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Journal ArticleDOI

Short-term control of hepatic lipogenesis by insulin

01 Nov 1980-Trends in Biochemical Sciences (Elsevier)-Vol. 5, Iss: 11, pp 288-290

TL;DR: Evidence that insulin acutely affects carbohydrate and lipid metabolism in isolated rat hepatocytes is presented and a coherent picture emerges of the concerted mechanism by which insulin sets the stage for lipogenesis in the hepatocyte.
Abstract: This article presents evidence that insulin acutely affects carbohydrate and lipid metabolism in isolated rat hepatocytes. A coherent picture emerges of the concerted mechanism by which insulin sets the stage for lipogenesis in the hepatocyte.
Topics: Lipogenesis (62%), Insulin (58%), Lipid metabolism (52%)

Content maybe subject to copyright    Report

288 TIBS -November 1980
Acknowledgement
We would like to thank Prof. W.D.
Bonner for his many useful contributions
and comments throughout the preparation
of this manuscript.
References
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Press, Amsterdam
21 Rich, P. R. and Bonner, W. D. Jr (1978) inFunc-
tions of Alternative Oxidases (Degn, G., Lloyd,
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Short-term control of hepatic
lipogenesis by insulin
Anton C. Beynen, Math J. H. Geelen and Simon G. Van den
Bergh
This article presents evidence that insulin acutely affects carbohydrate and lipid metabolism
in isolated rat hepatocytes. A coherent picture emerges of the concerted mechanism by
which insulin sets the stage for lipogenesis in the hepatocyte.
The liver of an animal ingesting a high-
carbohydrate diet must take up the glucose
in the blood, store some as glycogen and
transform the rest into fatty acids. If
starved rodents are re-fed a glucose-rich
meal, the liver may contribute as much as
50% to the fatty acid synthesis of the
body a.2. This increase in lipogenesis upon
refeeding is more pronounced and promp-
ter in the liver than in any other tissue ~,
thus emphasizing the fact that the liver is of
considerable importance as a lipogenic
organ. The fatty acids which are synthes-
ized de novo in the liver may either be
stored in the liver as triacylglycerols or
released into the blood stream as con-
stituents of lipoproteins.
Re-feeding induces in the liver the syn-
thesis of a number of lipogenic enzymes,
but it takes several hours before this results
in an increase in the amount of active en-
zyme molecules 3. The rate of fatty acid syn-
thesis is, however, greatly increased well
A. C. Beynen, M. J. H. Geelen and S. G. Van den
Bergh are at the Laboratory of Veterinary Biochemis-
try, State University of Utrecht, Biltstraat 172, 3572 BP
Utrecht, The Netherlands.
© Elsevier/North-Holland Biomedical Press 1980
before this long-term regulatory mechan-
ism is effective. Here we have restricted
our attention to the short-term control of
hepatic lipogenesis.
The acute acceleration of hepatic fatty
acid synthesis observed in rodents after
ingestion of glucose-rich meals 1 could be
explained by an increased availability of
the substrate, glucose in the portal blood*.
This is supported by our observation that
supplementing isolated hepatocytes with
glucose markedly enhances fatty acid
synthesis as measured by the incorporation
of label from tritiated water (Table I).
Like the blood level of glucose 4, that of
insulin increases dramatically and
promptly (within 30 min) during the switch
from starvation to refeeding 6. It has been
stated repeatedly that insulin, when pres-
ent as the sole hormone, has no acute
effect on hepatic lipogenesis 7, glycogen
metabolism 8 and gluconeogenesis 9. Addi-
tion of insulin to hepatocytes in suspension,
however, stimulates fatty acid synthesis
within 30 min 5'1° (see also Table I). Here
we present the evidence that insulin rapidly
affects carbohydrate and lipid metabolism
in isolated rat hepatocytes. A concerted
mechanism is described by which insulin
acutely stimulates the conversion of glu-
cose into fatty acids by the liver. Fig. 1
depicts the pathway of the hepatic syn-
thesis of triacylglycerol from glucose.
Glycolysis
Insulin increases the flux through
glycolysis in hepatocytes prepared from
meal-fed rats as judged by the hormone-
induced elevation of the sum of lactate and
pyruvate concentrations 5. In addition, insu-
lin was found to stimulate the incorpor-
ation of D-[U-x*C]glucose into lactate
(Table II).
Potential regulatory sites within the
glycolytic sequence are considered to be
phosphofructokinase and pyruvate kinase.
Insulin rapidly counteracts glucagon-
mediated inhibition of phosphofructokin-
ase in isolated hepatocytes, but is ineffec-
tive if added as the sole hormone n. In
hepatocytes, insulin decreases the concen-
tration of fructose 1,6-biphosphate
TM
which
is not compatible with activation of phos-
phofructokinase by insulin. Possibly the
TABLE I
Effects of glucose and insulin on fatty acid synthesis by
isolated hepatocytes. Hepatocytes were isolated from
meal-fed rats and incubated for 1 h with 3H~O (0.3
mCi/ml) exactly as previously described ~.
Additions
Fatty acid synthesis
(nmol acetyl units
incorp, h -1 mg protein -~)
None 16
Glucose (10 mM) 31
Glucose + insulin (85 nM) 45

TIBS - November 1980
289
stimulation of pyruvate kinase by insulin,
which has been demonstrated in the per-
fused rat liveP 3, is responsible for this.
Elevation of glycerol 3-phosphate levels
in
hepatocytes exposed to insulin 14 may be
the result of an increased cytoplasmic
(NADH)/(NAD +) ratio, which is caused
by addition of insulin (Table II).
Mitochondrial function
Insulin activates pyruvate kinase
TM
but
slightly, and significantly, lowers pyruvate
concentrations in hepatocyte suspensions
(Table II). This would suggest that activa-
tion of pyruvate kinase is not the overall
rate-limiting step of the pathway of fatty
acid synthesis in the liver. One could
explain this fall in pyruvate concentration
by the activation of pyruvate dehydrogen-
ase
in
mitochondria which, as we have
shown, is increased upon treatment of the
intact hepatocytes with insulin 15. Further-
more, in mitochondria from insulin-treated
hepatocytes the oxidation of succinate is
Glucose
I
I
I
I
F6P
TABLE II
Effect of insulin on glycolysis, glycogenesis and gluconeogenesis. Hepatocytes were isolated from meal-fed rats
and incubated for 1 h with 10 mM D-[U-14C]glucose (0.04 Ci/mol) or (last line) with unlabelled glucose and 1 mM
[3-14C]pyruvate (0.045 Ci/mol). as described previously 5~1°.
Control Insulin (85 nM)
Glucose incorporation into lactate (nmol h-' mg protein -1) 101 +_ 1
Lactate accumulation (mM) 1.86 --+ 0.12
Pyruvate accumulation (mM) 0.36 +-- 0.02
Lactate/pyruvate ratio 5.17
Glucose incorporation into glycogen (nmol h-' mg protein -1) 17 _+ 3
Pyruvate incorporation into glucose (nmol h-1 mg protein -1) 27 _+ 2
120 +_ 5 b
2.02
+- 0.05 b
0.31 _+ 0.01 b
6.52
34 _+ 6 a
18 -- 3 a
Each figure represents the mean -+ SD of three different incubations. Statistical analysis was performed accord-
ing to
Student's t
test. Versus control; a, p < 0.01 ; b, p < 0.02.
depressed
TM.
Slowing down the Krebs cycle
would lead to the formation and efflux of
citrate from the mitochondria, which in
turn would favor fatty acid synthesis, since
substrate availability would be increased.
Fatty acid synthesis
per se
In fact, however, insulin slightly lowers
citrate concentrations in hepatocytes pre-
pared from meal-fed rats 1°,1s. This points to
TG
t6
F-1,6-P 2
' DG -- ~" PC, PE
I
', ~ .... ~" GP &
T
\ ,
PEP
'
Pyr
~ Lctc
,L
Acyt -CoA
I
I
I
!
MQI-CoA
t4
Cit ~- AcCoA < Acetate
Pyr
~- Cit -I
Fig. 1. Enzymatic sites of acute control of hepatic triacylglycerol synthesis. F6P, fructose 6-phosphate; F-1,6-P~
fructose 1,6-bisphosphate; PEP, phosphoeno!pyruvate; Pyr, pyruvate; Lac, lactate; AcCoA, acetyl-CoA; OAA,
oxaloacetate; Cit, citrate; Mal-CoA, malonyl-CoA; Lyso-PA, lysophosphatidate; DG, diacylglycerol; TG,
triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolaraine; GP, glycerol 3-phosphate. The regulat-
ory enzymes: 1, phosphofructokinase (EC 2.7.1.11); 2, pyruvate kinase (EC 1.7.1.40); 3, pyruvate dehydrogen-
ase (EC 1.2.4.1); 4, acetyl-CoA carboxylase (EC 6.4.1.2); 5, glycerolphosphate acyltransferase (EC 2.3.1.15); 6,
diacylglycerol ac yltrans ferase ( E C 2.3.1.2 0).
an insulin-induced stimulation of a step in
fatty acid synthesis beyond the formation
of cytosolic citrate. This conclusion is also
supported by the observation that insulin
stimulates the incorporation of [1-14C] -
acetate into fatty acids, an effect which
cannot be explained by a hormone-
mediated alteration of the specific radioac-
tivity of the cytosolic acetyl-CoA precursor
poop °'~7. Furthermore, acetyl-CoA car-
boxylase, generally considered to be the
rate-limiting enzyme in fatty acid biosyn-
thesis, was indeed found to be subject to
short-term endocrine control. Insulin
rapidly stimulates acetyl-CoA carboxylase
activity as measured in homogenates of
hormone-treated rat hepatocytes s'l°. Our
results concerning insulin effects both on
fatty acid synthesis (as determined by the
aH,O procedure) and on acetyl-CoA car-
boxylase activity were recently confirmed
by Witters and co-workers
TM.
Upon a brief
exposure to insulin, isolated hepatocytes
display an increased cellular content of
malonyl-CoA
TM,
the product of acetyl-CoA
carboxylation. This substantiates the role
of acetyl-CoA carboxylase as a target in the
short-term control of fatty acid synthesis by
insulin. We propose that the rate of fatty
acid biosynthesis is determined by the level
of malonyl-CoA, which sets the velocity of
the reaction catalysed by fatty acid synth-
ase lo.
Triacylglycerol synthesis
Since long-chain acyl-CoA esters are
potent inhibitors of both pyruvate dehy-
drogenase and acetyl-CoA carboxylase,
lipogenesis is dependent upon the liver's
capacity to dispose of these CoA esters.
Insulin stimulates fatty acid esterification
in short-term incubations of rat
hepatocytes 14, thereby preventing feed-
back inhibition of lipogenesis by long-chain
acyl-CoA. The stimulation of fatty acid
esterification by insulin may be due to a
combination of an activation of glycerol-
phosphate acyltransferase, as demon-
strated in the perfused liver
TM,
and an elev-

290 TIBS - November 1980
ation of the intraceilular concentration of
glycerol 3-phosphate", the precursor for
the glycerol backbone of the glycerolipids.
The microsome-bound diacylglycerol
acyltransferase, the only enzyme in the
pathway exclusively concerned with
triacylglycerol synthesis, was recently
found to be inhibited by glucagon 2°.
Whether insulin activates this enzyme
remains to be established.
Interconversion of regulatory enzymes
between active and inactive forms by
phosphorylation-dephosphorylation cycles
Studies with partially purified enzyme
systems have demonstrated that the six
regulatory enzymes involved in the conver-
sion of glucose into triacylglycerols are sub-
ject to short-term regulation by covalent
modulation (for review, see Ref. 21). It
should be noted that in the case of
glycerolphosphate acyltransferase and
diacylglycerol acyltransferase there is only
suggestive evidence for regulation of
enzyme activity by a phosphorylation-
dephosphorylation mechanism. All en-
zymes are active in the dephosphory-
lated mode and inactive in their phosphory-
lated state. Immediate changes in enzyme
activity in vitro can be evoked by phos-
phorylation and dephosphorylation by pro-
tein kinases and phosphoprotein phos-
phatases, respectively.
For some enzymes evidence has been
presented that within the intact cell, phos-
phorylation-dephosphorylation cycles are
also involved in the endocrine control of
their activity. Glucagon causes phosphory-
lation of hepatic phosphofructokinase 22,
pyruvate kinase 28 and acetyl-CoA carb-
oxylase ~8'~a, which is accompanied by inac-
tivation of these enzymes. Insulin simul-
taneously induces activation and dephos-
phorylation of hepatic acetyl-CoA carb-
oxylase
TM
and of pyruvate dehydrogenase
in fat cells ~. It is attractive to speculate that
insulin via one common mechanism trig-
gers the dephosphorylation of all the reg-
ulatory enzymes of lipogenesis. Unfortu-
nately, experimental evidence for this
hypothesis is largely lacking at present.
Interrelationship between short-term
hormonal control of lipogenesis and of the
other major metabolic processes in the liver
It is now recognized that glycogen
metabolism, gluconeogenesis, glycolysis,
fatty acid oxidation (ketogenesis), fatty
acid synthesis, fatty acid esterification and
cholesterogenesis are all coordinately regu-
lated in the liver by short-term regulatory
mechanisms. This seems necessary in view
of the possible operation of futile cycles
© Elsevier/North-Holland Biomedical Press 1980
between glucose and glycogen and be-
tween acetyl-CoA and fatty acyI-CoA. With
regard to insulin-mediated control of liver
metabolism, a coherent picture is emerg-
ing.
In isolated hepatocytes, insulin stimu-
lates glycolysis (Table II), fatty acid syn-
thesis 5'1°, glycogen synthesis (Table II),
fatty acid esterification" and cholesterol
synthesis ~. On the other hand, fatty acid
oxidation" and gluconeogenesis (Table II)
are depressed by insulin. All these
metabolic pathways are affected by
glucagon in a manner opposite to that of
insulin (for review see Ref. 21 ).
In summary, the regulatory enzymatic
sites in the conversion of glucose into fat
are rapidly and coordinately activated by
insulin, possibly by modulation of their
covalent phosphorylation state. Further-
more, insulin induces a rapid decrease in
the rates of pathways opposing lipogenesis,
i.e. fatty acid oxidation and gluco-
neogenesis.
Acknowledgement
The investigations in the authors'
laboratory were supported in part by the
Netherlands Foundation for Chemical
Research (SON) with financial aid from
the Netherlands Organization for the
Advancement of Pure Research (ZWO).
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A role for oligosaccharides in
glycoprotein biosynthesis
Ron Gibson, Stuart Kornfeld and Sondra Schlesinger
Oligosaccharide chains can influence the ability of a protein to fold properly. The large size
of the precursor of asparagine-linked oligosaccharides may be essential if certain proteins
are to achieve the correct tertiary structure.
The formation of a functional protein often
includes many steps in addition to the
synthesis of peptide bonds; one of the most
R. Gibson, S. Kornfeld and S. Schlesinger are at the
Washington University School of Medicine, Depart-
ments of Microbiology and Immunology, Biochemis-
try, and Medicine, St. Louis, MO 63110, U.S.A.
elaborate and perhaps most extensively
studied steps is glycosylation. Many mem-
brane and secreted proteins contain
oligosaccharide chains covalently attached
either to asparagine (N-linked) or to serine
and threonine (O-linked). N-linked
oligosaccharides are divided into two
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Journal ArticleDOI
TL;DR: The fasted-to-fed transition of hepatic carbohydrate and lipid metabolism can be accomplished in vitro over a time frame similar to that operative in vivo, and the requirement for insulin in the reversal of the fasting state of liver metabolism in vivo can best be explained by its ability to offset the catabolic actions of glucagon.
Abstract: Studies were conducted to determine whether the direction of hepatic carbohydrate and lipid metabolism in the rat could be switched simultaneously from a "fasted" to a "fed" profile in vitro. When incubated for 2 h under appropriate conditions hepatocytes from fasted animals could be induced to synthesize glycogen at in vivo rates. There was concomitant marked elevation of the tissue malonyl-coenzyme A level, acceleration of fatty acid synthesis, and suppression of fatty acid oxidation and ketogenesis. In agreement with reports from some laboratories, but contrary to popular belief, glucose was not taken up efficiently by the cells and was thus a poor substrate for eigher glycogen synthesis or lipogenesis. The best precursor for glycogen formation was fructose, whereas lactate (pyruvate) was most efficient in lipogenesis. In both case the addition of glucose to the gluconeogenic substrates was stimulatory, the highest rates being obtained with the further inclusion of glutamine. Insulin was neither necessary for, nor did it stimulate, glycogen deposition or fatty acid synthesis under favorable substrate conditions. Glucagon at physiological concentrations inhibited both glycogen formation and fatty acid synthesis. Insulin readily reversed the effects of glucagon in the submaximal range of its concentration curve. The following conclusions were drawn. First, the fasted-to-fed transition of hepatic carbohydrate and lipid metabolism can be accomplished in vitro over a time frame similar to that operative in vivo. Second, reversal appears to be a substrate-driven phenomenon, in that insulin is not required. Third, unless an unidentified factor (present in protal blood during feeding) facilitates the uptake of glucose by liver it seems unlikely that glucose is the immediate precursor for liver glycogen or fat synthesis in vivo. A likely candidate for the primary substrate in both processes is lactate, which is rapidly formed from glucose by the small intestine and peripheral tissues. Fructose and amino acids may also contribute. Fourth, the requirement for insulin in the reversal of the fasting state of liver metabolism in vivo can best be explained by its ability to offset the catabolic actions of glucagon.

123 citations


Cites background from "Short-term control of hepatic lipog..."

  • ...sis in rat hepatocytes have also been reported (4, 32-34), but again were not of great magnitude....

    [...]


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TL;DR: It is concluded that the effects of insulin and glucagon on the overall process of triacylglycerol secretion are reflections of the hormone-determined rate of Triacyl Glycerol synthesis.
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TL;DR: Findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte metabolism.
Abstract: The effects of insulin on phospholipid metabolism and generation of diacylglycerol (DAG) and on activation of protein kinase C in rat hepatocytes were compared to those of vasopressin and angiotension II. Insulin provoked increases in [3H]glycerol labeling of phosphatidic acid (PA), diacylglycerol (DAG), and other glycerolipids within 30 s of stimulation. Similar increases were also noted for vasopressin and angiotensin II. Corresponding rapid increases in DAG mass also occurred with all three hormones. As increases in [3H]DAG (and DAG mass) occurred within 30–60 s of the simultaneous addition of [3H]glycerol and hormone, it appeared that DAG was increased, at least partly, through the de novo synthesis of PA. That de novo synthesis of PA was increased is supported by the fact that [3H]glycerol labeling of total glycerolipids was increased by all three agents. Increases in [3H]glycerol labeling of lipids by insulin were not due to increased labeling of glycerol 3-phosphate, and were therefore probably due to activation of glycerol-3-phosphate acyltransferase. Unlike vasopressin, insulin did not increase the hydrolysis of inositol phospholipids. Insulin- and vasopressin-induced increases in DAG were accompanied by increases in cytosolic and membrane-associated protein kinase C activity. These findings suggest that insulin-induced increases in DAG may lead to increases in protein kinase C activity, and may explain some of the insulin-like effects of phorbol esters and vasopressin on hepatocyte metabolism.

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01 Sep 1983-FEBS Journal
TL;DR: The stimulation of glycolysis by insulin was investigated in monolayer cultures of adult rat hepatocytes and dexamethasone acted both as a long-term and short-term modulator, and the stimulatory effects of insulin may in part be attributed to the activated pyruvate kinase.
Abstract: Evidence for a direct metabolic effect of insulin in isolated liver preparations is scarce. The stimulation of glycolysis by insulin previously demonstrated in monolayer cultures of adult rat hepatocytes [(1982) Eur. J. Biochem. 126, 271-278] was further investigated. The degree of stimulation varied with the age of the culture and amounted to 250%, 200%, 500% and 200% of the control value using cells at the culture age of 2 h, 24 h, 48 h, and 72 h, respectively. Half-maximal dose of insulin was 0.1 nM. Maximal stimulation was reached within 5 min and lasted for at least 4 h. Dexamethasone acted both as a long-term and short-term modulator. Long-term pretreatment of the cells with dexamethasone proved necessary to permit insulin action. In addition to this permissive action, pretreatment with dexamethasone reduced the insulin-independent basal glycolytic rate. In short-term experiments dexamethasone decreased the basal glycolytic flux, however, it did not affect the absolute increase in glycolysis brought about by insulin. The half-maximal dose of dexamethasone was 10 nM. The stimulatory effects of insulin may in part be attributed to the activation of pyruvate kinase. Insulin produced a left-shift of the substrate saturation curve, decreasing the K0.5 value for phosphoenolpyruvate.

32 citations


Journal ArticleDOI
Anton C. Beynen, Math J.H. Geelen1Institutions (1)
01 Jan 1982-Toxicology
TL;DR: It is proposed that all of the drugs exert an inhibitory action at the level of acetyl-CoA carboxylase, the enzyme generally considered to catalyse the rate-limiting step in hepatic fatty acid synthesis.
Abstract: An overview is presented of a selected number of mono-aromatic derivatives and their short-term effects on hepatic fatty acid biosynthesis. The compounds discussed in this paper are ortho-hydroxybenzoate (salicylate), meta-hydroxybenzoate, para-hydroxybenzoate, benzoate, para-t--butylbenzoate, para-aminosalicylate, clofibrate, halofenate, α-cyano-4-hydroxycinnmate and benfluorex. all of these drugs inhibit fatty acid biosynthesis by isolated rat liver cells, albeit with different effectiveness. In contrast, the compounds have differential effects on fatty acid esterification and oxidation by isolated hepatocytes. An attempt is made to describe in molecular terms the underlying mechanisms of the acute inhibitory effects of the mono-aromatic derivatives on hepatic lipogenesis. It is proposed that all of the drugs exert an inhibitory action at the level of acetyl-CoA carboxylase, the enzyme generally considered to catalyse the rate-limiting step in hepatic fatty acid synthesis. This inhibitory effect may be either direct, i.e. by an alteration of the enzyme's structure as a result of interaction between drug and enzyme, or indirect, i.e. through a drug-induced change in the cellular levels of allosteric effectors of acetyl-CoA carboxylase.

15 citations


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Journal ArticleDOI
TL;DR: Light is shed on the relative significance of liver and adipose tissue in fatty acid synthesis in mice, on the mino importance of glucose in hepatic lipogenesis, and on the alterations in the rate of fatty acids synthesis in genetically obese mice.
Abstract: 1. The synthesis of long-chain fatty acids de novo was measured in the liver and in regions of adipose tissue in intact normal and genetically obses mice throughout the daily 24h cycle. 2. The total rate of synthesis, as measured by the rate of incorporation of 3H from 3H2O into fatty acid, was highest during the dark period, in liver and adipose tissue of lean or obese mice. 3. The rate of incorporation of 14C from [U-14C]glucose into fatty acid was also followed (in the same mice). The 14C/3H ratios were higher by a factor of 5-20 in parametrial and scapular fat than that in liver. This difference was less marked during the dark period (of maximum fatty acid synthesis). 4. In normal mice, the total rate of fatty acid synthesis in the liver was about twofold greater than that in all adipose tissue regions combined. 5. In obese mice, the rate of fatty acid synthesis was more rapid than in lean mice, in both liver and adipose tissue. Most of the extra lipogenesis occurred in adipose tissue. The extra hepatic fatty acids synthesized in obese mice were located in triglyceride rather than phospholipid. 6. In adipose tissue of normal mice, the rate of fatty acid synthesis was most rapid in the intra-abdominal areas and in brown fat. In obese mice, all regions exhibited rapid rates of fatty acid synthesis. 7. These results shed light on the relative significance of liver and adipose tissue (i.e. the adipose 'organ') in fatty acid synthesis in mice, on the mino importance of glucose in hepatic lipogenesis, and on the alterations in the rate of fatty acid synthesis in genetically obese mice.

178 citations


Journal ArticleDOI
J B Blair1, M A Cimbala1, J L Foster, R A Morgan1Institutions (1)
TL;DR: The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclicAMP-stimulated protein kinase.
Abstract: A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungstrom, O., Hjelmquist, G. and Engstrom, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.

151 citations




Journal ArticleDOI
TL;DR: Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha and exogenous cyclic AMP mimics the effect of this hormone.
Abstract: Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.

121 citations


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