111 NH4 + plus glucagon was synergistic. This suggested that glucagon also stimulates the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U -~ 4 C] alanine into glucose by ~-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.

Introduction Isolated liver cells are capable of synthesizing glucose from various substrates [1--15] at rates comparable to those observed in isolated, perfused livers. They also appear to retain the normal metabolism of lipids [14,16--18] and protein [3,19]. When they are exposed to very high concentrations of glucagon, they respond by increasing their content of cyclic AMP [3,6,7], increasing glycogenolysis [6,7,20], and slightly increasing gluconeogenesis [6,7,9--12]. This communication characterizes the incorporation U -~ 4C-labeled substrates into glucose by hepatocytes from fed rats and the response to a maximally effective concentration of glucagon. The effect of various compounds on the response of these cells to glucagon was also investigated. A number of additions are made to the results of earlier studies and some results and interpretations differ from those previously reported. Another paper will describe the sensitivity of these hepatocytes to various hormones in relationship to both gluconeogenesis and cyclic AMP metabolism [21]. Materials and Methods

Isolation o f liver cells. Male rats (150--300 g, Sprague-Dawley, Madison, Wisconsin) fed Purina Laboratory Chow ad libitum were used. Their livers were perfused essentially according to the method of Berry and Friend [1] b u t with the following modifications: (1) The perfusion buffer used was Krebs-Henseleit bicarbonate buffer [5] without CaC12, but with 5 m M glucose and 0.25% Fraction V albumin (Pentex); (2) the medium was gassed by vigorously bubbling 02/CO2 (95 : 5, v/v) directly into the perfusion reservoir and no adjustments of pH were made during perfusion; (3) the flow rate was reduced to approx. 14--15 ml/min in order to prevent disruption of the liver by hydrostatic pressure [ 5]; (4) 150 mg]l crude collagenase (Worthington) and 400 mg/1 of hyaluronidase (Sigma) were used; (5) EDTA was omitted. After approx. 40 min perfusion, the cells were dispersed as described by Berry and Friend [1] and the suspension was shaken for 10 min with the enzyme-containing medium used for perfusion and gassed continuously with the 02/CO2 mixture. The cell suspension was filtered through nylon mesh and then washed twice with Krebs-Henseleit bicarbonate buffer containing normal Ca 2÷ concentration (2.5 mM) and 1% Fraction V albumin. The final cell pellet was resuspended to a volume of 80--120 ml with the same buffer and the cells were incubated with continuous gassing in a 250-ml Erlenmeyer flask without any added substrates for 20--40 min. This incubation, as well as the use of a dilute cell suspension, may be important to assure the presence of normal ATP to ADP ratios in the

112 cells (Claus, T.H. and Pilkis, S.J., unpublished). While the yield of cells was somewhat variable, 90--95% of them excluded 0.2% trypan blue and appeared to have normal morphology under light microscopy. Plastic labware was used throughout, and all procedures were performed at or near 37°C to minimize the loss of intracellular K ~ [22]. Incubation of cells and analyses of substrates and products. 1-ml aliquots of the final cell suspension were added to 17 × 100 mm plastic tubes (Falcon Plastic) that contained the appropriate additions (5--20 pl) of substrates and hormones made from concentrated solutions. The tubes were gassed for a minute with 02/CO2 (95 : 5, v/v) while shaking vigorously (200 cycles/min) in a 37°C bath, then stoppered and the incubation continued with vigorous shaking for a total of 30 min unless otherwise stated. Reactions were usually stopped with 0.5 ml 5% ZnSO4 ; 0.5 ml of 0.15 M Ba(OH)2 and 2 ml of water were then added and the precipitate removed by centrifugation. [14 C] Glucose was separated from charged substances by the procedure of Exton and Park [23], with the resin treatment repeated once. Aliquots of the resin-treated supernatants were added to 10 ml of Aquasol (New England Nuclear) and analyzed for [~ 4 C] glucose in a Packard Tri-Carb liquid scintillation counter. When radioactivity in glycogen as well as glucose was to be assayed, or when substrates and products were to be assayed, reactions were stopped with 0.5 ml of 7.5% HC104 [24]. The precipitate was washed three times with 1 ml of 2.5% HC104 and the combined supernatants neutralized with K2 CO3. An aliquot of the neutralized extract was assayed for radioactivity in glucose plus glycogen by the resin treatment described above. Lactate, glucose, pymvate, alanine and ~-hydroxybutyrate were assayed cnzymatically [25]. All enzymes were obtained from Boehringer, Mannheim. Glucagon was a gift from Eli Lilly and Co. DNA analysis. Aliquots of the final cell suspension were analyzed for DNA by a modification of the m e t h o d of Burton [26], using calf t h y m u s DNA as standard. Diphenylamine reagent was added to cells that had been precipitated in 0.2 M HC104 and the entire mixture was boiled for 10 min, cooled to room temperature, centrifuged and read at 595 rim. Expression of results. Rates of incorporation of U -J 4 C-labeled substrates into glucose were expressed as pmol of substrate (lactate, pyruvate, or alanine) converted to glucose per 30 min per mg DNA. The number of micromoles incorporated was obtained by dividing the total radioactivity incorporated into glucose by the specific activity of added uniformly ~4 C-labeled substrate. No correction was made for 16% of the radioactivity that is lost to 14 CO2 via randomization of oxaloacetate in the Krebs cycle and subsequent decarboxylation the phosphoenolpyruvate. The experiments presented are with cells from individual rats. All the data are the mean -+S.E. of three or more observations and each experiment has been vertified by repetition from two to four times. Results

Time and cell concentration dependence of [1 4 C] glucose synthesis Fig. 1A shows the time course of [ 14 C] glucose synthesis from physiologi-

113 0.7-

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+

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250

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DNA (u.g)

Fig. 1. ( A ) T i m e c o u r s e o f t h e i n c o r p o r a t i o n o f 2 m M [ U - 1 4 C ] l a c t a t e i n t o glucose. Cells were first i n c u b a t e d 30 m i n w i t h o u t any additions and then incubated with substrate for (B) I n c o r p o r a t i o n o f [ U -14 C] l a c t a t e i n t o g l u c o s e as a f u n c t i o n o f t h e c o n c e n t r a t i o n o f cells V a r i o u s c o n c e n t r a t i o n s o f cells w e r e f i r s t i n c u b a t e d 30 m i n w i t h o u t a n y a d d i t i o n a n d t h e r a i n w i t h s u b s t r a t e as i n d i c a t e d . In b o t h cases, t h e g l u c a g o n c o n c e n t r a t i o n w a s 10 riM.

f r o m fed r a t s various times. f r o m f e d rats. i n c u b a t e d 30

cal levels (2 mM) of [ U -'4 C] lactate by cells isolated from livers of fed rats. A maximally effective concentration of glucagon (10 nM) stimulated [,4 C] glucose synthesis more than 2-fold. Both the control and hormone-stimulated rates were linear for at least 60 min. (Longer times were not investigated.) Similar linear rates were observed with higher lactate concentrations and with [ U - ' 4 C ] p y r u v a t e or [U-' 4C]alanine as substrates (data n o t shown). In all subsequent experiments, rates of incorporation of substrate into glucose were determined over a 30-min period. The synthesis of [' 4 C] glucose from 1.4 mM [ U J 4 C]lactate was linear to approx. 175 pg DNA per ml of cell suspension, and nearly linear to 350 pg per ml, in the absence of glucagon (Fig. 1B). With 10 nM glucagon or with higher substrate concentrations the rate of [,4 C] glucose synthesis was linear to 350 pg DNA per ml. All subsequent experiments were performed at a cell concentration of less than 250 pg DNA per ml and usually with only 80--120 pg DNA per ml. A similar dependence on cell concentration existed when [U -'4 C]pyruvate and [ U-14 C] alanine were substrates.

Rates of [' 4 C] glucose synthesis from UJ 4C-labeled lactate, pyruvate, and alanine and the effect of NH4 +on [~ 4-C] glucose synthesis from lactate The relationship between [ ~4 C] glucose production and the concentration of U J 4-labeled lactate, pyruvate, or alanine was investigated (Fig. 2). The rates with lactate as substrate (Fig. 2A) were greater than those observed with an equivalent concentration of alanine (Fig. 2B) b u t were less than those observed with pyruvate (Fig. 2D). The addition of 2 mM NH4 C1 (Fig. 2C) raised the rates of incorporation into glucose from [ U J 4 C] lactate to those observed with [U-' 4 C] pyruvate. A maximally effective glucagon concentration (10 nM) stim-

114

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[ U - ' " C ] SUBSTRATE (raM) Fig. 2. I n c o r p o r a t i o n o f U -14 C - l a b e l e d were first incubated 30 rnin without a b s e n c e ( - ) o r p r e s e n c e (;,) o f 1 0 n M c o n c e n t r a t i o n ( m M ) a n d V is t h e r a t e o f

s u b s t r a t e s i n t o g l u c o s e as a f u n c t i o n o f t h e i r c o n c e n t r a t i o n s , C e l l s any additions and then incubated 30 min with substrate in the g l u c a g o n . I n s e r t : H a n e s p l o t o f t h e d a t a w h e r e S is t h e s u b s t r a t e conversion to glucose (t~mol/30 min per mg DNA).

ulated [' 4 C] glucose synthesis from all concentrations of each substrate. This result differs from some previous reports [10,11] where glucagon inhibited gluconeogenesis from high pyruvate concentrations. With each substrate, the greatest percent stimulation (2--4-fold) was observed at low substrate concen-

115

trations while only a 60--140% stimulation was observed at very high substrate concentrations. Analysis of the data by the use of Hanes plots (inserts, Fig. 2) indicated that the major effect of glucagon was to lower by 75% the concentration of U_l 4 C-labeled substrate necessary for half-maximal rates of incorporation into glucose. In the absence of glucagon, these concentrations of lactate, alanine, pyruvate, and lactate plus NH4 Cl were 5.0, 3.0, 5.7 and 15.4 mM, respectively. Glucagon lowered these concentrations to 1.1, 0.7, 1.4 and 3.8 mM, respectively. Glucagon also had a smaller effect (30%) on the maximum rates of conversion of U -~ 4 C-labeled substrates into glucose. It raised them from 1.6, 1.2, 3.6, and 5.1 p m o l / 3 0 min per mg DNA for lactate, alanine, pyruvate, and lactate plus NH4 C1, respectively, to 2.1, 1.7, 4.7, and 6.3 pmol/30 min per m g D N A . It appears from the data of Fig. 2 that NH4 C1 increased the conversion of labeled substrate into glucose only from high lactate concentrations. A comparison of the rates of incorporation from 2 and 20 mM [U- 14 C] lactate, with and without NH4 C1, in the same cell preparation confirmed this observation (Table I). The effect of NH4C1 is thought to be due to the stimulation of glutamate synthesis which is necessary for transamination of oxaloacetate to aspartate in the mitochondrion [11]. At physiological levels of lactate, 2 mM NH4 C1 did not increase the conversion of [ U J 4 C] lactate to glucose, presumably because sufficient glutamate was present to meet the demands of transamination. In contrast to the findings of others [ 1 1 ] , NH4 C1 was not necessary for glucagon to exert its maximum effects. The same percent stimulation by glucagon was observed with 20 mM [U -J4 C]lactate plus NH4 C1 as was observed without NH4 C1, even though NH4 C1 doubled the actual rates. The effect of NH4 C1 was not mimicked by preincubating cells with a full complement of amino acids at their normal plasma concentration, probably because they are not readily transported into the cell. The variation in rates of conversion of U -14 C-labeled substrate to glucose among cells prepared on separate days was a b o u t the same as that observed TABLE

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w i t h o u t a n y a d d i t i o n s or w i t h a f u l l c o m p l e m e n t o f a m i n o a c i d s ( A A ) at t h e i r rat c o n c e n t r a t i o n s f o r 3 0 r a i n . G l u c o n e o g e n e s i s w a s t h e n m e a s u r e d o v e r t h e n e x t 3 0 rain a f t e r t h e appropriate additions of substratc, NH4+ and glucagon. When present, the NH4+ concentration was 2 mM a n d t h a t o f g l u c a g o n w a s 1 0 m M . T h e r e s u l t s are e x p r e s s e d a s g m o l c o n v e r t e d t o g l u c o s e / 3 0 r a i n p e r m g Cells were incubated

plasma

DNA.

Lactate (raM)

AA

NH4CI

Without glucagon

With glucagon

2

+ +

+ +

0.65 0.69 0.51 0.57

± 0.03 +- 0 . 0 3 ± 0.01 ± 0.06

1.62 1.80 1.60 1.47

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20

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0.09 0.23 0.12 0.12

116 with the perfused liver system. With 2 mM [ U-~ 4 C] lactate as substrate, the rate of incorporation into glucose w i t h o u t glucagon was 0.62 _+ 0.03 pmol per 30 min per mg DNA for 33 cell preparations and in the presence of glucagon was 1.96 + 0.16 pmol per 30 min per mg DNA for 30 preparations. For 18 cell preparations with 0.8 mM [U -~ 4 C] alanine as substrate, the rates of conversion to glucose were 0.22 ± 0.03 and 0.66 ± 0.10 pmol/30 min per mg DNA, without and with glucagon, respectively. If it is assumed that there are 2.5 mg DNA per g liver [27], and also that ]6% of the radioactivity from ]U-' 4 C] lactate or [UJ 4 C] alanine is lost to ' 4 CO2, the rates of conversion to glucose of 2 mM [UJ 4 C] lactate were 0.06 ± 0.003 and 0.19 ± 0.02 pmol per min per g liver. With 0.8 mM [U -] 4 C] alanine, the rates were 0.021 -+ and 0.063 ± 0.01 pmol per min per g liver. When the concentration of lactate was 2 mM, [14 C] glucose synthesis accounted for about 30% of the lactate taken up in the absence of glucagon (2.0 ± 0.5 pmol/30 min per mg DNA). Glucagon almost doubled the uptake and [ ] 4 C] glucose synthesis accounted for about 50% of the lactate consumed.

Effects o f t3-hydroxybutyrate on the conversion o f [U -'4 C] alanine to glucose It has been proposed that the lactate dehydrogenase reaction provides the reducing equivalents required at the triose phosphate dehydrogenase step when lactate is the substrate for gluconeogenensis. However, when pyruvate or alanine is the substrate for gluconeogensis, the reducing equivalents are provided by the export of malate from the mitochondria [28--32]. It has also

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l

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Fig. 4. I n c o r p o r a t i o n of 0.8 m M [ U - 1 4 C ] a l a n i n e i n t o glucose as a f u n c t i o n of the c o n c e n t r a t i o n of o r a c e t a t e . C e i l s w e r e i n c u b a t e d a s d e s c r i b e d i n Fig. 2.

ethanol

117 been proposed that glucagon stimulates gluconeogenesis by enhancing fatty acid oxidation, which decreases the [ N A D ] / [ N A D H ] ratio in the mitochondria. This, in turn, favors malate formation and promotes malate export [ 3 0 - - 3 5 ] . It was of interest, therefore, to determine whether the addition of fi-hydroxybutyrate, which decreases the mitochondrial [ N A D ] / N A D H ] ratio [ 3 6 ] , would mimic the effect of glucagon. To this end, isolated hepatocytes were incubated with either 0.8 mM [U- 14 C] alanine or 13 mM [U -14 C] alanine and various concentrations of/3-hydroxybutyrate (Fig. 3). Increasing concentrations of ~-hydroxybutyrate caused an increase in both the basal and glucagonstimulated rates of conversion to glucose. With a physiological concentration of alanine (0.8 mM), the percent stimulation by ~-hydroxybutyrate was a b o u t the same in the absence or presence of glucagon. However, the ketone b o d y stimulated more in the presence of glucagon than in its absence when a saturating alanine concentration was used. With either concentration of [U -14 C] alanine, the effects of ~-hydroxybutyrate and glucagon were synergistic; the stimulation by ~-hydroxybutyrate plus glucagon was greater than the sum of the stimulation by each agent. Analysis of the data by Hanes plots indicated that glucagon reduced the concentration of fi-hydroxybutyrate necesary for half-maximal incorporation rates (data not shown). The effect of ~-hydroxybutyrate probably reflects a change in redox state and is n o t due to the increased ketone b o d y concentration per se, since incubation of cells with increasing concentrations of ~3-hydroxybutyrate plus acetoacetate maintained at a constant ratio of 2.5 (the value reported [37] for livers from well-fed rats) did n o t appreciably alter either the basal or glucagon-stimulated rates (data n o t shown). The addition of acetoacetate, which could increase the [ N A D ] / [ N A D H ] ratio, had no significant effect at any concentration with or without glucagon. Effects o f acetate and ethanol on the conversion o f [U -~ 4 C] alanine to glucose It has also been proposed that glucagon stimulates lipolysis and raises the level of acetyl-CoA. This activates pyruvate carboxylase and stimulates gluconeogenesis [33,38--46]. To examine this proposal, isolated hepatocytes were incubated with various acetate concentrations and the rates of incorporation into glucose of 0.8 mM [U -14C]alanine were examined (Fig. 4). Maximum stimulation was observed at 4 mM for both the basal and hormonaUy stimulated rates. Similar results were obtained when the alanine concentration was 13 mM (data n o t shown). Ethanol, which supplies reducing equivalents directly to the cytoplasm [ 4 7 ] , gave very similar results to those with acetate when either 0.8 mM [U -14C]alanine (Fig. 4) or 13 mM [U -14C]alanine (data not s h o w n ) w e r e substrate. With either acetate or ethanol, the absolute increases in the rate of incorporation into glucose from both alanine concentrations were a b o u t the same in the presence or absence of glucagon and the stimulatory effects of glucagon and acetate or ethanol were additive. Effect o f glucose on the incorporation o f [ U-14 C] lactate into glucose High perfusate glucose concentrations inhibited the incorporation of labelled alanine into labeled glucose plus glycogen in the perfused rat liver

118 15mM LACTATE T + GLUCAGON

w

O o z3 .J (.D O Ir~ z L~ a

2"

~ 0 re~ w ~ t-

] ~

15mM L A C T A T E

i

2.5 mM LACTATE

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z"-----.-........~

2'0

GLUCOSE ( m M ) F i g . 5. I n c o r p o r a t i o n o f [ U - l 4 C ] l a c t a t e i n t o g l u c o s e p l u s g l y c o g e n as a f u n c t i o n g l u c o s e . C e l l s w e r e i n c u b a t e d as d e s c r i b e d i n F i g . 2.

of the concentration

of

[48]. However, Haft [49] and Exton and Park [23] showed that high concentrations of glucose did n o t suppress labeled lactate incorporation into glucose plus glycogen. It was of interest, therefore, to determine whether the incorporation of labeled lactate into glucose plus glycogen was inhibited by high glucose concentrations in isolated hepatocytes. It was of interest also to determine whether the stimulation by 10 nM glucagon was affected by glucose. Isolated hepatocytes were incubated with either 2.5 or 13 mM [U -]4 C] lactate and various concentrations of glucose and the incorporation of label into glucose plus glycogen was determined. With increasing glucose concentration, a progressive inhibition of incorporation was observed in the absence of glucagon at both the low and high lactate concentration (Fig. 5). This inhibition was not due to shunting of labeled glucose into glycogen as both glucose and glycogen were extracted (see Materials and Methods) and measured. The inhibition also was not due to dilution of gluconeogenic intermediates generated by increased glycolysis because the same percent inhibition was observed (e.g. 60% at 30 mM glucose) when the rate of incorporation was doubled by increasing the [U -]4 C] lactate concentration 5-fold. Glucose did not affect the rate of incorporation of labeled lactate into glucose when glucagon was present. Similar results have been reported by Clark et al. [50]. Discussion

Hepatocytes were prepared from fed rather than fasting rats because of the greater response of gluconeogenesis to stimulation by glucagon. With fasting rats, the basal rates of gluconeogenesis in vivo [51], as well as in isolated hepatocytes (Claus, T.H. and Pilkis, S.J., unpublished), are elevated 4-fold. This is probably the result of chronic exposure of the liver to elevated plasma glucagon concentrations. The addition of glucagon to hepatocytes from fasting

119

rats does n o t greatly stimulate gluconeogenesis, particularly when high substrate concentrations are used (Claus, T.H. and Pilkis, S.J., unpublished). Therefore, it appears that hepatocytes from fed rats are the cells of choice to study the stimulation of gluconeogenesis by glucagon. However, glucose is produced both by gluconeogensis and glycogenolysis in cells from fed rats and it is difficult to distinguish one source from the other by chemical analyses. The only sensitive method available to study gluconeogenesis in fed animals is the incorporation of radioactive substrate into glucose. This incorporation involves a complex series of reactions with several points where labeled carbon can be diverted from the pathway and several points where endogeneous substrates can enter the pathway [52,53]. Thus the rate of incorporation of U_14 C-labeled substrate into glucose is not the true rate of gluconeogenesis. It is, however, an estimate of gluconeogenesis that will reflect changes in gluconeogenesis brought about by hormones or other agents*. The rates of labeled substrate conversion to glucose reported here are in general agreement with those reported for perfused livers of fed rats or in vivo. The basal rate of incorporation into glucose of 2 mM [U -~ 4 C] lactate by hepatocytes {0.06 pmol per min per g liver) is the same as that reported for fed rats in vivo [51]. However, at saturating lactate concentrations (20 mM), the basal rate of incorporation is less than half that reported for perfused liver (0.5--0.6 pmol per min per g liver; refs 55 and 56) or in vivo (0.4 pmol per min per g liver; ref. 51). The addition of NH4 *, however, as described by Zahlten et al. [ 1 1 ] , restores the rate to that observed with pyruvate and to a rate nearly equal to that in vivo [51]. This accords with the view of Zahlten et al. [11] that the rate is limited in the absence of added ammonia by a deficiency of glutamate which is necessary for transamination of oxaloacetate to aspartate. [14C] Glucose synthesis from high concentrations of [U- 14 C] pyruvate, which is much less dependent on transamination, is not stimulated by NH4 ÷ and the rate of label incorporation is greater than from lactate. Alanine gives rates of incorporation that are slightly less than those observed with lactate at comparable concentrations. With a concentration of 0.8 mM, the basal rate of label incorporation of alanine in the cells (0.021 pmol per min per g liver) is similar to that in the perfused liver (0.014 pmol per min per g liver; ref. 56), but, at high concentrations it is lower [56]. Studies with the perfused liver [56] suggest that gluconeogenesis from alanine at physiological concentrations is significantly limited by transport across the plasma membrane and becomes appreciably limited at high concentration by cytoplasmic amino transferase. Presumably,

* A n y f a c t o r w h i c h p r o m o t e s g l u c o n e o g e n e s i s (e.g. i n c r e a s e d p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y ) will f a v o r t h e i n c o r p o r a t i o n of l a b e l e d s u b s t r a t e i n t o glucose. I n a d d i t i o n , s o m e f a c t o r s u n r e l a t e d to g l u e o n e o g e n e s i s will i n f l u e n c e i n c o r p o r a t i o n . ~I n c r e a s e d c o n v e r s i o n of l a b e l e d p y r u v a t e t o a c e t y l - C o A will i n c r e a s e o x a l o a c e t a t e specific a c t i v i t y a n d t h u s i n c o r p o r a t i o n i n t o glucose, while i n c r e a s e d c o n v e r s i o n of e n d o g e n o u s s u b s t r a t e s t o a c e t y l - C o A will d e c r e a s e o x a l o a c e t a t e s p e c i f i c a c t i v i t y a n d the i n c o r p o r a t i o n i n t o glucose. In p e r f u s e d liver, an analysis of [ 1 - 1 4 C ] - a n d [ 2 -14C] lac t a t e c o n v e r s i o n to glucose a n d CO 2 s h o w e d b o t h o f these " n o n - g l u e o n e o g e n i c " f a c t o r s to be a l t e r e d b y g l u c a g o n in such a w a y as t o diminish i n c o r p o r a t i o n while in fact g l u c a g o n i n c r e a s e d it [ 5 4 ] . Thus, i n c o r p o r a t i o n d a t a u n d e r e s t i m a t e d s o m e w h a t t h e d e g r e e to w h i c h g l u c o n e o g e n i c factors e n h a n c e d i n c o r p o r a t i o n in r e s p o n s e to glucagon. T h e possible i n f l u e n c e of " n o n - g l u c o n e o g e n i e " f a c t o r s h a s n o t b e e n f o r g o t t e n in the i n t e r p r e t a t i o n of the d a t a , b u t t h e i r p r e s e n c e d o e s n o t invalidate the conclusions.

120 these steps also limit utilization of alanine in the hepatocytes and reduce the gluconeogenic rate relative to that from lactate or pyruvate. The exact site(s) and mechanism of action of glucagon on gluconeogenesis remain unknown. The data presented here show that the hormone stimulates incorporation from all concentrations of U -14 C-labeled lactate, pyruvate or alanine, but that the greatest effect is seen with low (physiological) concentrations of substrate. With each substrate, the major effect of a maximum concentration of glucagon was to lower the concentration of substrate necessary for a half-maximal incorporation rate. A physiological glucagon concentration (0.5 nM) also reduced the half-maximal substrate concentrations by 50% but did not alter the m a x i m u m rate (Claus, T.H. and Pilkis, S.J., unpublished). Thus, the glucagon effect on the m a x i m u m rate of incorporation may not be important under physiological conditions. These observations do n o t imply that the K m of a rate-limiting enzyme is decreased by glucagon. They can be explained in several ways. For example, glucagon may increase the V of a step prior to the saturable step. Moreover, saturation of the pathway may represent depletion of a cofactor as rates increase rather than saturation of an enzyme site. This appears to occur in the absence of NH4 C1. A more definitive explanation must await metabolite analysis. It should be noted that the physiological concentrations of these substrates are less than those necessary for half-maximal incorporation. Were this not the case, the system would be poorly responsive to glucagon. It is also interesting to note that activation of gluconeogenesis by glucagon about doubles the percent of lactate converted to glucose as well as increasing uptake. Suppression of pyruvate dehydrogenase activity by glucagon, as suggested by Claus et al. [54] and Zahlten et al. [10], presumably contributes to this greater efficiency, since one of the three carbons is converted to CO2 by the e n z y m e and much of the acetyl-CoA formed will enter pathways other than Krebs cycle oxidation. In isolated hepatocytes, glucagon has also been reported to inhibit gluconeogenesis from high pyruvate concentrations, presumably by inhibiting pyruvate dehydrogenase which limits the supply of NADH available to triose phosphate dehydrogenase [ 10,11]. While it seems possible that p y m v a t e dehydrogenase activity may be suppressed by glucagon, as noted earlier, it seems unlikely t h a t this would so greatly reduce the supply of NADH that gluconeogenesis would be inhibited, for there are other dehydrogenases that can supply NADH. It seems more likely that this inhibition reflects depletion of oxidizable metabolites during preparation of the cells [11], although the use of cells from fasting rather than fed animals may also be a factor. As with our cell preparation, glucagon stimulated gluconeogenesis from high pyruvate concentrations in the perfused liver system [ 57--59]. Several suggestions regarding the mechanism whereby glucagon stimulates gluconeogensis were tested in the present study. Our studies support the supposition [11,60] that one action of glucagon may be to promote transamination of oxaloacetate to aspartate, which is an essential step in gluconeogenesis from lactate. The argument is as follows (with reference to the data of Table I): (1) In the absence of glucagon, the addition of NH4 CI stimulates incorporation into glucose of 20 mM [U -'4 C]lactate, presumably by increasing the level of glutamate necessary for the oxaloacetate-aspartate shuttle system [11]. NH~ ~

121

do not stimulate incorporation of 2 m M [U-14C]lactate in the absence of hormone, presumably because sufficient glutamate is present to maintain the relatively slow rate. The rate of incorporation from this concentration of substrate is presumably limited at a step other than transamination. (2) Glucagon stimulated incorporation into glucose of 2 or 20 mM lactate in the absence of ammonia to rates distinctly faster than that from 20 mM lactate without ammonia or glucagon, where the rate is presumably limited by a lack of glutamate. Thus glucagon must mobilize sufficient glutamate to maintain an adequate flux through the shuttle. Previous studies by Ui et al. [60] pointed to a rapid (within 2 min) effect of glucagon to mobilize glutamate in the perfused liver preparation. (3) However, if NH4 C1 is assumed to satisfy completely the requirements for optimal operation of the shuttle, glucagon must have stimulatory effects in addition to promoting shuttle activity. This is evidenced by the strong glucagon stimulation of incorporation of both 2 and 20 mM [U -14 C] lactate in the presence of NH4 C1. In fact, the data of Table I suggests that ammonia and glucagon are synergistic: the stimulation of incorporation into glucose of 20 mM [U -~4 C]lactate by NH4 C1 plus glucagon is greater than the sum of the stimulation by each agent. The synergistic effect of glucagon and fi-hydroxybutyrate on the incorporation into glucose of [U-~4C]alanine (Fig. 3) seems to suggest that glucagon may also increase flux through the oxaloacetate-malate shuttle, which is a necessary step in gluconeogenesis from this substrate. A glucagon action independent of its ability to promote the activity of the oxaloacetate-aspartate shuttle is strongly supported by the stimulatory effect of glucagon on the incorporation of [U -14 C]pyruvate into glucose, which is thought to employ the oxaloacetate-malate shuttle instead of the oxalo-acetateaspartate shuttle [28--32]. Likewise, the effect of glucagon on the incorporation of [U -~ 4C]lactate into glucose suggests the hormone stimulates the process independently of its ability to promote the activity of the oxaloacetate-malate shuttle. Thus it appears that glucagon has several actions which stimulate gluconeogenesis from three carbon substrates. The exact sites and mechanisms of these effects remain unknown. The stimulation of [U -~4 C] alanine incorporation into glucose by glucagon does not appear to be mediated significantly by provision of cytoplasmic reducing equivalents, since stimulation by ethanol is trivial in magnitude compared to that by the hormone. Similarly, provision of acetate, an excellent precursor for acetyl-CoA (which has been postulated to mediate the effect of glucagon by activation of pyruvate carboxylase), has only a small effect in comparison to that of the hormone. The additive effects of glucagon and ethanol or acetate suggest these agents acted by separate mechanisms. The mechanism whereby glucose inhibits the incorporation of [U -~4 C]lactate into glucose is unknown. However, it has been reported that the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase were depressed when rat livers were perfused with a high glucose concentration [61]. The ability of glucagon to overcome the inhibition by glucose may suggest that glucagon and glucose exert their effects at the same site{s).

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