetate and P-pyruvate was provided by perfusions with [Wlaspartate.
These showed that glucagon decreased the radioactivities of aspartate, malate, oxalacetate, and citrate and increased the radioactivity of P-pyruvate at both 246 * This work was supported by project program Grant AM07462 from the National Institutes of Health, United States Public Health Service. Preliminary reports of some of this work have appeared (1,2). ,t Present address, Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
$ Investigator of the Howard Hughes Medical Institute. and 835 min, despite the fact that the specific radioactivities of P-pyruvate precursors were reduced. Although the interpretation of these data is subject to reservation because of the existence of multiple pools of metabolic intermediates, they provide consistent support for the view that P-pyruvate synthesis from oxalacetate is a major site of action of glucagon on gluconeogenesis in the liver.
Several studies have shown that glucagon activates gluconeogenesis in the perfused liver (3-6).
The effect is mediated by adenosine 3' : 5'-monophosphate (5) by an unknown mechanism. It appears to be exerted primarily on a reaction(s) located in the gluconeogenic pathway between pyruvate and P-pyruvate (6,7). Although early studies suggested that it was secondary to increased lipolysis (3,8), this view has been disputed (9,10).
This report presents the results of a study of the effects of glucagon on the labeling of glucose and metabolic intermediates in livers perfused with 14C-labeled lactate, pyruvate, aspartate, and bicarbonate in the absence or presence of tryptophan.
The data are consistent with the view that the gluconeogenic action of glucagon involves activation of P-enolpyruvate carboxykinase. were consistent with carboxylation of pyruvate and conversion of oxalacetate to other Krebs cycle intermediates and P-pyruvate. Glucagon decreased the specific radioactivities of pyrvuate, malate, and I?-pyruvate and increased that of aspartate (Table I).
As will be discussed, these changes are consistent with stimulatory effects of glucagon on t,he conversion of 4-carbon dicarbosylic acids to P-pyruvate, on the conversion of glutamate and a-ketoglutarate to 4-carbon dicarboxylic acids, and on the transamination of pyruvate to alaninc and of oxalacetate to aspartate' Effects  [W]Pyruvate in the Absence or Presence of Tryptophan-In the preceding experiments, it was possible that activation of P-pyruvate synthesis from 4-carbon dicarboxylic acids might have obscured a simultaneous stimulatory effect of glucagon on pyruvate carboxylation.
To examine this point, the effects of glucagon were examined in livers in which P-cnolpyruvate carboxykinase was inhibited by addition of 2.4 rnM tryptophan to the perfusion medium.
In one series of experiments, the exposure to substrates and hormone was of relatively long duration, i.e. 20 min. Table IT presents the results.
In the absence of tryptophan, glucagon lowered the tissue levels of pyruvate and citrate and raised those of malate and I'-pyruvatc.
The hormone also dccrcascd the radioactivities of pyruvate, oxalacctate, malate, and citrate and increased those of P-pyruvate and glucose. The specific radioactivities of pyruvate and malatc were decreased, whereas those of aspartate, citrate, and Ppyruvate were unchanged.
These changes support the view that glucagon activates gluconeogenesis at a site between the 4-carbon dicarboxylic acids and P-pyruvate.
Tryptophan treatment increased the tissue levels of pyruvate, malate, aspartate, and citrate and markedly lowered the level of P-pyruvate (Table II).
In the presence of the inhibitor, glucagon increased aspartate but did not alter the other intermediates. As expected, tryptophan increased the radioactivities of intermediates prior to P-pyruvate in the gluconeogenic pathway and lowered that of P-pyruvatc (Table II).
Labeling of glucose was diminished by 94%. In tryptophan-blocked livers, glucagon decreased malate and citrate radioactivities but did not significantly change the isotope content of other intermediates.
The hormone also reduced the specific radioactivities of malate and citrate under these conditions.
These results provide no evidence that glucagon stimulates pyruvatc carboxylation.
The increased levels of malate and aspartatc induced by the hormone by guest on March 22, 2020 http://www.jbc.org/ vate in control perfusions and abolished the increases induced by glucagon (Fig. 1). The inhibitor also increased the tissue levels of malate and aspartate and, in its presence, glucagon produced further large increases (Fig. 1). As expected, the radioactivities of malate and aspartate were increased in the tryptophan-treated livers (data not shown).
In such livers, glucagon decreased malate radioactivity and did not alter aspartate radioactivity (Fig. 2). Consequently glucagon markedly decreased the specific radioactivities of malate and aspartate (Fig. 2) substrates, e.g. glutamate and protein (see 11). The possibility that an effect of glucagon on pyruvate carboxylation might have been discernible at shorter time periods was also examined in experiments in which livers were exposed to substrates and hormones for 2,L$ min in the absence or presence of tryptophan.
The results obtained were very similar to those seen in the perfusions of longer duration.
Tryptophan treatment markedly inhibited the labeling of glucose and P-pyru-EJecl of glucagon on tissue levels arvl W conlent of inlerrne~liales in livers perfused wilh [WI bicarbonale Livers from fed rats were perfused for 1 hour with rccircLdat,ing medium and then for 11 min with I,-lactate-pyruvate (1 mM-0.1 rnM) in the presence or absence of glucagon in a flow-through system.
[%]Bicarbonate was infused at a rate of 29 X lo6 cpm for 1 min before the liver was excised. Tissue powder from six COW trol or six glucagon-treated livers was combined and analyzed for intermediates in duplicate.
Values for chemical amount are t,he means of duplicate estimations which differed by less than 5%. in livers exposed for a short period (1 min) to HW03. Table  III shows t,hat glucagon pretrcatmcnt for 10 min produced changes in the levels of mctabolites which were consistent with increased conversion of 4-carbon dicarboxylic acids to Ppyruvate and aspartate.
The labeling of intermediates in control livers was indicative of WO2 fixation into a small pool of oxalacctate which turned over extremely rapidly leading to labeling of malatc aspartate, citrate, P-pyruvate, and other Krcbs cycle intcrmediates during the I-min time period (Table III).z Glucagon produced marked changes in the labeling pattern.
The radioactivities of a-ketoglutarate, malate, oxalacetate, citrate, and pyruvate were decreased, whereas those of succinatc, fumaratc, aspartate, P-pyruvatc, and glucose were increased. These changes are similar to those observed with [14C]lactatc plus [Wlpyruratc (Table I) and with ['%]glutamate (11) and arc consistent with multiple effects of the hormone, namely, activation of the conversion of oxalacetate to I'-pyruvatc and aspartate and of a-ketoglutarate to succinate.

Eflects of Glucagon on Gluconeogenesis and Labeling of Metaboiic Intermediates in Livers Perfused with [W]
Aspartate-Since the preceding results pointed to a possible effect of glucagon on P-enolpyruvate carboxykinase, this was tested more directly by using [l*C]aspartate, a substrate which could yield oxalacetatc in the cytosol without first participating in mitochondrial mctabolism. Table IV shows the changes in livers from fasted rats perfused with lactate, pyruvate, and [14C]aspartate and exposed to glucagon for 2j$ or 8:s min. It is seen that the effects produced at S>$ min were generally more striking than those seen at 2;.6 min. The hormone increased the tissue concentration of P-pyruvate but had little effect on the other intermediates mcasured. It also reduced the radioactivitics of aspartatc, malatc, oxalacetate, and citrate and increased those of P-pyruvatc and Livers from rats fast,ed for 18 to 24 hours were perfused as described in Table I in the presence of absence of glucagon (5 X 10-P M) in a flow-through system. In  Glucagon produced similar alterations in livers from fed rats perfused with [14C]aspartatc except that the iucrease in [14C]glucase synthesis was more marked (4-fold) and there were no significant changes in the specific radioactivitics of intermediates (data not shown).
The data with this labeled amino acid are thus again consistent with an action of glucagon on P-pyruvate synthesis from oxalacctate.
The present results provide several lines of evidence indicating that glucagon stimulates gluconeogcnesis in part by activating P-pyruvate synthesis from oxalacctatc.
First are the changes in the tissue concentrations of intermediary metabolites (Figs. 1 and 2, Tables I to IV) which are similar to those found in previous studies (6,7,11). These indicate a site of interaction b&w-ten the 4-carbon dicarboxylic acids and P-pyruvatc which is discernible at early time periods and is abolished by tryptophan ( Fig. 1 (Table III and References 6 and 7), the stimulation must involve a mechanism such as activation of pyruvate carboxylase (6) or facilitation of mitochondrial pyruvate uptake (13). However, efforts to demonstrate effects of glucagon on pyruvate carboxylation were unsuccessful in the present study. When Pcnolpyruvate carbosykinase was blocked by treatment with tryptophan, no effect of glucagon to increase the conversion of [%]pyruvate to osalacetate, aspartate, or malate was discerned at early or late time periods.
There was also no indication that the hormone increased the total radioactivity in oxalacetate, malate, aspartate, and citrate in livers perfused with H14C03 for 1 min.
It could be argued that the negative results in the tryptophanblocked livers were due to the fact that the flow of substrate beyond oxalacetate was impeded.
The increased levels of malate, aspartate, and citrate could conceivably have inhibited pyruvate carboxylation.
However, studies with purified pyruvate carboxylasc do not indicate that product inhibition is an important control mechanism for this enzyme (14,15).3 Another explanation could be that stimulation of carboxylation is dependent on changes in the gluconeogenic pathway at or beyond P-cnolpyruvate carboxpkinase or on other changes resulting from increased flus through the gluconeogenic pathway.
Many schemes can be developed which link pyruvate carboxylation with other gluconeogenic reactions, e.g. coupling pyruvate entry with movement of other metabolites across the mitochondrial membrane or linking changes in adcnine nucleotides or CoA esters with alterations in pyruvate carboxylase activity (14)(15)(16)(17).
As noted in our studies of glutamate metabolism in the perfused liver, the present findings indicate an effect of glucagon to increase the transamination of oxalacetate to aspartatc and of pyruvate to alanine.
The explanation for these changes is uncertain, but they could bc secondary to the enhancement of aketoghitaratc oxidation by glucagon (11). This increases the transamination of glutamate to cu-ketoglutarate (ll), and it is likely that oxalacetate and pyruvate scrvc as acceptors of amino vroups. D Gl ucagon stimulation of gluconeogcnesis from a physiological mixture of lactate and pyruvate, as in the present study, would require an increase in the mitochondrial rfflux of aspart.ate rather than malatc, since there is minimal need for hydrogen transfer iuto the cytosol.
An increase in the transamination of glutamate relative to its oxidation would therefore be more consistent with the enhanced gluconeogenesis.
Since GTP is an inhibitor of glutamate drhydrogenasc (18), its increased production in the succinyl thiokinase reaction could be a factor in the apparent enhancement of glutamate transamination by glucagon.4 Stimulation of l'-pyruvate synthesis from oxalacetato was suggested as a mechanism for the gluconeogenic action of glucagon in 1965 (20). The effect could be due to an activation of l'-cnol- pyruvate carboxykinase or to a stimulation of the efflux of aspartate, malate, or oxalacetate from the mitochondrion.
Regarding the first mechanism, it has not been possible to observe any rapid increase in the activity of P-enolpyruvate carboxykinase in livers perfused with glucagon (2l).5 In addition, no changes in the enzyme have been observed in cell-free extracts incubated with adenosine 3': 5'-monophosphate under a wide variety of conditions (22).5 Thus, if activation of P-enolpyruvate carboxykinase occurs, it would appear to involve a mechanism different from phosphorylase activation.
It has been suggested that the efflux of dicarboxylic acids from mitochondria may be rate-limiting for gluconeogenesis in liver (2,23). This would not be inconsistent with measurements of metabolite levels in livers perfused with concentrations of lactate which were saturating or supersaturating for gluconeogenesis (7). Facilitation at this site, if limiting, would lead to decreased over-all levels of 4-carbon dicarboxylic acids since these would be rapidly metabolized in the cytosol.
However, in the present study glucagon had little effect on the tissue concentration of malate and increased the level of asparate.
Furthermore, glucagon increased [14C]glucose formation from [14C]aspartate, a substrate which can yield oxalacet'ate directly in the cytosol.
Attention has been focused on the region of the gluconeogenic pathway between pyruvate and P-pyruvate in the present report Yince earlier studies indicated that this was the principal site at which glucagon stimulated gluconeogenesis from physiological substrates (5,24). These studies showed, for example, that maximal glucose production from substrates entering the pathway at 2-P-glycerate or above (e.g. fructose and dihydrosyacetone) was much greater than that from lactate, pyruvate, or amino acids and was not increased by glucagon (5,24). More recently, however, Veneziale has found that glucagon csln produce a large stimulation of glucose production from fructose in the perfused liver if subsaturating concentrations of the hexose are employed, and that the stimulation is not diminished by prior perfusion with quinolinate (25). Although the possibility remains that the quinolinate may not have completely blocked gluconeogenesis from lactate derived from fructose (see Footnote 6), these data suggest that glucagon may exert an additional effeet on a reaction located at the upper end of the gluconeogenic pathway or involved in the metabolism of fructose prior to entry into the gluconeogenic pathway (see Footnote 3 in Reference 7). The former location seems more likely since many studies of the effects of glucagon on the perfused liver have shown metabolite changes consistent with activation of the conversion of triose-P to glucose-6-P (6,7,26). The mechanism(s) by which glucagou affects this portion of the gluconeogenic pathway and the possible physiological importance of such an effect(s) remain to be clarificd.6 5 M. Ui and T. 1-T. Claus, unpublished observations. 6 Veneziale has also raised the intriguing possibility that pyruvatc may be converted to 2-P-glycerate in the liver by an unknot\-n pathway which is not sensitive to inhibition by quinolinate (27,28