Effects of Fatty Acids, Glucagon and Anti-insulin Serum on the Control of Gluconeogenesis and Ketogenesis in Rat Liver

EFFECTS OF FATTY ACIDS, GLUCAGON AND ANTI-INSULIN SERUM ON THE CONTROL OF GLUCONEOGENESIS AND KETOGENESIS IN RAT LIVER JOHN R. WILLIAMSON The Johnson Research Foundation, University of Pennsylvania,Philadelphia, Pennsylvania

THE control of gluconeogenesis is a problem which remains unsolved despite extensive studies with tissue homogenates, tissue slices, mitochondrial preparations, purified enzyme systems, and the measurement of adaptive enzyme changes. Previous investigations of this problem focused attention upon three major sites in the glycolytic and gluconeogenic pathways in which control is thought to be exerted. Figure 1 summarizes these three control sites. The first site represents the conversion of glucose to glucose-6-P in the glycolytic pathway and the reversal of this reaction by hydrolysis of glucose-6-P by glucose-6-phosphatase. The second site represents the phosphorylation of fructose-6-P by phosphofructokinase and reversal by fructose-l,6-diphosphatase, while the third site depicts the formation of P-enolpyruvate from pyruvate, with oxaloacetate as an intermediate, and reversal of the overall reaction by pyruvic kinase. Recycling Reactions Of Gluconeogenesis I

Glucokinose , ~ p ATP VVI

Glucose L

2

GSP-o.

ATP +F6P P-Fruct°kin°SelFDP ÷ADP

t

FoP-o.

;

Pyruvole

3

PEP

Corboxylose Pyruvate

ATP

'

OA~ J

Corboxykinose, PFl::) GTP

------

/

Pyruvote Kinase

FIG. 1 Thermodynamicallyirreversible steps of glycolysis and gluconeogenesis. 229

230

J O H N R. W I L L I A M S O N

An alternative approach to the elucidation of control mechanisms of gluconeogenesis is to measure the concentration of metabolic intermediates in the intact liver either in vivo or after perfusion. By this means it is possible to identify control sites in vivo and ascertain whether changes in the level of control metabolites are compatible with postulated control mechanisms based on in vitro observations. This approach reveals that the carboxylation of pyruvate during the switch from glycolysis to gluconeogenesis is of particular importance, while G6P-ase and FDP-ase are of less significance. Results reported here describe the changes of metabolic intermediates in liver upon stimulation of gluconeogenesis by the administration of fatty acids or glucagon.

MATERIALS

AND

METHODS

Animals. Male albino rats of Wistar or Holtzman strain, 200-220 g in weight, were used. Fed animals had unrestricted access to food and water, while food was withheld from "unfed" rats for 18-24 hr. The rats were anaesthetized with sodium pentobarbital (50-60 mg/kg body wt) by subcutaneous or intraperitoneal injection. Liver perfusion. Livers for perfusion were taken only from unfed rats. The method of liver perfusion was adapted from that of Miller et al., ~1) with modifications as described elsewhere32~ The perfusion medium was Krebs bicarbonate buffer containing 4% bovine serum albumin (fraction V, Sigma Chemical Company) and 10mM L(+)-alanine as substrate. Oleic acid was added as an emulsion by continuous infusion after the liver had equilibrated in the perfusion apparatus for 30 min. Initially, the recirculating vol. of medium was 100 ml, and 2 ml aliquots were removed periodically for chemical analyses. At the end of the experiment the liver was frozen with tongs precooled in liquid N2. In vivo studies. Several methods of glucagon administration were used. In the first method, the rat was anesthetized and glucagon infused continuously through the femoral vein at the rate of 50/xg/min for the first min, followed by 5/xg/min for the duration of the experiment. In the second method, a single dose of 250/xg glucagon was injected intraperitoneally into the unanaesthetized animal. The third method involved the injection of glucagon (250/xg/200 g rat) into the tail vein at the same time as administration of anti-insulin serum. Acute insulin insufficiency was induced by injecting 2 ml (equivalent to 6 units of beef insulin) guineapig anti-insulin serum (AIS) into the tail vein33) Insulin (5.5 units/rat) was also administered through the tail vein. Livers were frozen rapidly in situ with tongs precooled in liquid N2 30 min after injection of sodium pentobarbital. Blood sample was taken at the time of death.

231

C O N T R O L OF G L U C O N E O G E N E S I S A N D K E T O G E N E S I S

Carbon dioxide fixation into blood glucose was measured after intraperitoneal injection of 50 /zc of 14C-bicarbonate (specific activity 1 mc per mmole) in 1 ml ofNaCl. ~7) Analytical. Tissue extracts from pulverized frozen liver samples were prepared as described previously. (4) Metabolic intermediates were measured by enzymic methods (5) modified for fluorometric assays (4,6) or by procedures outlined elsewhere. (4"7"s) Materials. Crystalline glucagon (lot no. 258-234B-167-1) and beef insulin (lot no. PJ-4609) were obtained through the courtesy of Dr. W. Shaw of Lilly Research Laboratories. Enzymes, nucleotides and other cofactors were purchased from the Boehringer Mannheim Corporation, N e w York, and the Sigma Chemical Company.

RESULTS

AND

DISCUSSION

Effects of Fatty A cids on Glucose Production in the Perfused Rat Liver Previous work with liver slices, (9) kidney cortex slices (1°) and with the perfused rat liver
O/ec

//

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i

so-

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n~

l

Pyruvote Production

c

, Control

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IO 20 30 40 50 60

Time Of Liver Petfuslo~(min)

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Time Of Liver Perfumon (min)

Control %

/

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0

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Time Of Liver Perfusion (min)

FIG. 2 Effect of oleic acid on the rates of glucose, lactate, and pyruvate production in the perfused rat liver. The substrate was L-(+)-alanine present initially at a concentration of 10 mM in 100 ml perfusate.

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J O H N R. W I L L I A M S O N

followed by a maintenance rate of 3/~moles/min. Figure 2 shows that the rate of glucose production was approximately doubled by the addition of fatty acid. The Figure also shows that lactate and pyruvate production increased over the first 45 min of perfusion in control livers, and that both substrates were used at an accelerated rate after addition of oleic acid.

5rain Oleic Acid

200-

150" "e 0

¢,,.) I00

=-,?. Crossover

50-

0

:c, P~rT O~AMol T PiP2 PT3&A' o~PFOP T F~ G6P ' G,~,~ G A GAP ~B

~ / 3 0 m i n Oleic Acid

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o

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50

Crossover

FIG. 3 Crossover plot of gluconeogenic intermediates in perfused rat livers, 5 and 30 rain after addition of oleic acid. The vertical bars represent two standard errors of the percentage change of intermediate levels after oleic acid infusion relative to the mean control value, with six to eight livers in each group. Abbreviations used: Lact, lactate; Pyr, pyruvate; 0,4.4, oxaloacetate; Mal, malate; PEP, P-enolpyruvate; 2PGA, 2-P-glycerate; 3PGA, 3-P-glycerate; GAP, glyceraldehyde-3-P; DAP, dihydroxyacetone-P; FDP, fructose- 1,6-di-P; F6P, fructose-6-P; G 6 P , glucose-6-P.

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

233

Changes in the levels of glycolytic intermediates in livers following administration of oleic acid are shown in Fig. 3. The data are presented in the form of a crossover plot, in which the intermediates are placed along the abscissa in the order in which they are currently thought to be formed in the gluconeogenic enzyme sequence. The ordinate gives the percentage changes of each intermediate relative to the mean control value. Within 5 rain of oleic acid addition, pyruvate and lactate levels decreased, while the level of malate increased. These changes indicate possible control in the conversion of pyruvate to oxaloacetate, despite unchanged or depressed oxaloacetate levels. This is because oxaloacetate and malate remain in equilibrium determined by the D P N H / D P N + ratio, since malate dehydrogenase is highly active in liver. Levels of P-enolpyruvate and P-glyceric acids also decreased, and a second forward crossover is observed between 3-P-glyceric acid and glyceraldehyde-3-P, indicating the existence of a site of interaction at the glyceraldehyde-Pdehydrogenase:P-glyceric kinase enzyme combination. Hexose monophosphates and fructose- 1,6-di-P remained higher than controls, indicating that the rate of the fructose-1,6-diphosphatase reaction was sufficient to support the elevated flux associated with the enhanced rate of glucose formation. Oxidation of fatty acids by mitochondria is accompanied by changes in the ratios of pyridine nucleotide-linked substrate redox couples which are present in both mitochondrial and cytoplasmic compartments. Figure 4 shows that the ratios of lactate:pyruvate and malate:oxalo-

Lact/Pyr Ratio

.a-Glycero-P/DAP MoIote/OAA Ratil Ratio

Oleic Acid 20-

-20

t00

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Oleic Acid

I I

50

I0 Control Control b

'

'

~o

b

'

'

3'o

b

'

'

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Minutes After Oleic Acid Addition FIG. 4 Effect o f oleic acid on substrate redox pairs in the pei'fused rat liver.

234

JOHN R. WILLIAMSON

acetate increased approximately 2-fold upon the addition of oleic acid. The ratio of a-glycerophosphate:dihydroxyacetone-P also increased, but to a lesser degree, possibly because of an enchanced rate of ot-glycerophosphate utilization by esterification with fatty acids, (12)or oxidation by the cytochrome-linked mitochondrial a-glycerophosphate oxidase. (13) The increased ratio of the substrate redox pairs is indicative of a more negative redox potential of the cytoplasmic DPN-system, and suggests that facilitation of glyceraldehyde-P-dehydrogenase could occur by virtue of an increase in the concentration of cytoplasmic D P N H . Pi t

~.Gl~gen ATP Acetyl CoA

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-ATP GTP

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:--i.Fatty ACids

FIG. 5 Control sites in the gluconeogenic pathway. Abbreviations used: LDH, lactic dehydrogenase; PYR Carb, pyruvate carboxylase; PEP CK, P-enolpyruvate carboxykinase; PGK, P-glycerate kinase; GAPDH, glyceraldehyde 3-P dehydrogenase; UDPG, uridine diphosphoglucose; Gly Syn, glycogen synthetase; Phos, phosphorylase; G6PDH, glucose-6-P dehydrogenase; CS, citrate synthase; A cetyl CoA Carb, acetyl CoA carboxykinase.

The effects of increased availability of fatty acids on liver metabolism may be summarized with reference to the general control diagram shown in Fig. 5. Increased oxidation of fatty acids increases ketone body formation, D P N H , T P N H , and fatty acyl-CoA levels. (2) Acetyl-CoA levels are also increased in livers from starved (~,14,1s) or diabetic-cortisoltreated rats,(16) conditions each associated with elevated plasma free fatty acid levels and enhanced ketogenesis. Acetyl-CoA levels probably increased in the present experiments with the perfused rat liver, but this has not been ascertained. The increased levels of the cofactors have secondary effects on many metabolic pathways in the cell, which contribute to the altered metabolic state of the organ when fatty acid availability is increased.

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

235

Reducing equivalents formed during fatty acid oxidation are reoxidized by the electron transport chain, and therefore may compete with reductive steps of the citric acid cycle, and reduce flux in the latter pathway. Of the D P N H thus formed by fatty acid oxidation, part is used in the reductive step of gluconeogenesis and part for the rephosphorylation of nucleotide diphosphates produced by the pyruvate carboxylase, PEPcarboxykinase, and P.-glyceric kinase reactions. Acetyl-CoA is, itself, an activator of pyruvic carboxylase, t17) and hence it may be envisioned that the rate of fatty acid oxidation facilitates the gluconeogenic pathway by coordinating control at the input step (pyruvic carboxylase) with that of the reversible triose-P dehydrogenase step. Ketone bodies may be regarded as a by-product of fatty acid oxidation, since the rate of production of acetyl-CoA exceeds its rate of disposal by the pathways of the citric acid cycle and fatty acid synthesis. These alternative pathways for acetyl-CoA utilization may, in fact, be inhibited as pointed out previously by Wieland,OS) owing to inhibition of citrate synthetase and acetyl-CoA carboxylase by long chain fatty acyl-CoA. The relevance of these and other postulated control mechanisms based on the kinetic behavior of enzyme systems in vitro to the metabolic changes in the intact liver will be discussed later. In summary, the present findings show that increased availability of fatty acids provides an effective means of rapidly increasing the rate of glucose production by gluconeogenesis from suitable 3-carbon precursors. Effects of Glucagon on Hepatic Gluconeogenesis and Ketogenesis in Fed Rats in vivo Glucagon has a well-defined glycogenolytic effect in liver. "9) It is evident from recent work of several laboratories that glucagon also increases the rate of gluconeogenesis, o1'2°22) This effect presumably accounts for the earlier observations of Miller t2a) that urea production by the liver is increased by glucagon. However, the effects of glucagon on lipid metabolism are more controversial3 ~9) The remainder of this paper will be devoted to an examination of the possibility that the gluconeogenic and ketogenic effects of glucagon are secondary to enhanced lipolysis. (4, s, 24, 25) Selection of an in vivo system has the advantage of being more physiological than the isolated liver, but the disadvantage that the rates of glucose production and utilization are more difficult to measure. Figure 6 shows that glucagon (administered by intraperitoneal injection) greatly stimulated the rate of fixation of 14C-bicarbonate into blood glucose. The count-incorporation into glucose with glucagon was maximum after 30 rain, and declined abruptly to control levels after 60-90 rain. These

236

JOHN

R. WILLIAMSON

10to '_o

Glucogon -..

o

~6-

m

2-

Control

0

I

0

0

J i

I

i

60 Minutes

90

120

FIG. 6

Effect of gluca$on (250 v-g added by intraperitoneal injection) on the incorporation of 14C-bicarbonate into blood glucose in rats in vivo. Each point represents the mean +SEM of four determinations.

results indicate that the effect of glucagon on gluconeogenesis in the normal fed rat is rapid, but transient. A similar transient effect of glucagon on ketogenesis is illustrated in Fig. 7. The level of ketones in the liver is more than doubled 5 min after glucagon addition and further increases after 30 min, but declines toward control levels after 60 min. The increase in the tissue levels of ketones with time in the control animals, however, may represent an effect of anesthesia. Fatty acyl-CoA and acetyl-CoA levels are also elevated by glucagon, particularly after 30 min, and CoA levels follow a reciprocal pattern. By 60 min, acetyl-CoA and CoA levels are similar to the controls. The activity of a triglyceride lipase of liver has recently been shown to increase with glucagon.(25) Therefore, it is probable that the elevated concentrations of CoA esters and ketones observed after glucagon treatment are caused by enhanced availability of free fatty acids, both in the blood (4) and in the intracellular environment. Measurement of the changes in the levels of the gluconeogenic intermediates 5, 30, and 60 min after glucagon infusion (Fig. 8) show that the onset of glycogenolysis, like lipolysis, is rapid. This is indicated by the greatly elevated levels of hexose monophosphates 5 min after glucagon administration. Lactate, pyruvate, and oxaloacetate levels decrease,

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

Control

300-

J

200I00O

Acetyl Co A o

I00-

~

50. 0

237

Glucagon.

i

Total Ketones

2000I0000

5min

50min FIG. 7

60min

Effects o f glucagon, added by c o n t i n u o u s intravenous infusion to anesthetized

rats, on the hepatic levels of CoA, acetyI-CoA, fatty acyl-CoA and total ketones. The glucagon dose was 50 #.g/min for the first rain followed by 5/zg/min for the duration of the experiment. The vertical bars represent two standard errors of the mean with four rats in each group. while the intermediates b e t w e e n P - e n o l p y r u v a t e and triose-P are elevated. A f t e r 30 min these changes are, for the m o s t part, m o r e pronounced, and a clear-cut c r o s s o v e r is o b s e r v e d b e t w e e n oxaloacetate and malate. A f t e r 60 min, lactate, p y r u v a t e , oxaloacetate and malate levels are similar to controls, but other intermediates remain elevated. Since CO2 fixation is increased b y glucagon, o b s e r v e d changes of metabolic intermediates are most readily interpreted as indicating a facilitation of the p y r u v a t e - c a r b o x y l a s e step b y the elevated levels of a c e t y l - C o A . T h e failure of o x a l o a c e t a t e levels to rise as its rate o f formation is increased m a y be explained b y the c o n v e r s i o n of part o f the o x a l o a c e t a t e so formed into malate, according to the following reactions: Pyruvate+ATP+COz

Mg~*

Acety~Co^ ~ O x a i o a c e t a t e + A D P + P i

O x a l o a c e t a t e + D P N H + H + ~ Malate + D P N + Pyruvate + ATP+ CO2+ DPNH+

(1)

(2)

H + ---> Malate + D P N + A D P + Pi

238

JOHN R. WILLIAMSON

300

2 200

8 I00

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Mol

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FDP

G~oP

30min Glucogon 200" ¢_)

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|

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•

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!

mm

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PEP I SPGAI DAP I F 6 P ~Gtucose Mal 2P'GA GAP F[~° G6P

300"

IO0-

OL;c," oiA" Pyr

Mol

"3P'GA"oiP" F;P "G&ose 2PGA GAP

FDP

G6P

FIG. 8 Crossover plots of the gluconeogenic intermediates after 5, 30 and 60 min treatment of anesthetized rats with glucagon. The experimental conditions were the same as those of Fig. 7.

The equilibrium of eq. (2) is pushed from left to right because of a shift of the redox potential of the DPN system to a more negative state, (~4) and malate accumulates instead of oxaloacetate. The fall of malate, acetyl-CoA and ketones towards control levels 60 min after glucagon infusion, and the sharp fall in 14C-bicarbonate incorporation into blood glucose between 30 and 60 min, probably reflect a decrease in the rate of lipolysis. The anti-lipolytic effect of insulin in adipose tissue is well documented(~6"~) and therefore the transience of some of the glucagon effects in the normal fed animal may be due to endogenous insulin release from the islet cells elicited either directly

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

239

by glucagon or indirectly by the elevated blood glucose levels. The glycogenolytic effect of glucagon is apparently less sensitive to reversal by insulin than the lipolytic, ketogenic, and gluconeogenic effects. This is indicated by the greatly elevated levels of glucose, hexose phosphates, and other glycolytic intermediates in livers 60 min after the start of glucagon infusion (Fig. 8). In summary, glucagon has a rapid, but transient, stimulatory effect on the rate of gluconeogenesis and ketogenesis in livers of normal fed rats. Changes of the metabolic intermediates are consistent with these effects being caused by an enhanced rate of lipolysis, which increases the tissue levels of long- and short-chain fatty acid CoA esters. Increased levels of these CoA esters may provide the basis for the gluconeogenic action of glucagon, since they stimulate the carboxylation of pyruvate to oxaloacetate and inhibit the conversion of pyruvate to acetyl-CoA. The transient effects of glucagon reported here, and the numerous contradictory reports of glucagon effects on lipolysis and ketogenesis in the literature,~19) may be explained by the release of insulin which suppresses glucagon-induced lipolysis.

Effects of Glucagon on Rats Treated with Guinea-pig Anti-insulin Serum In order to circumvent the effects produced by the secondary release of insulin in response to the glucagon-induced hyperglycemia, rats were treated with guinea-pig anti-insulin serum at the same time as glucagon administration38) The serum from each rat at the time of death was assayed for unneutralized insulin antibody to insure that the AIS was given in sufficient amount to neutralize all the endogenous circulating insulin. The metabolic effects in rats 30 and 60 min after injection of glucagon plus AIS are compared with those produced by AIS alone, using rats treated with normal guinea-pig serum as controls. In order to investigate further the effects of glucagon and insulin interaction in vivo, excess insulin was injected intravenously to rats 30 min after prior treatment with AIS plus glucagon, and the livers were frozen 30 or 60 min later. Figure 9 shows that AIS treatment alone significantly increases plasma glucose levels, and that glucagon added with the AIS causes a further elevation. Insulin added to the AIS plus glucagon-treated rats causes a large decrease of plasma glucose, presumably through a combination of decreased hepatic production and increased peripheral utilization. The more prolonged effect of glucagon, when added together with the AIS, is to be contrasted with the relatively transient effect noted when glucagon is added to the normal fed rat. This is also illustrated by the time course of the count incorporation from 14C-bicarbonate into blood glucose shown in Fig. 10. Anti-insulin serum alone causes a 2-fold increase

240

J O H N R. W I L L I A M S O N

400 E

200

0

Normal

(30') (60') AIS

(30') (60") AI5 + Glucogon AIS + Glucagon (30') Followed By Insulin

FIG. 9 Effects of guinea-pig anti-insulin serum (AIS), AIS plus glucagon, and insulin reversal of these effects on plasma glucose levels in the rat. Three different treatments are shown: (1) AIS-treatment alone for 30 and 60rain; (2) AIS plus glucagon treatment for 30 rain and 60 min; (3) AIS plus glucagon treatment for 30 min followed by insulin, which was allowed to act for 30 rain and 60 min. The vertical bars represent two standard errors of the mean while the number of rats in each group is shown in parentheses. of counts in glucose (expressed as glucose per ml of blood), and is further increased by glucagon over a period o f 2 hr. Butyrate added to normal rats also increases 14C-glucose formation, thereby giving support to the concept that the stimulation of CO2 fixation into glucose observed with AIS or glucagon is secondary to the release o f free fatty acids. Figure 11 shows plasma F F A levels in the same series of experiments as those illustrated in Fig. 9. T h e levels are increased by AIS, further elevated by AIS plus glucagon, and subsequently decreased by insulin. One interesting difference between the changes of plasma glucose and F F A is that while glucose levels increased significantly, after 30 and 60 min treatment with AIS, F F A levels showed little further rise. This difference is also revealed by the changes in the total ketone content of the liver (Fig. 12) which are similar to those of the F F A , except that the initial high rate of ketogenesis induced by glucagon was not maintained for as long as the elevated F F A levels. Changes in the tissue levels of fatty acyl-CoA (Fig. 13) follow the same general pattern as the plasma F F A changes during the different experimental treatments, with the exception that the increase of fatty a c y l - C o A after 30-min exposure of the rat to A I S is less than might be expected from the increase of plasma F F A . T h e administration of glucagon with AIS increases fatty acyl-CoA levels after a 30- or 60-min

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

12-

AIS +Glucagon ( 250Fg - i,p. )

2 '_o E O=

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Butyrate AI$

8 4"10 0 O0

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) ) i 60 90 120 Minutes FIG. 10 Incorporation of ~C-bica.'bonate into blood glucose in rats treated with AIS, AIS plus glucagon, and with sodium butyrate (1 remote by intraperitoneal injection). The vertical bars represent two standard errors of the mean with four rats in each group. 30

12C

cr

sc E

4C

Normal

(50") (60") (50') (60") AI5+ Glucocjon AIS AtS + Glucogon (50') Followed By Insulin

FIG. 11 Effects of AIS, AIS plus glucagon and reversal of these effects by insulin, on plasma free fatty acid (FFA) levels. The experimental conditions were the same as those of Fig. 9.

241

242

JOHN R. WILLIAMSON

o

Insulin (30'1 (60')

.

0 ~~

Normal

(30') (60') AIS

(30') (60') AIS+Glucocjon AIS + Glucogon (30') Followed By Insulin

Fro. 12 Effects of AIS, AIS plus glucagon and reversal of these effects by insulin on the hepatic levels of total ketones in rats in vivo. The experimental conditions were the same as those of Fig. 9.

160

120

80 0

E 4O

0

Normal

(30') (60') (30') (60') AIS+Glucacjon AIS AIS + Glucacjon (30') Followed By Insulin

FIG. 13 Effects of AIS, AIS plus glucagon and reversal of these effects by insulin on the hepatic levels of long-chain fatty acyl-CoA derivatives in rats in vivo. The experimental conditions were the same as those of Fig. 9.

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

243

time interval. Although these changes are compatible with the postulated activation of a triglyceride lipase by glucagon, (25) the increase of fatty acyl-CoA can also be ascribed to the elevated plasma F F A levels. The changes of fatty acyl-CoA are to be contrasted with those of acetyl-CoA in a similar series of experiments (Fig. 14). Acetyl-CoA levels were increased significantly 30 min after administration of AIS, but were not increased further either by more prolonged treatment with either AIS or AIS plus glucagon. The elevated acetyl-CoA levels returned to normal or below normal values with the administration of insulin (Fig. 14). A comparison of Figs. 12 and 14 shows that ketone body production was not proportional to the concentration of acetylCoA, in agreement with previous findings (4) and the studies of Garland e t al., with isolated rat liver mitochondria32s) The discrepancy between

>, 2 0 0

-6 E I00

Normol

(30') (60') AIS

(30') (60') AIS+ Glucogon AIS + Glucogon (50') Follow By Insulin

FIG. 14 Effects of AIS, AIS plus glucagon and reversal of these effects by insulin on the hepatic levels of acetyl-CoAin rats in vivo. The experimentalconditionswere the same as those of Fig. 9. the tissue ketone content and acetyl-CoA content was particularly striking after 30-min treatment with AIS plus glucagon. These results provide further evidence supporting the existence of a control step in the conversion of acetyl-CoA to acetoacetate, as suggested previously by Garland. (~s) However, the nature of the control process is, at present, unknown. Changes of the gluconeogenic intermediates with AIS alone and with AIS plus glucagon are shown in Figs. 15 and 16. The data are expressed as a percentage of the values found in livers of control rats after treatment

244

JOHN R. WlLLIAMSON

300-

;-. AIS + Glucogon

., \

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30min

200

~"

/

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ol

30rain

\

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Pyr

i

i

•

l

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I

i

i

I

i

OAA i PEP [3PGA i FDP [ G6P i Mol 2PGA DAP F6P Glucose

FIG. 15 Crossover plots of the gluconeogenic intermediates in the livers of rats after treatment with AIS and AIS plus glucagon for 30 rain.

for 30 or 60 min with normal guinea pig anti-insulin serum. After 30 min treatment with AIS (Fig. 15) the changes were small, but two forward crossover sites can be distinguished; one between pyruvate and malate and another between fructose-l,6-di-P and fructose-6-P. The first crossover probably represents activation of pyruvic carboxylase by acetyl-CoA in the manner discussed earlier, while the second could represent either inhibition of phosphofructokinase or activation of fructose-l,6-diphosp~atase. The most dramatic change with glucagon plus AIS after 30 rain was an,]accumulation of hexose monophosphates

400 AIS + Glucogon ~'-,.,,,=~ / 60min " ~ /I

300-

8 200-

/. ~ , \

//'~

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Glucose

FIG. 16 Crossover plots of the gluconeogenic intermediates in the livers of rats after treatment with AIS and AIS plus glucagon for 60 rain.

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

245

(Fig. 15), caused by the stimulation of glycogenolysis. Fructose-l,6di-P and dihydroxyacetone-P levels remained depressed relative to the controls, but the reverse crossover was shifted from a site between malate and P-enolpyruvate (observed with AIS alone) to one between 3-P-glyceric acid and dihydroxyacetone-P. After 60 rain, AIS caused a further elevation of malate, while lactate and pyruvate levels remained lower than the controls (Fig. 16). Penolpyruvate, 2-P-glycerate, and 3-P-glycerate were the same or slightly higher than the controls, while the triose phosphates and fructose-1,6di-P levels remained depressed. Hexose monophosphates, however, increased more than 2-fold. These changes are identical to those reported recently by Kalkhoff e t ai.,t2a) in livers from rats treated with AIS for 3 hr. The effects produced by AIS plus glucagon after 60 min were similar to those observed after 30 min, except that the reverse crossover between 3-P-glycerate and dihydroxyacetone-P was less pronounced.

400I _ 300J

o

AIS+ Glucogj~60min AIS+ Glucogon30rain FollowedBy InsulinFor 30min

\

Followed By InsulinFor 60min

0Loect r O~Ad0IP~P ~ 5~GA ~ F~P ~ G;P Pyr 2P'GA DAP F6P Glucose FIG. 17 Effect of insulin following AIS plus glucason administration on the gluconeogenic intermediates in rat livers in vivo. The experimental conditions for insulintreatmentwere the sameas those of Fig. 9. Figure 17 shows the effect of insulin given to rats after 30-min treatment with giucagon plus AIS. Effects produced by AIS plus giucagon after 60 min are shown again in this Figure for comparative purposes. Insulin was allowed to act for 30 or 60 min before the livers were frozen. The general tendency of the changes produced by insulin was alteration of the levels of the intermediates toward control levels. The glycogenolytic effect of glucagon, however, was not abolished by insulin, since the levels of glucose-6-P and fructose-6-P remained elevated relative to the controls.

246

JOHN R. W I L L I A M S O N

Contrary to the results of Kalkhoff et a13TM the ratio of lactate:pyruvate in the liver was increased by AIS treatment (Fig. 18). The ratio of malate: oxaloacetate also increased under these conditions (Fig. 19). Glucagon produced little further change of either the lactate:pyruvate ratio or the malate:oxaloacetate ratio. The elevated ratios of these metabolic redox couples were not diminished appreciably by insulin. In fact, Fig. 18 shows that the lactate:pyruvate ratio was greater 30 min after insulin was given following AIS plus glucagon, than when AIS plus glucagon was given alone. Since insulin effectively decreased the plasma F F A level, it is clear that factors other than the rate of fatty acid oxidation also determine the redox potential of the intracellular pyridine nucleotides.

Insulin (3o') (6o') 50" 40. 302010. 0

Normol

(30') (60' AIS

(3o') (6o') AIS +Glucogon

AIS+ Glueogon (:50') Followed By Insulin

FIG. 18 Changes in the ratio of lactate to pyruvate in rat livers in vivo. The experimental conditions were the same as those of Fig. 9.

The rapid development of hyperglycemia in AIS-treated rats was caused mainly by an increased rate of glucose release by the liver, but a decreased rate of glucose utilization by peripheral tissues (3°) may also be a contributing factor. An increased rate of lipolysis by adipose tissue was also evident from the elevated plasma F F A and tissue fatty acylCoA levels. The cause of these changes was probably not due to insulin insufficiency p e r se, but to a derangement of the hormonal homeostatic balance mechanisms, so that the effects of hormones such as gIucagon, epinephrine, A C T H , and corticosteroids, which are normally counteracted by insulin in vivo, are more fully revealed in the acute insulin-

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

247

deficient statefl's) Of these hormones, glucagon may be the most important in producing the initial response to acute insulin deficiency. A comparison of the changes in the intermediates of the gluconeogenic pathway when glucagon was added to the normal fed rat (Fig. 8) with those produced by AIS after 60 min (Fig. 16) supports this theory. The most noticeable difference between glucagon in the presence and absence of AIS was in the changes of the fructose-l,6-di-P and triose phosphate levels, which increased with glucagon but decreased with AIS treatment. Unambiguous interpretation of the changes of the metabolic intermediates is difficult because of branch points and feed-back loops in the sequence of glycolytic or gluconeogenic intermediates. Thus, glycogenolysis and glucose phosphorylation directly augment the hexose monophosphate pool, while the depletion of this pool is caused by the actions ofglucose-6phosphatase, phosphofructokinase, glycogen synthetase and enzymes of the hexose monophosphate shunt. Flux through the glucose-6-phosphatase reaction is quantitatively the greatest of the four pathways removing glucose-6-P, but a precise distribution of fluxes between the pathways cannot be made. However, available evidence suggests that the hexose monophosphate shunt may be inhibited under the present experimental conditions because of inhibition of glucose-6-phosphate dehydrogenase by long chain fatty acyl-CoA derivatives. (31) Likewise, it is also possible that glycogen synthesis by the uridine diphosphoglucose pathway is inhibited in the insulin deficient state, t~2) Glucose-6-P is therefore channeled toward the production of glucose. Insulin (30') 160') 300250200.

II

150I00500

Normol

130'1 (60'1 (30'1 (60'1 AIS+Glucogon AIS AIS +Glucagon 130') Followed By Insulin

Fig. 19 Changes in the ratio of malate to oxaloacetate in rat livers in vivo. The experimental conditions were the same as those of Fig. 9.

248

JOHN R. WILLIAMSON

Fructose-1,6-di-P and the triose phosphates usually remain fairly close to the equilibrium ratios determined by the aldolase and triose-P isomerase reactions. When flux is in the direction of glucose to pyruvate, the level of fructose-l,6-di-P in the cell is determined by the relative rates of phosphofructokinase and glyceraldehyde-3-P-dehydrogenase. An increase of the hexose monophosphate levels in conjunction with a decrease of the fructose-l,6-di-P level and glycolytic flux is indicative of inhibition at the phosphofructokinase site, as discussed previously in relation to the control of glycolysis in cardiac musclefl ) When flux is in the direction of pyruvate to glucose, similar changes of the glucose-6-P and fructose1,6-di-P levels can be interpreted as indicating facilitation of fructose1,6-diphosphatase. In liver, isotopic evidence indicates that the glycolytic and gluconeogenic pathways operate simultaneously. {33,34) Hence, the results obtained with AIS-treated rats can be interpreted as indicating an inhibition of glycolysis at the phosphofructokinase site as suggested by Kalkhoff, et al3 TM However, alternative explanations, such as diminished triose-P formation by the hexose monophosphate shunt, or facilitation of fructose-l,6-diphosphatase, cannot be ruled out. In the absence of more precise knowledge concerning fluxes in the various pathways, it is only possible to state that the differences observed between the various experimental treatments probably reflect different degrees of coordination between the various control sites in the gluconeogenic pathway (Fig. 5).

GLYCOLYSIS

~

GLUCONEOGENESIS

PFK,FDP.oSe i . . .]. . .Control F~nvitro

FIG. 20 Control characteristics of phosphofructokinase and fructose-l,6-diphosphatase in vitro.

Figure 20 depicts postulated control at the sites of phosphofructokinase and fructose-1,6-diphosphatase discussed by Gevers and Krebs t35) and Newsholme and Underwood3 a6) Phosphofructokinase in vitro is inhibited by a high A T P : A D P ratio and by high citrate, while fructose1,6-diphosphatase is activated by a high A T P : A D P ratio. From the kinetic behavior of these enzymes in vitro, it is apparent that if control

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

249

of flUX is exerted in this portion of the glycolytic and gluconeogenic pathways, increased gluconeogenesis should be accompanied by an increase of the A T P : A D P ratio and/or an increase of citrate. Figure 21 shows the observed changes of the A T P : A D P ratio in the livers of rats treated with AIS and AIS plus glucagon. The general tendency was towards a decrease in the ratio, which was greater in the

Insulin (30') (60') I

I t

6

5 452I0

(13] Normal

(30') (60') (30')(603 AIS+Glucogon (30') AIS AIS+Glucogon

Followed By Insulin

FIG. 21 Changes in the ratio of ATP and A D P in rat livers in vivo. The experimental conditions were the same as those of Fig. 9.

AIS plus glucagon treated animals than in those treated with AIS alone. Insulin after 60 min, but not after 30 min, reversed the changes brought about by glucagon. It is apparent, therefore, that the observed change of the A T P : A D P ratio is in the direction opposite to that anticipated from the postulated control mechanism. Figure 22 shows changes of citrate in the same series of experiments. Anti-insulin serum produced a slight increase of the citrate level, which was statistically significant after 60 min but not after 30 min. Glucagon added with AIS had no effect after 30 min, but increased levels 2-fold above normal values after 60 min. Insulin after 30 min had the curious effect of decreasing citrate levels relative to those found after 60 min treatment with AIS plus glueagon, but subsequently increasing the citrate content if allowed to act for a further 30 rain. It must be coneluded from these studies that the observed changes of citrate appear to

250

J O H N R. W I L L I A M S O N

be too inconsistent to provide effective control of the phosphofructokinase and fructose- 1,6-diphosphatase sites. The concept that some intracellular metabolite exerts reciprocal control at these two sites, thereby preventing the energetically wasteful cycle of fructose-1,6-di-P formation and degradation, is very attractive. However, it appears that neither adenine nucleotides nor citrate fulfill the requirements for the control metabolite under the present experimental conditions, unless compartmentation is involved. Clearly, difficulties arise in the extrapolation from measurements of the total

Insulin (30') (60')

'~

~

1.5.

0.5 O,

Normol 130'1 160'1 130') 160') AIS + Glucooon AIS AIS + Glucogon (30') Followed By Insulin Fig. 22 Changes in the level of citrate in rat livers in vivo. The experimentalconditions were the same as those of Fig. 9. tissue content of metabolites to their concentration in the same phase as the enzymes whose activities they modify. The present results, therefore, cannot directly exclude the possibility that the changes of adenine nucleotides and citrate concentration in the cytoplasm are opposite in direction to those in the mitochondria. Alternatively, some other intracellular metabolite may modulate the activity of the enzymes. The recent findings of Weber e t al. (3~" as) of direct fatty acid control of key enzymes of gluconeogenesis and glycolysis are consequently of great pertinence to the present discussion. Changes in the levels of intermediates of the citric acid cycle in livers from rats treated with AIS, AIS plus glucagon and AIS plus glucagon followed by insulin are shown in Fig. 23. Treatment with anti-insulin serum for 60 min produced an increase in the levels of aspartate, acetylCoA, citrate and malate, a 50% decrease of a-ketoglutarate and a small

CONTROL OF GLUCONEOGENESIS AND KETOGENESIS

251

decrease of oxaloacetate. The changes after AIS plus glucagon treatment were qualitatively similar, except for glutamate, which also decreased. After insulin treatment, the changes were generally smaller than the corresponding changes observed when AIS plus glucagon was allowed to act for the full 60 min. Information about possible changes of flux in

AIS + Glucagon (60') ,~f

250

s

(60'

. . . . .

0

l

I

I

Asportote i AceIy~-CoA i =- ~<¢j ~ M~lote 3170 OAA 159 Citrate429Glutomote1325 12.8 862 9940 Control Values (mp.moles/g Dry Wt.)

FIG. 23 Effects of AIS, AIS plus glucagon and AIS plus glucagonfollowedby insulin, on intermediates of the citric acid cycle. The experimentalconditions were the same as those of Fig. 9. the citric acid cycle is not available directly from the present observations, but the increase of citrate and decrease of ot-ketoglutarate levels suggest that either aconitase or isocitric dehydrogenase may be inhibited under the experimental conditions. However, since many of the intermediates shown in Fig. 23 are present in both cytoplasmic and mitochondrial spaces, other interpretations of the data are possible.

SUMMARY AND CONCLUSIONS Results of the measurement of the total content of intracellular intermediates of metabolism in liver are reported for three types of experimental treatments, each of which increased the rate of glucose production. Fatty acids were added to the isolated perfused rat liver, glucagon was added to normal fed rats in viva, and glucagon was added to rats simultaneously treated with guinea-pig anti-insulin serum (AIS). Fatty acids

252

J O H N R. W I L L I A M S O N

stimulated the rate of gluconeogenesis from 3-carbon precursors. The proposed mechanism for this effect (Fig. 5) involves activation of pyruvic carboxylase by acetyl-CoA and facilitation of glyceraldehyde-3-P dehydrogenase by DPNH. Fructose-l,6-diphosphatase and glucose-6phosphatase were not rate-limiting steps. Glucagon stimulated glycogenolysis, lipolysis, and ketogenesis in rats in vivo. Stimulation of gluconeogenesis contributed to the high plasma glucose levels in addition to an enhanced rate of hepatic glycogenolysis. Experimental measurements of the changes of metabolic intermediates were consistent with the hypothesis that glucagon-induced gluconeogenesis was secondary to increased lipolysis. Stimulation of the activity of pyruvic carboxylase by acetyI-CoA appeared to be the most important step in this process. Glucagon effects in normal rats in v i v o were transient compared with similar but more prolonged effects produced by glucagon in rats treated with AIS, suggesting that insulin secretion, elicited either directly by glucagon or in response to glucagon-induced hyperglycemia, counteracted glucagon-induced metabolic responses. This hypothesis was tested directly by adding excess insulin to rats after prior treatment with AIS plus glucagon. Most of the metabolic effects produced in the AIS plus glucagon-treated rats were reversed by insulin. AIS alone produced metabolic changes which were greatly similar, but less pronounced, than the changes observed with glucagon. It is therefore suggested that the acute effects of insulin deficiency are a manifestation of the effects produced by endogenous glucagon and other lipolytic hormones. Insulin--~--~ Gtucose

Fatty Acids

t

Pyruvate t . ~ MalonylCo A

a-Glycero-P

("

I

=-

....

Triglycerides---Tr.Fatty Acids--~ Fatty Acyl Co AAc- -e t y l ATP G~I

ATP ®

/ Insulin

Fumarole

Succinate

~ I ~ OAA\I

I

~

Citrate . . . .

s cin,,:co Ko. ,Titro

.I

Co A-~ Ac Ac Co A I I II AcAc

= L-oP..

t

I~o HB J

ADP, AMP

FIG.24 Controldiagramoflipolysisand ketogenesis.A negativesigndepicts an inldbidon, and a positive sign an activation. The controls shown are taken from reports in the literature, some of which are based on findingswith in vitro crude enzyme systems, and have not been proved to functionin vivo.

C O N T R O L OF G L U C O N E O G E N E S I S A N D K E T O G E N E S I S

253

The proposed mechanism of glucagon action may be summarized with reference to Fig. 24, which also gives a symbolic representation of the possible control interactions. Glucagon stimulates the formation of cyclic 3',5'-AMP by the adenylic cyclase system present in many tissues. Production of cyclic 3',5'-AMP is counteracted by insulin. Cyclic 3',5'-AMP then triggers several activation processes. In liver, glycogen is mobilized to glucose by activation of the phosphorylase cascade. In adipose tissue, but also in liver and possibly other tissues, triglycerides are mobilized to free fatty acids and glycerol by activation of triglyceride lipase. Of the total free fatty acids thus released intracellularly in liver, part is re-esterified to triglyceride, and part is oxidized to acetyl-CoA with subsequent formation of acetoacetate and fl-hydroxybutyrate. Elevated tissue levels of fatty acyl-CoA or acetyl-CoA, produced by the increased availability of intracellular free fatty acids, inhibit pyruvate decarboxylation and malonyl-CoA synthesis. Fatty acyl-CoA derivatives and ATP have also been reported to inhibit citrate synthase. In the present experiments with glucagon treatment, fatty acyl-CoA levels increased while A T P levels decreased, and citrate levels were elevated. Increased citrate levels with glucagon could reflect a balance between the various factors controlling the activity of citrate synthase. Alternatively, they may be the result of a more powerful inhibition at an enzymic site betwen citrate and o~-ketoglutarate in the citric acid cycle, since c~-ketoglutarate levels were depressed. Since the DPN-linked isocitric dehydrogenase is activated by ADP, control at this site would require an increase of the mitochondrial A T P / A D P ratio. Gluconeogenesis appears to be enhanced primarily by the stimulatory effect of acetyl-CoA on pyruvic carboxylase. Facilitation of glyceraldehyde-3-P-dehydrogenase by elevated cytoplasmic levels of D P N H also contributes to the switch from glycolysis to gluconeogenesis induced by glucagon or AIS treatment. It is probable that the switch mechanism is aided by inhibition of pyruvic kinase, phosphofructokinase, and hexose kinase in order to prevent the operation of the energetically wasteful cycles depicted in Fig. 1. Observed changes of adenine nucleotides and citrate in the tissue are opposite to those expected for effective control at these sites and suggest that other factors are involved.

ACKNOWLEDGMENTS

This work was supported by the U. S. Public Health Service Grant G M 12202-03 and by the American Heart Association Grant No. 66-707. It is a pleasure to acknowledge the invaluable assistance of Dr. R. A. Kreisberg, Dr. P. H. Wright, Dr. W. J. Malaisse, Dr. S. R.

254

JOHN R. WILLIAMSON

W a g l e a n d D r . J. A s h m o r e in v a r i o u s p h a s e s o f this w o r k . C o n s c i e n t i o u s a n d highly s k i l l e d t e c h n i c a l a s s i s t a n c e w a s p r o v i d e d b y M r s . B a r b a r a H e r c z e g , M i s s H e a t h e r C o l e s , M r . R o b e r t D a n i s h a n d Mr . S t a n l e y Schwartz, and excellent editorial assistance by Miss Kathryn Rader.

REFERENCES 1. L. L. MILLER,C. G. BEY, M. L. WATSONand W. F. BALE,The dominant role of the liver in plasma protein synthesis, J. Exptl. Med. 94, 431-453 (1951). 2. J. R. WILL1AMSON,R. A. KREISBERGand W. P. FELTS, Mechanism for the stimulation of gluconeogenesis by fatty acids in perfused rat liver, Proc. Natl. Acad. Sci. 56, 247-254 (1966). 3. P. H. WmGHT, Experimental diabetes induced by insulin antibodies, pp. 354-360 in On the Nature and Treatment o f Diabetes (B. S. LEIBELand G. A. WRENSHALL,eds.), Excerpta Medica Foundation, New York (1965). 4. J. R. WILLIAMSON,B. HERCZEG, H. COLES and R. DANISH, Studies on the ketogenic effect of glucagon in intact rat liver, Biochem. Biophys. Res. Communs. 24, 437-442 (1966). 5. H. U. BERGMEYER(ed.), Methods o f Enzymatic Analysis, Academic Press, New York (1963). 6. J. R. WILLIAMSON,Glycolytic control mechanisms. I. Inhibition ofglycolysis by acetate and pyruvate in the isolated, perfused rat heart, J. Biol. Chem. 240, 2308-2321 (1965). 7. S. R. WAGLE and J. ASHMORE,Studies on experimental diabetes. II. Carbon dioxide fixation, J. Biol. Chem. 238, 17-20 (1963). 8. J. R. WILLIAMSON,P. H. WRIGHT,W. J. MALAISSEand J. ASHMORE,Control of gluconeogenesis by acetyl CoA in rats treated with glucagon and anti-insulin serum, Biochem. Biophys. Res. Communs. 24, 765-770 (1966). 9. R. C. HAYNES, JR. The control of gluconeogenesis by adrenal cortical hormones, Advances in Enzyme Regulation 3, 111-119 0965). 10. H. A. KgEns, R. N. SPEArd~ and R. HEMS, Acceleration of renal gluconeogenesis by ketone bodies and fatty acids, Biochem J. 94, 712-720 (1965). I 1. E. STRUCK,J. AsnMOl~ and O. WmLAND, Kurze mitteilung Stimulierung der Gluconeogenese dutch Langkettige Fetts~iuren und Glucagon, Biochem. Z. 343, 107-1 l 0 (1965). 12. D. STEINBERG, Catecholamine stimulation of fat mobilization and its metabolic consequences, Pharmacol. Revs. 18, 217-235 (1966). 13. TH. BiJCHER,Metabolic significance of the glycerophosphate cycle, Abstracts, International Congress o f Biochemistry, 491-492 (1964). 14. W. M. BORTZ and F. LYNEr~, Elevation of long chain acyl CoA derivatives in livers of fasted rats, Biochem. Z. 339, 77-82 (1963). 15. P. K. TunEs and P. B. GARLAND,The long-chain fatty acid acyl CoA concentration in rat heart and liver, Biochem. J. 89, 25P (1963). 16. O. WIELArqD and L. WEiss, Increase in liver acetyl-coenzyme A during ketosis, Biochem. Biophys. Res. Communs. 10, 333-339 (1963). 17. D. B. KEECH and M. F. UTTER, Pyruvate carboxylase. II. Properties, J. Biol. Chem. 238, 2609-2614 (1963). 18. O. WmLArqD, L. WEXSSand I. ECER-NEUFELDT,Enzymatic regulation of liver acetyl CoA metabolism in relation to ketogenesis, Advances in Enzyme Regulation 2, 85-89 (1964). 19. P. P. FOA, Giucagon, pp. 531-556 in The Hormones 4 (G. PINCUS, K. V. TruMAN and E. B. ASTWARD,eds.), Academic Press, New York (1964). 20. J. H. ExTorq and C. R. PARle, The stimulation of gluconeogenesis from lactate by epinephrine, glucagon and adenosine 3',5'-monophosphate in the perfused rat liver, Pharmacol. Revs. 18, 181-188 (1966).

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21. J. H. EXTON, L. S. JEFFERSON, R. W. BUTCHER and C. R. PARK, Gluconeogenesis in the perfused liver: The effects of fasting, alloxan diabetes, glucagon, epinephrine, adenosine Y,5'-monophosphate and insulin, Am. J. Med. 40, 709-715 (1966). 22. A. GARCIA, J. R. WILLIAMSON and G. F. CAHILL, Studies on the perfused rat liver. II. Effect of glucagon on gluconeogenesis, Diabetes 15, 188-193 (1966). 23. L. L. MILLER, Some direct actions of insulin, ghicagon, and hydrocortisone on the isolated peffused rat liver, Recent Progress in Hormone Research 17, 539-568 (1961). 24. J. R. WILLIAMSON,Mechanism for the in vivo stimulation of hepatic gluconeogenesis by glucagon, Bioehem. J. 101, 11c-14c (1966). 25. P. D. BEWSHER and J. ASHMORE, Ketogenic and lipolytic effects of glucagon on liver, Biochem. Biophys. Res. Communs. 24, 431-436 (1966). 26. R. MAHLER, W. S. STAFFORD, M. E. TARRANT and J. ASHMORE, The effect of insulin on lipolysis, Diabetes 13, 297-302 (1964). 27. R. L. JUNGAS and E. G. BALL, Studies on the metabolism of adipose tissue. XII. The effects of insulin and epinephrine on fatty acid and glycerol production in the presence and absence of glucose, Biochem. 2, 383-388 (1963). 28. P. B. GARLAND, D. SHEPARD and D. W. YATES, Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long chain fatty acyl-coenzyme A in rat liver mitochondria oxidizing palmitate, Biochem. J. 97, 587-606 (1965). 29. R. K. KALKHOFF, K. R. HORNBROOK, H. B. BURCH and D. M. KIPNIS, Studies of the metabolic effects of acute insulin deficiency III. Changes in hepatic glycolytic and Krebs-cycle intermediates and pyridine nucleotides, Diabetes 15, 451-456 (1966). 30. W. H. GREGOR, J. M. MARTIN, J. R. WILLIAMSON,P. E. LACY and D. M. KIPNIS, A study of the diabetic syndrome produced in rats by anti-insulin serum, Diabetes 12, 73-81 (1963). 31. I. EGER-NEUFELDT, A. TEINZER, L. WEISS and O. WIELAND, Inhibition of glucose6-phosphate dehydrogenase by long-chain acyl-coenzyme A, Biochem. Biophys. Res. Communs. 19, 43-48 (1965). 32. D. F. STEINER and J. KING, Induced synthesis of hepatic uridine diphosphate glucose glycogen glucosyltransferase after administration of insulin to alloxan-diabetic rats, J. Biol. Chem. 239, 1292-1300 (1964). 33. J. R. WILLIAMSON, A. GARCIA, A. E. RENOLD and G. F. CAHILL, Studies on the perfused rat liver. I. Effects of glucagon and insulin on glucose metabolism, Diabetes 15, 183-187 (1966). 34. P. R. DAVONEAU and J. BORNSTEIN, Effect of glucagon on metabolism of glucose and acetate by isolated rat liver, Am. J. Physiol. 197,887-892 (1955). 35. W. GEVERS and H. A. KREBS, The effects of adenine nucleotides on carbohydrate metabolism in pigeon-liver homogenates, Biochem. J. 98, 720-735 (1966). 36. E. A. NEWSrIOLME and A. H. UNDERWOOD, The control of glycolysis and gluconeogenesis in kidney cortex, Biochem. J. 99, 24c-26c (1966). 37. G. WEBER, H. J. HIRD CONVERY, M. A. LEA and N. B. STAMM, Feedback inhibition of key glycolytic enzymes in liver: Action of free fatty acids, Science 154, 1357-1360 (1966). 38. G. WEBER, M. A. LEA, H. J. HIRD CONVERY and N. B. STAMM, Regulation of gluconeogenesis and glycolysis: Studies of mechanisms controlling enzyme activity, Advances in Enzyme Regulation 5, 257-298 (1967).