Download Isoforms of acetyl-CoA carboxylase

Biochemical Society Transactions
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lsoforms of acetyl-CoA carboxylase: structures, regulatory properties and metabolic
functions
I232
R. W. Brownsey’, R. Zhande and A. N. Boone
Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia,
Canada V6T I 2 3
Acetyl-CoA carboxylase (ACC; EC 6.4.1.2) catalyses the formation of malonyl-CoA, which is
essential as a metabolic substrate and as a modulator of specific protein activity. Malonyl-CoA is
a substrate for fatty acid synthase (FAS), for
polyketide synthases (in plants, fungi and
bacteria) and for fatty acyl chain-elongation
systems. As an allosteric modulator, malonyl-CoA
potently inhibits carnitine palmitoyltransferase-I
(CPT-I), with far-reaching effects on intermediary metabolism and cell secretory functions.
ACC contributes importantly to flux control in
fatty acid biosynthesis and b-oxidation and is
subject to a range of interacting mechanisms that
dictate tissue-specific expression and specific
enzyme activity. Detailed reviews of ACC enzymology appeared in the late 1980s [l-31, so we
focus on selected developments since 1990, with
most emphasis on the properties of the mammalian multifunctional ACC polypeptides. We discuss the structure and regulation of the ACC
genes only briefly, since these aspects are dealt
with elsewhere [4].
ACC reaction and mechanism
The overall reaction catalysed by ACC is a twostep process that involves ATP-dependent formation of carboxybiotin followed by transfer of
the carboxyl moiety to acetyl-CoA to form malonyl-CoA. ACCs possess distinct active sites for
biotin carboxylation and for carboxyl transfer
together with a ‘mobile’ biotin that acts as a
carboxyl carrier between the two active sites. The
first half-reaction of biotin carboxylation probably
involves initial biotin-independent activation of
bicarbonate, because ATP/ADP nucleotide
exchange is not inhibited by avidin and a formal
carboxy-phosphate
intermediate
has been
Abbreviations used: ACC, acetyl-CoA carboxylase;
FAS, fatty acid synthase; CPT-I, carnitine palmitoyl
transferase I; BCCP, biotin carboxyl carrier protein;
AMP-PK, AMP-activated protein kinase; PKA, CAMPdependent protein kinase; MAP, mitogen-activated
protein; JNK, c J u n N-terminal kinase; PI 3-K,
phosphatidylinositol 3-OH kinase; ERK, extracellular
signal-related protein kinase.
‘To whom correspondence should be addressed.
Volume 25
observed, at least in studies with pyruvate carboxylase [5,6]. The carboxyl transferase halfreaction proceeds via proton extraction, which
generates a reactive carbanion on the methyl
carbon of acetyl-CoA [7]. Detailed structural
studies, which are most advanced for the Escherichiu coli ACC subunits, should provide further
definition of the details of the ACC reaction
mechanisms [8].
Roles of ACC
Biotin-dependent carboxylases emerged early in
evolution and are well conserved between eubacteria, unicellular eukaryotes, plants and animals.
The importance of malonyl-CoA in cell metabolism is most directly reflected by the fact that
ACC expression is essential for normal growth of
bacteria, yeast and isolated animal cells in culture. For example, diploid yeast cells deficient in
ACC show aberrant mitosis [9], and corresponding haploid spores fail to enter vegetative growth
[lo]. Specific ablation of the ACC gene in
animals has not yet been reported, although inhibition of fatty acid synthesis leads to apoptosis in
cultured mammalian cancer cell lines [ 1 13.
Roles ofACC in fatty acid synthesis
In unicellular organisms, long-chain fatty acids
are most important for membrane lipid synthesis; regulation of ACC therefore reflects control
of phospholipid biosynthesis and of overall cell
growth. In multicellular organisms, de novo biosynthesis of long-chain fatty acids makes an
important contribution to the synthesis of energy
stores as well as to membrane lipid structure; so
regulation of energy homoeostasis also impacts
importantly on ACC expression and activity.
The most prominent product of FAS is
palmitate (16:0), but longer chain lengths are
essential for the production of eicosanoids,
sphingolipids and other products important in
intra- and inter-cellular communication and in
specific organelle membrane functions [ 121. Acyl
chain elongation is achieved by enzyme systems
in mitochondria and endoplasmic reticulum but
only the latter uses malonyl-CoA as a carbon
donor [13].
Molecular Aspects of the Regulation of Lipid Biosynthesis
A third general metabolic fate for malonylCoA occurs predominantly in plants, fungi and
bacteria with the biosynthesis of polyketides [ 141,
where the polyketide synthases may be regarded
as a family of diverse and highly adapted variants
of FAS.
Finally, it is important to note that ACC
itself may be a target for feedback regulation by
malonyl-CoA if consumption by FAS or other
systems is restricted. Therefore, in cells that
express low levels of enzymes that consume malonyl-CoA, removal must be effected by alternative routes - notably by decarboxylation back to
acetyl-CoA [ 151.
Roles of ACC in fatty acid oxidation
Fatty acid oxidation within mitochondria is influenced by ACC, because malonyl-CoA is a potent
inhibitor of the entry of fatty acyl chains into the
mitochondrial matrix. This role of malonyl-CoA
was first noted in the context of the regulation of
hepatic ketogenesis [ 161 and involves direct
binding of malonyl-CoA to the catalytic subunit
of CPT-I [17]. In some situations, the direct
effect of malonyl-CoA may be compounded by
other mechanisms which alter the sensitivity of
the CPT-I to malonyl-CoA [18]. Fatty acid
degradation also occurs in peroxisomes, in particular to shorten very long fatty acyl chains
before mitochondrial oxidation, but the entry of
fatty acyl chains into peroxisomes is apparently
not dependent on carnitine, even though a malonyl-CoA-sensitive CPT-I does appear to be
present [19].
In addition to its role in the regulation of
hepatic CPT-I, malonyl-CoA is also critical in
the regulation of fuel selection in muscle cells
[20,21]. Furthermore, generation of malonyl-CoA
is important in the nutrient-induced increase in
insulin secretion from pancreatic /]-cells
[21-231. It has been proposed that increases in
malonyl-CoA in /]-cells may lead to re-direction
of fatty acyl-CoA esters to sites that impact on
the secretory process.
Distinct forms of ACC
Two broad classes of ACC have so far been
detected, in one of which the three major functional domains exist on two or more distinct
polypeptides. In contrast, all eukaryotic cells
studied so far express forms of the second major
class of ACC in which all the functional domains
are contained within a contiguous polypeptide
sequence.
Forms of ACC with functional domains on two or
more distinct polypeptides
In E. coli, four component polypeptides of ACC
are encoded by distinct genes accA-accD. Biotin
carboxyl carrier protein (BCCP, 17 kDa) is
encoded by accB; biotin carboxylase (which
assembles as a dimer of two 49 kDa subunits) by
accC; and the cy- and P-subunits of carboxyl
transferase (which assemble as a 130 kDa cy2//jz
tetramer) by accA and accD respectively. Other
bacteria which have been studied, including Anabeana sp. and Pseudomonas sp. also express
ACC with multiple subunits similar to those in
E. coli. ‘Prokaryotic’ ACCs are also found in the
plastids of certain (dicotyledenous) plants,
although these plant ACC forms have larger
BCCP subunits and assemble into larger active
complexes than bacterial ACC [24-261.
In several biotin carboxylases, including rat
and human propionyl-CoA carboxylase, biotin
carboxylase and BCCP are encoded by a single
gene. This raises the possibility of forms of ACC
that comprise only two functional polypeptides,
as suggested for Corynebacterium glutamicum
~271.
Forms of ACC with all fimctional domains
assembled on a single polypeptide
Yeast ACC, encoded by the single-copy ACCI
gene, is highly related to other eukaryotic forms
of the enzyme and is expressed as a tetrameric
protein with a subunit size of -265 kDa [28].
Multifunctional ACC has also been characterized in a number of photosynthetic eukaryotes,
firstly in the alga Cyclostella ciyptica and also in
a variety of plant species, including brassica,
maize, wheat, rice, pea, alfalfa, tobacco and arabidopsis [24,26]. T h e large-subunit forms of ACC
in plants are homodimers with subunits of
220-240 kDa, and in most species examined,
there is evidence for distinct cytoplasmic and
plastid forms. De novo fatty acid synthesis occurs
exclusively in the plastids of plants (which is also
the site of expression of the ‘prokaryotic’ form of
ACC when it is present).
Two major isoforms of multifunctional
animal ACC have so far been detected, and each
may exist in one of two possible forms based on
the presence or absence of a distinct motif. No
animal species has yet been found which
expresses a ‘prokaryotic’ form of ACC. The
animal ACCs display subunit sizes of 265-280
kDa on denaturing gels, exist minimally as
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dimers under non-denaturing conditions and,
unlike any other ACC described, are activated by
di- and tri-carboxylic acids, which may induce
aggregation into elongated polymers consisting of
5-20 dimers. The rest of this report will focus
on a comparison of the properties of the two
major forms of animal ACC, designated here as
ACC-1 (for the 265 kDa or ACC-a isoform) and
ACC-2 (for the 275-280 kDa or ACC-/? isoform).
Properties of the major animal ACC
isoforms
Predicted sequences of ACC isoforms
ACC-1 has been purified and characterized from
a number of animal tissues, notably from liver,
adipose tissue and mammary gland. Sequences of
ACC-1 have now been deduced for the enzyme
from chicken [29], rat [30], goat [31] and human
[32]. ACC-2 has been recognized as a distinct
isoform in heart [33], liver [34,35] and skeletal
muscle [36], and the sequences of the two variants of human ACC-2 have been deduced
[37,38]. The variant forms of ACC-1 and ACC-2
are caused, respectively, by the addition of eight
amino acid residues beginning at Pro-1 196 and
the deletion of 101 residues beginning at Arg1114 [38,39].
The genes for the human ACC-1 and -2
isoforms are located on distinct chromosomes
(chromosomes 17q12 or 17q21 and 12q23
respectively) and show approx. 60% overall
identity, ACC-2 exceeding ACC-1 in length by
more than 100 amino acid residues. The two
isoforms differ most at the N-termini, where
residues 1-217 of ACC-2 correspond to residues
1-74 of ACC-1. The extension of ACC-2 near
the N-terminus accounts for most of the difference in molecular size, and has been argued to
provide a sequence with the potential to target
ACC to mitochondria [37]. Many other specific
sequence differences between ACC-1 and ACC-2
are scattered throughout the two sequences, as
predicted on the basis of earlier comparative
peptide analysis [35]. Among the sequences that
are particularly well conserved in human ACC-1
and ACC-2 are several that have catalytic or
regulatory significance, for example: (i) the motif
AE(I/M)EVMKMIMT, which is essential for
binding biotin, begins at Ala-780 of ACC-1 and
Ala-924 of ACC-2; (ii) SEGGGGKGIRK,
required for ATP binding, begins at Ser-313 of
ACC-1 and Ser-456 in ACC-2; (iii) the sequence
SFKRIMAPWAQTWTGRARLGGIPVGVI,
is
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involved in acetyl-CoA binding, beginning at Ser1959 of ACC-1 and Ser-2095 of ACC-2; (iv) the
sequence RSS(W/M)SGLHLVK, is a site for
regulatory phosphorylation (of the underlined S),
beginning at Arg-76 of ACC-1 and Arg-217 of
ACC-2.
Differential distribution of ACC isoforms in
mammalian tissues
Individual tissues express predominantly one isoform of ACC [34-36,401. The most dramatic
pre-eminence of one ACC isoform is found in rat
white adipose tissue, which expresses ACC- 1
exclusively [ 1,34,35]. In liver, brown adipose
tissue, lactating mammary tissue, brain and pancreas, expression of ACC-1 exceeds that of ACC2 by a factor of 3-10 [34,40]. Heart and skeletal
muscle are distinctive in that the expression of
ACC-2 is 10 times that of ACC-1 [33,34,40]. The
highest levels of ACC expression are found in
lipogenic tissues such as white and brown fat,
liver and lactating mammary gland. Assuming the
specific activity of homogeneous ACC is approx.
20 unitslmg of protein, the expression of the
enzyme in lipogenic tissues is in the range
50-250 pglg wet weight. In contrast, ACC
expression in heart, skeletal muscle and pancreas
is in the range 1-2 pglg wet weight of tissue.
Regulation of animal ACC isoform expression
Earlier classical studies had established that the
turnover of ACC-1 in liver and adipose tissue
had a half-life in excess of 48 h in animals that
were fed normally on a typical chow diet [1,25].
Strong ACC-1 repression occurs in adipose
tissue and liver in starvation, during feeding
high-fat diets (including suckling in mammals)
and in diabetes. In these situations, ACC-1
repression is reversed, respectively, by feeding
low-fat diet (by weaning) or by insulin treatment.
The regulation of ACC-1 expression shown
by studies of protein turnover is reflected in
appropriate changes in gene transcription as
identified by studies of mRNA expression
[4,41,42]. Comparison of the time courses for
expression of mRNA and of protein suggest that
there are important controls of message stability
and/or of protein translation as well as control at
the level of transcription [41,42].
Isoform analyses have indicated that the
repression of ACC-1 and ACC-2 occurs in parallel in the liver during starvation and in experi-
Molecular Aspects of the Regulation of Lipid Biosynthesis
mental diabetes, but that cardiac and muscle
ACC-2 is not significantly repressed [40,43].
In addition to the studies of diet and diabetes, a number of other model or developmental
systems deserve close scrutiny, because they may
shed important light on factors that may be critical in regulating ACC isoform expression in
diverse physiological settings. For example, the
differentiation of adipose cells from precursors,
the onset of lactation in the mammary gland and
myelination in the brain all represent developmental switches that are accompanied by large
increases in ACC- 1 expression, coupled with
repression of ACC-2 [ 1,25,41,42,44]. Analogous
changes in ACC-2 expression occur during myodifferentiation and in the neonatal heart [43,45].
Specific hormones and intracellular gene
regulatory mechanisms that account for the
diverse aspects of control of ACC isoform
expression are still poorly understood. It is likely
that cardinal extracellular signals that play a role
in regulating ACC expression during dietary
modulation and diabetes include insulin, glucagon, catecholamines, thyroid hormones, steroid
hormones and lactogenic hormones [41,42] as
well as nutrients, notably glucose [42]. At the
intracellular level, key regulatory elements that
control the ACC genes are still being defined,
and understanding of the relevant family of critical transcription factors is still emerging [4].
Post-tronslational regulation of animal ACC
isoforms
T h e ACC isoforms purified by avidin affinity
chromatography from liver, adipose tissue, heart
and skeletal muscle show broadly similar kinetic
properties [33,34]. Both enzymes show similar
substrate kinetics with respect to bicarbonate,
ATP and MgZ+,although ACC-2 has a K , for
acetyl-CoA approximately twice that of ACC-1.
Both isoforms are markedly activated by citrate,
with K , values in the millimolar range and V,,,
values of 1-3 units/mg of protein. Both isoforms
show similar sensitivity to inhibition by avidin,
malonyl-CoA and palmitoyl-CoA. The sensitivity
of ACC-2 to free CoA (which is a potent inhibitor of ACC-1) has not yet been tested. Citrate
activation leads to marked increase in the molecular size of ACC-1 but so far there are no
reports on the effects of citrate on the molecular
size of ACC-2.
The regulation of ACC-1 by reversible phosphorylation has been studied in considerable
detail, and a number of critical ACC-1 phos-
phorylation sites have been identified and the
cognate protein kinases proposed [ 1-31. An
important inhibitory phosphorylation site at Ser79 in rat ACC-1 is phosphorylated by AMP-activated protein kinase (AMP-PK). Additional sites
at Ser-1200 and Ser-1215 may be phosphorylated
by either AMP-PK or CAMP-dependent protein
kinase (PKA), and although these latter two sites
are phosphorylated more slowly in vitro [3,46], all
three are occupied following elevation of cAMP
levels in intact cells [l-31. Because another site
for PKA (Ser-77) is not appreciably phosphorylated by increases in intracellular cAMP levels, it
has been proposed that the AMP-PK is the
principal inhibitory ACC kinase [46,47]. A somewhat different view of the relative importance of
ACC-1 phosphorylation sites emerges from
studies of the ACC-1 variant and of ACC-1 in
which specific phosphorylation sites were altered
by site-directed mutagenesis; these results indicate that phosphorylation of Ser-1200 also contributes importantly to inhibition of ACC-1 [39,48].
Like ACC-1, ACC-2 is a substrate for AMPPK and PKA in vitm but direct inhibition of
ACC-2 activity has only been observed in the
presence of the AMP-PK [35,49]. An important
difference between the two ACC isoforms is that
ACC-2 appears to be a far better substrate for
PKA in vitro than ACC-1. This was first
observed for the rat liver ACC preparation [3S]
and has been confirmed using preparations of
ACC-2 from rat heart and skeletal muscle [SO].
Is the phosphorylation of ACC-2 important
physiologically? ACC-2 is rapidly inactivated in
intact heart following ischaemia/reperfusion [S 11
and in skeletal muscle stimulated electrically or
by exercise [52]. Under these conditions the
inactivation of ACC-2 occurs in parallel with
activation of AMP-PK, although a causal relationship has not yet been established. We have
examined the effects of isoprenaline on intact
cardiac ventricular myocytes and found that
ACC-2 is rapidly phosphorylated at sites corresponding to the sites of purified ACC-2 phosphorylated by PKA [SO]. These observations are
based on comparative phosphopeptide mapping;
the sequences of ACC-2 phosphorylation sites
have not yet been reported. The role of PKA in
the regulation of ACC-2 may therefore be more
significant than its role in the regulation of ACC1, a situation reminiscent of the distinct regulation of the hepatic and cardiac isoforms of
phosphofructo-2-kinase. The molecular basis for
the enhanced phosphorylation of ACC-2 by PKA
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has not yet been established. Interestingly, the
Ser-1200/Ser-1215 motif is markedly different in
the two isoforms and in ACC-2 does not appear
to present a canonical PKA consensus phosphorylation site. This suggests that alternative
and quite distinct sites for phosphorylation by
PKA exist in ACC-2.
T h e effects of insulin on ACC isoforms are
likely to be complex, probably involving: (i) the
activation of the phosphodiesterase-I11 isoform
of cyclic nucleotide phosphodiesterase; (ii) the
activation of protein phosphatase-1 or -2.4; and
(iii) the activation of an ‘ACC-kinase’ that is able
to phosphorylate the distinct, insulin-directed
phosphorylation site of ACC-1 [l]. So far, no
effects of insulin on ACC-2 have been demonstrated that persist with cell fractionation. Malonyl-CoA levels have been shown to increase in
skeletal muscle [53] and heart [54] in the
presence of glucose plus insulin, but, so far, no
changes in activity of isolated ACC-2 have been
found. Rather, it has been argued that increased
flux through ACC-2 is promoted through
insulin-mediated increases in citrate and/or
acetyl-CoA [ZO].
A number of well-characterized protein
kinases have been tested in the search for
insulin-activated ‘ACC kinases’, but no identified
protein kinases have been found that are able to
phospholylate the insulin-directed site of ACC-1.
Examples of protein kinases that have been
tested include conventional isoforms of protein
kinase C, casein kinases, CDKS and glycogen
synthase kinase-3 ([l-31; and R. W. Brownsey,
unpublished work). We have also explored the
possible roles of three major classes of mitogenactivated protein (MAP) kinase as putative ACC
kinases. Unlike the sea-star homologue [55],
preparations of extracellular signal-related protein kinase (ERK)-1 purified to homogeneity
from rat adipose tissue do not phosphorylate the
appropriate insulin-directed phosphorylation site
of ACC-1 and do not induce ACC activation.
Moreover, in contrast with studies with isolated
hepatocytes [56], exposure of fat cells to hypoosmotic extracellular media does not induce activation of ACC-1 or of fatty acid synthesis, even
though the ERKs do become activated [57].
Indeed, insulin induces activation of fatty acid
synthesis even when the activation of the ERKs
is blocked in hyper-osmotic media [57]. MAP
kinases related to the ERKs include p38/RK (the
mammalian homologue of the yeast HOG-1 protein kinase) and the c-Jun N-terminal kinases
Volume 25
(JNKs). We found no evidence for activation of
p38/RK in rat white adipose tissue following
insulin treatment, and although we did observe
insulin-stimulated activation of JNKs, this did
not correlate with activation of fatty acid biosynthesis [57]. T h e mechanism by which insulin
activates ACC therefore remains poorly defined,
although it does appear to require activation of
phosphatidylinositol 3-OH kinase (PI 3-K), or a
related protein sensitive to wortmannin [58].
Protein kinase B is one important target which
acts downstream from PI 3-K but we found no
evidence that this protein kinase acts directly on
purified ACC-1 or ACC-2. As noted earlier, the
regulation of ACC by insulin and other hormones
may be dependent upon allosteric factors or
other proteins as well as appropriate regulation
of the phosphorylation of specific sites by hormones [59].
Conclusions
T h e two isoforms of animal ACC so far
described have distinct subunit structures, display subtle differences in kinetic properties and
potentially important differences in regulation.
T h e expression of the ACC isoforms is tissuespecific, ACC-2 being most evident in cells
which do not carry out substantial de novo synthesis of fatty acids (the function for which ACC1 appears to be the dominant isoform). A
number of dietary manipulations, hormone
imbalances and developmental processes illustrate that the two ACC isoforms are regulated
by distinct and complex mechanisms. It is
becoming increasingly evident that malonyl-CoA
plays significant roles, not only as a substrate for
fatty acid biosynthesis, but also in the regulation
of metabolic fuel selection, insulin secretion and
formation of atypical fatty acids that may be critical for membrane architecture and function.
These observations further underline the importance of gaining a full understanding of the structure, function and regulation of ACC isoforms.
The work carried out in our laboratory and described
in this report was supported by funds from The
Medical Research Council of Canada and from The
Canadian Diabetes Association (an award in the name
of Dorothea Riddell Minnis).
1 Brownsey, R. W. and Denton, R. M. (1987) in The
Enzymes: Control by Phosphorylation, Part B
(Boyer, P. D. and Krebs, E. G., eds.), vol. XVIII,
pp. 123-146, Academic Press, Orlando
Molecular Aspects of the Regulation of Lipid Biosynthesis
2 Kim, K.-H., Lopez-Casillas, F., Bai, D. I I . , Luo, X.
and Pape, M. E. (1989) FASEB J. 3, 2250-2256
3 Hardie, D. G. (1989) Prog. Lipid Res. 28, 117-146
4 Kim, K.-€I. and Tae, H.-J. (1994) J. Nutr. 124,
1273S-3283S
5 Fry, D. C., Fox, T., Lane, M. D. and Mildvan, A. S.
(1985) Ann. N.Y. Acad. Sci. 447, 140-151
6 Phillips, N. F. B., Snoswell, M. A., ChapmanSmith, A., Keech, D. R. and Wallace, J. C. (1992)
Biochemistry 31,9445-9450
7 Kuo, D. J. and Rose, I. A. (1993) J. Am. Chem.
SOC.115, 387-390
8 Waldrop, G. I,., Rayment, I. and IIoldcn, 11. M.
(1994) Biochemistry 33, 10249-10256
9 Saitoh.. S... Takahashi.. K... Nabeshima.. K...
Yamashita, Y., Nakaseko, Y., IIirata, A. and
Yanagida, M. (1996) J. Cell. Riol. 134, 949-961
10 Hasselacher, M., Ivessa, A. S., Paltauf, F. and
Kohlwein, S. D. (1993) J. Biol. Chem. 268,
10946- 10952
11 Pizer, E. S., Jackisch, C., Wood, F. I)., Pasternack,
G. R., Davidson, N. E. and Kuhajda, F. P. (1996)
Cancer Res. 56, 2745-2747
12 Schneiter, R. and Kohlwein, S. D. (1997) Cell 88,
43 1-434
13 Saggerson, E. D., Ghadiminejad, 1. and Awan, M.
(1992) Adv. Enzyme Regul. 32,285-306
14 Shen, R. and Hutchinson, C. R. (1993) Science
262, 1535-1540
15 Courchesne-Smith, C., Jang, S.-lI., Shi, Q.,
DeWille, J., Sasaki, G. and Kolattukudy, P. E.
(1992) Arch. Biochem. Biophys. 298, 576-586
16 McGarry, J. D., I’akabayashi, Y. and Foster, D. W.
(1978) J. Hiol. Chem. 253, 8294-8300
17 Brown, N. F., Esser, V., Foster, D. W. and
McGarry, J. D. (1994) J. Riol. Chem. 269,
26438-26442
18 Velasco, G., Guzman, M., Zammit, V. A. and
Geelen, M. J. €1. (1997) Biochem. J. 321, 211-216
19 Derrick, J. P. and Ramsay, R. R. (1989) Biochem. J.
262,801-806
20 Lopaschuk, G. D., Belke, I). D., Gamble, J., Itoi,
T. and Schoenekess, B. 0. (1994) Biochim.
Hiophys. Acta 1213, 263-276
21 Brown, N. F. and McGarry, J. I). (1997) Eur. J.
Biochem. 244, 1-14
22 Prentki, M., Vischner, S., Glennon, M. C., Rcgazzi,
R., Deeney, J. T. and Corkey, 13. E. (1992) J. I3iol.
Chem. 267,5802-5810
23 Prentki, M. (1996) Eur. J. Endocrinol. 134,
272-286
24 Toh, H., Kondo, II. and Tanabe, 1’.(1993) Eur. J.
Biochem. 215, 687-696
25 Volpe, J. J. and Vagelos, P. R. (1976) Physiol. Rev.
56, 339-417
26 Sasaki, Y., Konishi, ‘r. and Nagano, Y. (1995) Plant
Physiol. 108, 445-449
27 Jager, W., Peters-Wendisch, P. G., Kalinowski, J.
and Puhler, A. (1996) Arch. Microbiol. 166, 76-82
28 AI-Feel, W., Chirala, S. S. and Wakil, S. J. (1992)
Proc. Natl. Acad. Sci. U.S.A. 89, 4534-4538
29 ‘I’akai, T., Yokoyama, C., Wada, K. and Tanabe, T.
(1988) J. Biol. Chem. 263, 2651-2657
30 Lopez-Casillas, F.,Bai, I). H., I m ) , X., Kong, 1 . 3 ,
IIermodsen, M. A. and Kim, K.-Il. (1988) Proc.
Natl. Acad. Sci. U.S.A. 85, 5784-5788
31 Barber, ,M.C. and ‘I’ravers, M. T. (1995) Gene
154, 271-275
32 Abu-Elheiga, I,., Jayakumar, A., Baldini, A.,
Chirala, S. S. and Wakil, S. J. (1995) Proc. Natl.
Acad. Sci. U.S.A. 92, 4011-4015
33 ‘I’hampy, K. G. (1989) J. Biol. Chem. 264,
17631 - 17634
34 Bianchi, A., Evans, J. I,., Iverson, A. J., Nordlund,
A.-C., Watts, ‘I.. D. and Witters, I,. A. (1990)
J. B i d . Chem. 265, 1502-1509
35 Winz, R., IIess, D., Aebersold, R. and Brownsey,
R. W. (1994) J. Hiol. Chem. 269, 14438-14445
36 Trumble, G. E., Smith, M. A. and Winder, W. W.
(1995) blur. J. Biochem. 231, 192-198
37 IIa, J., I x e , J.-K., Kim, K.-S., Witters, I,. A. and
Kim, K.-H. (1996) Proc. Natl. Acad. Sci. 1J.S.A.
93, 11466- 1 1470
38 Abu-Elheiga, I,., Almarza-Ortega, D. H., Baldini, A.
and Wakil, S. J. (1997) J. Riol. Chem. 272,
10669-10677
39 Kong, 1 . 3 , Lopez-Casillas, F. and Kim, K.-H.
(1990) J. B i d . Chcm. 265, 13695-13701
40 Iverson, A. J., Bianchi, A., Nordlund, A.-C. and
Witters, 1,. A. (1990) Biochem. J. 269, 365-371
41 Iritani, N. (1992) Eur. J. I3iochem. 205, 433-442
42 Girard, J., Perdereau, D., Foufelle, F., Prip-Buus,
C. and Ferre, 1’. (1994) FASEB J. 8, 36-42
43 Winder, W. W., MacIxan, P. S., I,ucas, J. C.,
Fernlcy, J. E. and Trumble, G. E. (1995) J. Appl.
Physiol. 78, 578-582
44 Garbay, H., Bauxis-Lagrave, S., Roiron-Sargucil, F.,
Elson, G. and Cassagnc, C. (1997) Dev. Brain Res.
98, 197-203
45 Lopaschuk, G. I)., Witters, I,. A., Itoi, T., Barr, K.
and Barr, A . (1994) J. I3iol. Chem. 269,
25871 -25878
46 Davies, S. P., Sim, A. ‘1’. K. and IIardie, I). G.
(1990) Eur. J. I3iochem. 187, 183-190
47 Cohen, P. and IIardie, D. G. (1991) I3iochim.
Hiophys. Xcta 1094, 292-299
48 Ila, J., Daniel, S., Hroylcs, S. S. and Kim, K.-11.
(1994) J. Biol. Chcm. 269, 22162-22168
49 Winder, W. W., Wilson, I I . A., Hardie, D. G.,
Rasmussen, B. R., Hutbcr, C. A, Call, G . l3.,
Clayton, K. D., Conely, I,. M., Yoon, S. and Zhou,
13. (1997) J. Appl. Physiol. 82, 219-225
50 Boone, A. N. and Rrownsey, R. W. (1997)
Proceedings of the 40th CFBS Congress, Quebec
City (abstract #200), CFBS Press, Ottawa
I997
I237
Biochemical Society Transactions
I238
51 Kudo, N., Barr, A. J., Barr, R. L., DeSai, S. and
Lopaschuk, G. D. (1995) J. Biol. Chem. 270,
17513-17520
52 Hutber, C. A., Hardie, D. G. and Winder, W. W.
(1997) Am. J. Physiol. 272, E262-E266
53 Duan, C. and Winder, W. W. (1993) J. Appl.
Physiol. 74, 2543-2547
54 Awan, M. M. and Saggerson, E. D. (1993)
Biochem. J. 295,61-66
55 Pelech, S. L., Sanghera, J. S., Padden, H. B.,
Quayle, K. A. and Brownsey, R. W. (1991)
Biochem. J. 274, 759-767
56 Hue, L. (1994) Biochem. SOC.Trans. 22, 505-508
57 Zhande, R. and Brownsey, R. W. (1996) Biochem.
Cell. Biol. 74, 513-522
58 Moule, S. K., Edgell, N. J., Welsh, G. I., Diggle,
T. A., Foulstone, E. J., Heesom, K. J., Proud, C. G.
and Denton, R. M. (1995) Biochem. J. 311,
595-601
59 Quayle, K. A., Denton, R. M. and Brownsey, R. W.
(1993) Biochem. J. 292,75-84
Received 11 July 1997
Signalling pathways involved in the stimulation of fatty acid synthesis by insulin
R. M. Denton', K. J. Heesom, S. K. Moule, N. J. Edgell and P. Burnett
Department of Biochemistry, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 ITD, U.K.
An important effect of insulin after a carbohydrate meal is to increase the conversion of
glucose into fatty acids in fat, liver and mammary
cells. Both short-term and long-term mechanisms are involved. A main interest of this laboratory for a number of years has been the
short-term mechanisms involved in insulin signalling in rat epididymal fat cells, and this will be
the subject of this brief article. Three different
steps in the pathway are activated in parallel
within 5 min of exposing these cells to insulin,
and the overall result is a 10-fold increase in the
rate of fatty acid synthesis. These steps are:
glucose transport across the cell membrane,
pyruvate dehydrogenase (PDH) in the mitochondria, and the cytoplasmic enzyme acetyl-CoA carboxylase (ACC) [l]. It is now becoming clear
that different signalling pathways are involved
although, as with other metabolic actions of
insulin, important gaps in our knowledge remain.
Signalling pathways involved in the
activation of glucose transport, PDH
and ACC by insulin
Figure 1 summarizes the work of many different
laboratories on the early events in insulin signalling [2-51. In essence, the binding of insulin
activates the intrinsic tyrosine kinase activity of
Abbreviations used: ACC, acetyl-CoA carboxylase;
PDH, pyruvate dehydrogenase; ERK, extracellular
signal-related protein kinase; EGF, epidermal growth
factor.
'To whom correspondence should be addressed.
Volume 25
the insulin receptor. This results in autophosphorylation of the receptor and then phosphorylation of intracellular proteins, including
insulin-receptor substrate-1, and -2, SHC and
GAB-1. T h e regions of these proteins that contain phosphotyrosine then form specific docking
sites for a number of other proteins that contain
SH2 domains. Important consequences are the
activation of Ras and of PtdIns 3-kinase. In turn,
these events lead to the activation of a number of
protein kinases. T h e activation of Ras initiates
the activation of a well-established kinase cascade leading to the stimulation of the mitogenactivated protein kinases ERK-1 and ERK-2.
T h e activation of PtdIns 3-kinase results in an
increase in the product PtdIns(3,4,5)P3, and it
seems likely that this results in the activation of
other protein kinases including protein kinase B
(also known as RAC or akt) and p70 S6 kinase.
PtdIns(3,4,5)P3 may directly influence protein
kinase B by binding to the P H domain in the
kinase, but it seems that its main effect may be
to activate other kinases (PtdIns(3,4,5)P3dependent kinase-1 and -2), which in turn phosphorylate and activate protein kinase B [6].
Downstream of protein kinase B may lie a
number of important enzymes and other proteins
involved in the metabolic actions of insulin.
These may include glycogen synthase, because
protein kinase B is able to phosphorylate, and
hence inhibit, GSK-3 (the kinase that phosphorylates the main inhibitory sites in glycogen
synthase) [7]. Also downstream of protein kinase
B may be p70 S6 kinase, but not as a direct
substrate [8].