A8
Biochemical Society Transactions (2000) Volume 28, part I
D1
D3 The use of liver-specific targeting of both insulin and acyl
'
moieties to study the effects of the hormone on hepatic
triacylglycerol(TAG) secretion in vivo.
Victor A. ZAMMIT and Susan M. RENNIE
Hannah Research Institute, Ayr, KA6 5HL, Scotland
The acyl-CoA oxidase gene family in Arabidopsis thuliuna:establishing gene function through reverse genetics
'I'.J.Eastmond, *M.A. Hooks, 'LA. Graham
Department of Biology, University of York, York, YO1 5 W , 2School of
Biological Sciences, Universiry of Wales, Bungor Gwynedd, LLS7 2UW
The first step in peroxisomal P-oxidation of fatty acids is carried out by the
acyl-CoA oxidase enzyme. In plants there is a family of acyl-CoA oxidases
that are specific for different chain length fatty acyl-CoAs. We have
recently cloned and characterised medium-long and long chain acyl-CoA
oxidases from Arabidopsis thuliana by heterologous overexpression in
Eschcrichu colt. A short chain acyl-CoA oxidase has also recently been
characterised by other workers. Based on the substrate specificities of these
enzymes and gel filtration chromatography we speculated that a medium
chain acyl-CoA oxidare must also exist in order for fany acids to be efficiently P-oxidised. T-DNA promoter trapping in Arubidopsis has led us to
discover a fourth acyl-CoA oxidase gene. Functional analysis of this gene
by expression of a full length cDNA in E. coli demonstrated that it encodes
an enzyme with medium-chain acyl-CoA oxidase activity. In agreement
with this data, analysis of the Arabidojxis knockout mutant ( a d ) showed
that germinating seedlings conuined less than 3 % of wild type mediumchain acyl-CoA oxidase activity.
The importance of these different enzymes in controlling P-oxidation of
fatty acids thoughout plant development will be discussed. Particular
emphasis will be placed on evaluation of these enzymes as targets to control
the futile cycling of unusual fatty acids in genetically engineered crop
plants.
The elucidation of the effects of insulin on hepatic TAG secretion
has been controversial because of hitherto conflicting observations
on the activation or inhibition produced by insulin either on
different in vitro systems or in different species in vivo.
Examination of the literature indicated that this controversy may be
due to the effect of insulin being dependent on the metabolic state
of the tissue or preparation prior to insulin challenge. Therefore, we
studied the effects of acute exposure to insulin on TAG secretion in
(i) the liver in vivo (ii) the isolated perfused liver, and (iii) cultured
rat hepatocytes, to determine which in v i m preparation better
represents the effects observed in vivo. The latter were studied
through simultaneous exposure of awake, unrestrained animals to a
glucose clamp, specific labelling of the main hepatic fatty acid pool,
and selective insulinisation of the liver by infusion of hepatocytetargeted liposome-encapsulated insulin. The data show that the
direction of the effect of insulin in vivo on the utilisation of fatty
acyl moieties for synthesis of secreted TAG changes from
srirnulatory in the fed, normoinsulinaemic state, to tnhibirory in
either the fasted or insulin-deficient state. This switch in direction
of insulin action on TAG secretion was reproduced by the isolated
perfused liver preparation. By contrast, insulin invariably inhibited
TAG secretion by 24h-cultured rat hepatocytes irrespective of the
physiological state of the donor animals. The experimental,
physiological and pathological implications of these observations
will be discussed.
D2
Studying metabolic regulation in human muscle
G.J.Kemp
Department of Musculoskeletal Science, University of Liverpool,
Liverpool L69 3GA, UK
Muscle metabolism can be studied in vivo by several methods. Whereas
arteriovenous difference and biopsy techniques are invasive, phosphorus
magnetic resonance spectroscopy (MRS) can noninvasively measure several
relevant cellular metabolites: phosphocreatine, orthophosphate, 'phosphomonoesters', and (indirectly) free protons, ADP and AMP. Despite its
limitations (poor sensitivity and fibre-type averaging), MRS is a useful
window on metabolism and its regulation. For example, in ischaemic
exercise ATP is produced only anaerobically, and we can use MRS to study
the regulation of glycolysis. Despite some closed-loop feedback elements
(e.g. stimulation of glycogen phosphorylase by orthophosphate), this is
dominated by open-loop ('feed-forward') mechanisms (e.g. calciumdependent phosphorylase b-to-a conversion, and other, unknown,
mechanisms). In purely aerobic exercise, and also in recovery from exercise,
we can study the regulation of oxidative ATP synthesis. This is dominated
by closed-loop mechanisms (e.g. stimulation by ADP); however the contribution of open-loop elements and the relationship to formal metabolic
control analysis remain controversial. The co-regulation of anaerobic and
aerobic mechanisms in 'mixed' exercise is not well understood. To advance,
we will need richer dausets: the combination of phosphorus MRS with
measurements of muscle oxygenation (by near-infrared spectroscopy) and
blood flow has considerable potential. We also need better understanding of
the conceptual tools appropriate to these studies, where information is
incomplete and steady state assumptions often inapplicable.
0 2000 Biochemical Society
D4
The use of hemoglobinfree perfused liver in metabolic control
analysis
S.,
G.C. Brown
Institut fur Physiologische Chemie I, Heinrich-Heine-Universitat
Diisseldorf. Germany
Whcn studying bioenergetics in intact cells or organs, difficulies are
encountered of the complex network of ATP-consuming and ATPproducing reactions of the cell on one hand, and of the limited possibility to
measure changes in subcellular intermediates, on the other hand. Top-down
metabolic control analysis (1) regards a whole pathway o r a network of
pathways, and analyses the control exerted by whole pathways or parts of
pathways interacting via common intermediates. A top-down approach
was applied to in sit# perfused rat liver from 24h fasted rats. We used a nonrecirculating perfusing system with a carbogen gassed Krehs-Henseleit
bicarbonate buffer, p H 7.4, supplied with substrates and inhibitors, respectively (2). By this way it is possible to estimate metabolic pathway rates via
perfusate metabolite analyses. We analysed oxidative phosphorylation (as
ATP-producing pathway), urea synthesis, gluconeogenesis and the sum of
other (maintenance) ATP- consuming pathways, linked via the cytosolic
ATPIADP Pi system.
(1) Brown, G., Hafner, R.P.&Brand, M.D.(1990) Eur. J. Biochcm. 188,
321-325
(t)Soboll, S., Oh, M-H. & Brown, G. (1998) Eur. J. Biochem. 254, 194 201
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