The substrate specificity of hormone-sensitive lipase from adipose tissue of the Antarctic fish Trematomus newnesi
1 Department of Biology, Arizona State University, Tempe, AZ 85287-1501,
USA
2 School of Marine Sciences, University of Maine, Orono, ME 04469-5751,
USA
* Author for correspondence (e-mail: bsidell{at}maine.edu)
Accepted 3 December 2003
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Summary |
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Key words: hormone-sensitive lipase, triacylglycerol lipase, Antarctic fish, Trematomus newnesi, adipose tissue, substrate specificity
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Introduction |
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A considerable body of evidence suggests that the obligately aerobic energy
metabolism of Antarctic fishes is fueled predominantly by the catabolism of
fatty acids derived from TAG stores in liver and adipose tissue. For example,
the maximal activity of carnitine palmitoyl transferase (CPT; a marker enzyme
for fatty acid oxidation) assayed at 1°C is elevated to a greater degree
than activities of citrate synthase and cytochrome oxidase in tissues of polar
compared with temperate fish species
(Crockett and Sidell, 1990).
Furthermore, in the Antarctic fish Gobionotothen gibberifrons, fatty
acids were more effective substrates of energy metabolism in oxidative
skeletal muscle and heart than either glucose or lactate
(Sidell et al., 1995
). Not
only is there a high capacity for fatty acid catabolism in polar species but
also, in several instances, the biochemical machinery is best able to
metabolize unsaturated fatty acids. For example, aerobic skeletal and
ventricular muscle of G. gibberifrons displays a clear preference for
the catabolism of monounsaturated fatty acids (particularly palmitoleic acid),
a preference that is mirrored by that of CPT, the rate-limiting enzyme in the
ß-oxidation pathway (Sidell et al.,
1995
). Similarly, in G. gibberifrons, mitochondria showed
a marked preference for the oxidation of palmitoleoyl-CoA and two
polyunsaturated fatty acids (PUFAs; 20:5 and 22:6), suggesting that in this
species both monoenes and PUFAs may support aerobic energy metabolism.
Metabolism of fats by vertebrates requires a complex suite of integrated
processes that encompass dietary acquisition, assimilation, synthesis of
storage depots (TAGs), mobilization of stored fats and, ultimately,
catabolism. Although catabolism of fats has been partially characterized in
Antarctic fish, processes of fat mobilization have not been characterized. The
rate-limiting enzyme responsible for initiating the mobilization of stored
fats from adipose tissue reserves is HSL (alternatively called TAG lipase). A
limited survey of Antarctic fish has previously demonstrated that, even when
measured at physiological temperature (0°C), the activity of adipose
tissue HSL is comparable to that observed in tissues of temperate-zone fishes
at their much higher body temperatures
(Sidell and Hazel, 2002). Some
limited data suggest that the process of lipolysis in Antarctic fish tissues
is nonrandom so that some fatty acids (perhaps those that are not
preferentially oxidized) are over represented in their abundance in storage
TAG compared with the composition of fats transported to other tissues for
oxidation. For example, the percentage of 14:0 in neutral lipid deposits of
adipose tissue exceeds that in pools of serum or oxidative skeletal muscles in
both G. gibberifrons and Trematomus newnesi
(Lund and Sidell, 1992
).
Likewise, in mammals, the mobilization of fatty acids from adipose tissue is
nonrandom, and chemically different fatty acids are mobilized at different
rates: rapidly mobilized fatty acids include C1620 fatty
acids with 45 double bonds, whereas slowly mobilized fatty acids
include C2024 fatty acids with 01 double bonds
(Raclot et al., 2001
).
We undertook the present study to characterize partially the substrate specificity of HSL isolated from adipose tissue of the Antarctic fish Trematomus newnesi and to determine whether this enzyme biases the composition of fatty acids released from adipose tissue and made available to other tissues for catabolism.
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Materials and methods |
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Just prior to experiments, fish were killed by a sharp blow to the head, followed by severance of the spinal cord posterior to the braincase. Body mass and standard length of all animals were measured and recorded. The fish used in these experiments averaged 79.3±3.46 g (mean ± S.E.M.) in mass, were 18.5±0.23 cm in length and possessed 0.22±0.03 g of mesenteric adipose tissue.
Tissue and enzyme preparation
Mesenteric adipose tissue was finely diced using a single-edged razor blade
and homogenized (20% w/v) in 0.35 mol l1 sucrose, 1 mmol
l1 EDTA, 25 mmol l1 Tris-HCl, pH 7.5 at
0°C employing a hand-held, ground-glass Tenbroeck tissue grinder (Wheaton,
Millville, NJ, USA). The homogenate was centrifuged at 10 000g for 30 min and the supernatant brought to 0.5 mol
l1 NaCl by the addition of an appropriate mass of solid
NaCl. HSL activity (which does not bind to heparin under the conditions
employed) was separated from lipoprotein lipase (LPL) activity (which does
bind to heparin) by heparinSepharose affinity chromatography as
described by Sheridan and Allen
(1984). The 10 000g supernatant was loaded onto a 1.5 cmx20 cm
heparinSepharose affinity column (prepared as described in a technical
bulletin provided by Sigma Chemical Company, St Louis, MO, USA) previously
equilibrated with homogenizing medium containing 0.5 mol l1
NaCl. Adipose tissue HSL eluted immediately after the void volume of the
column (Fig. 1); fractions
containing this activity were pooled and concentrated from 1525 ml to
approximately 5 ml by membrane ultrafiltration (Amicon, Millipore Corp.,
Billerica, MA, USA; 10 kDa cutoff) under a head of nitrogen pressure. Enzyme
activity was determined immediately after concentration, without prior
freezing.
|
Measurement of HSL activity and assessment of substrate specificity
We employed a modification of the radiometric assay of Sheridan and
coworkers (Sheridan and Allen,
1984; Sheridan et al.,
1985
) to measure HSL activity. This assay employed
14C-labeled triolein (isotope was present at the carboxyl carbon of
the oleic acid esterified to each of the three carbons of glycerol) as a
substrate, and activity was measured as radioactivity recovered in free fatty
acid after exposure to lipase.
A stock solution of radiolabeled triolein was prepared by drying solvent (ethanol) from 14.8 MBq of carboxyl-[14C]triolein [New England Nuclear, Perkin-Elmer, Boston, MA, USA; catologue no. NEC-674 (4.22 GBq mmol1)] under a gentle stream of nitrogen gas, followed by dissolving the radiolabel and 48 µmoles of nonradiolabeled triolein in 4 ml of absolute ethanol. This stock solution was stored at 04°C in an amber vial prior to use in experiments. The substrate specificity of the enzyme was inferred from the effects of the addition of nonradiolabeled competing substrates on the rate of hydrolysis of radiolabeled triolein; substrates more effective than triolein are expected to reduce the amount of radioactivity recovered in the assay. The final reaction mixture was prepared by diluting the above stock solution in buffer so that the final reaction mixture contained: 125 µmol l1 [14C]triolein, 1% (w/v) fatty-acid-free bovine serum albumin, 25 mmol l1 Tris-HCl, pH 7.5 at 0°C, and 25 µmol l1 nonradiolabeled competing substrate [the total triacylglycerol concentration in the assay was 150 µmol l1, with 83.3% of this amount comprised of radiolabeled triolein and 16.6% comprised of nonradioactive TAG (triolein in the case of control experiments or other species of TAG in the case of competition experiments)].
Execution of the experimental series, in some cases, required more than a single preparation of post-column enzyme. We observed insignificant differences in specific activity among the preparations of enzyme when measured with reference triolein substrate. Measurements with each preparation were made on all substrates within an experimental series (e.g. monoenoic, saturated or polyunsaturated TAGs) and, as indicated below, referenced to a corresponding estimate of activity in the presence of triolein. This method of normalization should ensure correction for even small variance in specific activities between preparations.
Reactions were initiated by the addition of 1 ml of post-column,
concentrated, enzyme preparation to 3.075 ml ofotherwise complete assay
buffer. Immediately after addition of enzyme, a small aliquot (50 µl) of
the reaction mixture was sampled for the determination of specific
radioactivity. The assay temperature was 0°C. Reactions were stopped at
240 min (previous experiments had established linearity of product release
over this time period; Sidell and Hazel,
2002) by the addition of organic solvents
[chloroform:methanol:benzene (1:2.4:2, v/v)], and free fatty acids were
extracted as described previously (Sidell
and Hazel, 2002
). Radioactivity was determined by the method of
external standard quench correction employing an LKB-Wallac 1409 liquid
scintillation spectrometer (Perkin-Elmer, Boston, MA, USA). HSL activities
were expressed in units of pmoles [14C]oleate released min
g1 wet mass. For assessment of substrate specificity, rates
of oleate release in the presence of 25 µmol l1 of the
competitive substrate being tested were compared with those induced by the
addition of 25 µmol l1 triolein.
Statistical analyses
Comparisons between substrates were performed by one-way analysis of
variance (ANOVA) using the StatisticaTM (Statsoft, Tulsa, OK, USA)
software package. To capture the natural variance associated with the addition
of nonradiolabeled triolein as a control (which is lost when results are
expressed as a ratio relative to triolein addition), statistical comparisons
were made for all TAG additions relative to the addition of a comparable
volume of assay buffer (devoid of TAG). Post-hoc tests for
significance between substrates employed the least significant difference test
for planned comparisons.
Chemicals and reagents
Sepharose 6MB, heparin and all species of both tri- and diacylglycerols
were from Sigma Chemical Company. All other chemicals were of reagent grade or
better.
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Results |
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Substrate specificity of adipose tissue HSL activity
Inferences about relative substrate specificity were made by determining
the effects of adding nonradiolabeled TAGs of varying chain length and degree
of fatty acyl unsaturation, or in some cases by the addition of defined
molecular species of diacylglycerols (DAGs), on lipase-catalyzed liberation of
oleic acid from triolein. All additions of competitive substrates were
compared to the effects of adding a similar concentration of nonradiolabeled
triolein (in each case, the nonradiolabeled TAG is in excess concentration to
the labeled compound). A significant reduction in HSL activity in the presence
of a competitive substrate (compared with triolein) indicates that the
substrate competes more effectively for the enzyme than triolein. ANOVA
indicated a highly significant effect (P<0.00001) of competitive
substrate addition on rates of triolein hydrolysis.
Saturated species of TAGs
Saturated species of TAGs significantly stimulated rather than inhibited
rates of triolein hydrolysis (Fig.
2). The stimulatory effect of trisaturated species of TAG on rates
of triolein hydrolysis was inversely related to chain length: in the presence
of tri-14:0, tri-16:0 and tri-18:0 species of TAG, rates of triolein
hydrolysis were 3.7-fold (P<0.0001), 2.1-fold
(P<0.001) and 1.9-fold (P<0.002) greater,
respectively, than that observed with triolein as the sole substrate. The
particularly pronounced stimulation of triolein hydrolysis in the presence of
tri-14:0 suggests that this compound is a particularly poor substrate for HSL,
thus effectively elevating the specific radioactivity of the triolein pool in
the assay.
|
Monounsaturated species of TAGs
Rates of triolein hydrolysis were significantly influenced by only two of
the tri-monounsaturated species of TAG varying in chain length from 14 to 24
carbons (Fig. 3). The longer
chain (C22 and C24) monoenoic species of TAG, similar to
saturated species, significantly (P<0.04) stimulated rather than
inhibited rates of triolein hydrolysis. Although other differences were not
significant, tri-16:1 and tri-18:1 molecular species yielded the lowest rate
ratios.
|
Polyunsaturated species of TAGs
Rates of triolein hydrolysis were significantly (P<0.04)
depressed in the presence of polyunsaturated (tri-20:4 and tri-18:2) molecular
species of TAG (Fig. 4).
|
Positional isomers of DAGs
DAGs are intermediate products in the hydrolysis of TAGs to monoglycerides
or glycerol and fatty acids. None of the positional isomers of either 18:1 or
16:0 DAG significantly influenced the rates of triolein hydrolysis
(Fig. 5). Two trends in these
data are, however, noteworthy: (1) rates of triolein hydrolysis in the
presence of 16:0 DAGs were significantly lower than those observed with
tri-16:0 TAG (compare Figs 5
and 2) and (2), although not
significantly different, for DAG species of both 18:1 and 16:0, the
1,2-molecular species consistently depressed rates of triolein hydrolysis to a
greater extent than the 1,3-molecular species.
|
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Discussion |
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In mammals, it is clear that the release of fatty acids from adipose tissue
is not random. For example, in rabbit adipose tissue, mobilization of fatty
acids into the plasma was not proportional to their abundance but rather was
determined by their molecular structure; rates of mobilization varied over a
range of 46-fold and correlated positively with degree of unsaturation and
negatively with chain length (Connor et
al., 1996). Indeed, eicosapentaenoic acid (20:5n3) and
arachidonic acid (20:4n6), important precursors in prostaglandin
biosynthesis, were mobilized at the highest rates. Since HSL is a key and
rate-limiting enzyme in lipolysis, we hypothesized that the substrate
specificity of this enzyme in Antarctic fish might result in the hydrolysis of
selected fatty acids from storage reserves. In particular, we wished to
determine whether the previously established preferences in the capacities for
fatty acid catabolism by muscle and liver tissues (Crockett and Sidell,
1990
,
1993a
,b
;
Sidell et al., 1995
) were
matched by a corresponding pattern of fatty acid release from adipose tissue
reserves.
We have previously reported that mass-specific activities of HSL in
Antarctic fish, when measured at 0°C, are roughly comparable to those of
temperate zone species assayed at their physiological temperatures
(Sidell and Hazel, 2002). This
observation most probably arises from one, or a combination of, unexplored
adaptations that might include: (1) evolution of a lipase that is more
efficient at low temperatures, as has commonly been reported for a variety of
other enzymes (Somero, 1995
),
(2) elevated titers of the enzyme in tissues of Antarctic fish or (3) a
reduced temperature sensitivity for HSL compared with other enzymes. With
regard to the third possibility, it is interesting to note that human HSL
displays significant sequence homology to an Antarctic bacterial lipase and
displays significant hydrolytic capacity even at low temperatures
(<10°C), causing this enzyme to be classified as psychrotolerant
(Laurell et al., 2000
).
However, aside from demonstration of significant catalytic activity at low
temperature, the potential role of this enzyme in determining the nature of
the fatty acids released for catabolism in Antarctic fish has not been studied
previously.
To characterize the substrate specificity of HSL from the adipose tissue of Antarctic fish, it was first necessary to separate HSL from other members of the same gene family with different substrate preferences and kinetic properties. The other activity of concern is LPL, which functions to degrade serum lipids destined for deposition in adipose tissue and possesses a heparin-binding domain not present in HSL. HeparinSepharose affinity chromatography (Fig. 1) clearly resolved HSL and LPL activities present in total tissue extracts of adipose tissue from T. newnesi. Of these two activities, HSL accounted for approximately 75% of the total activity (LPL 25%).
To assess the substrate specificity of HSL, a competition assay was
employed. Control assays were used to define the maximal release of oleic acid
from radiolabeled triolein. Substrate specificity was assessed by adding a
second nonradiolabeled substrate to the reaction mixture and determining the
extent to which the liberation of oleic acid was inhibited. The degree to
which the release of oleic acid is decreased in the presence of the
non-radiolabeled substrate reflects the ability of HSL to hydrolyze one
species of TAG preferentially over another. The substrate causing the greatest
degree of inhibition is presumably the one most readily hydrolyzed
(Crecelius and Longmore, 1984).
Conversely, elevation in the rate of radiolabeled release from triolein by the
added substrate would indicate either a true stimulation of HSL activity or,
alternatively, that the added TAG was such a poor substrate for HSL that it
was effectively `transparent' to the enzyme, thus elevating the specific
radioactivity of the labeled triolein pool.
To our surprise, totally saturated molecular species of TAG caused an apparent stimulation rather than inhibition in rates of oleate release from triolein (Fig. 2). The magnitude of this activation varied between 2- and 4-fold and decreased as a function of increasing chain length for species of TAG bearing fatty acids between 14 and 18 carbons in length. By contrast, molecular species of TAG containing monoenoic fatty acids varying between 14 and 20 carbons in length had no significant effect on rates of oleic acid release (Fig. 3) compared with triolein. The observation that activity ratios were somewhat lower for tri-16:1 and tri-18:1 TAG than tri-14:1 or tri-20:1 is suggestive of a slight preference for monoenoic species of TAG with chain lengths of 1618 carbons. Longer chain (C2224) monoenoic species of TAG resembled totally saturated species more than shorter chain monoenoic species since they significantly stimulated rather than inhibited oleate release (Fig. 3). More highly unsaturated species of TAG (containing di- and tetraenoic fatty acids) significantly depressed rates of oleate release (Fig. 4), indicating that these species are preferentially hydrolyzed compared with triolein.
Taken collectively, the above data support the following rank order of
preference for the hydrolysis of molecular species of TAG by adipose tissue
HSL from the Antarctic fish T. newnesi: polyenoic species >
monoenoic species > saturated species. This order corresponds roughly with
that established for the rates of fatty acid release from adipose tissue in
mammals (Connor et al., 1996).
Among monoenes, the preferential release of oleic acid also resembles the
substrate selectivity of rat and human HSL
(Raclot et al., 2001
);
however, the preferential release of PUFAs is a unique feature of the T.
newnesi HSL that is not shared by either the rat or human enzymes. Thus,
in contrast to the situation for human and rat HSL, for which fatty acid chain
length is the major determinant of rates of hydrolysis, the degree of fatty
acid unsaturation appears to be the primary determinant of HSL activity in
T. newnesi. The substrate specificity of T. newnesi HSL also
meshes well with the pattern of differential peripheral utilization of fatty
acids by liver and muscle (Crockett and Sidell,
1993a
,b
;
Sidell et al., 1995
).
The failure of fully saturated and long-chain monounsaturated species of
TAG to inhibit the hydrolysis of triolein was unanticipated and a novel
feature of the present data set. The pronounced 3.7-fold increase in triolein
hydrolysis in the presence of trimyristin (tri-14:0) is particularly
noteworthy, given the disproportionate representation of this TAG in adipose
tissues of Antarctic fish species (Lund
and Sidell, 1992). As indicated above, this observation could
suggest that tri-14:0 either specifically stimulates HSL activity or,
alternatively, is a poor substrate for this enzyme. The percentage of 14:0 in
adipose tissue TAG of T. newnesi (15.1±1.0%) is vastly greater
than in pools of TAG from either oxidative skeletal muscle (5.7±0.6%)
or combined serum TAG and cholesterol esters (4.5±0.4%;
Lund and Sidell, 1992
). Since
fully saturated fatty acids possess significantly higher melting points than
oleic acid (Gurr and Harwood,
1991
), one possibility is that TAG species containing these acids
may phase-separate from triolein at 0°C and, thus, might not be readily
accessible to the enzyme. However, our data indicate significant differences
among saturated fatty acids in their abilities to stimulate triolein
hydrolysis, which is not consistent with a phase separation argument (i.e. the
ability to stimulate triolein hydrolysis is inversely related to fatty acid
melting point, exactly the opposite trend predicted by a phase-separation
argument; Fig. 2). Thus, it
seems most likely that the differences in rates of triolein hydrolysis
observed in the presence of competing saturated and other species of TAG
reflect the true substrate specificity of the enzyme rather than a biophysical
effect of low temperature on the state of the substrate. In light of the above
observations, we favor the interpretation that tri-14:0 (and, by inference,
the other saturated TAGs) is a particularly poor substrate for lipolysis by
HSL when more suitable alternative TAG substrates are available to the enzyme.
It is worth noting, however, that regardless of the mechanisms in play, the
physiological implications remain the same.
Although HSL is the most likely determinant of the mix of fatty acids
released from adipose tissue, it should be acknowledged that two other
proteins may play a secondary role in regulating this process: (1) perilipins
are proteins located on the surface of lipid droplets in unstimulated
adipocytes that presumably block the access of HSL to substrate in
unstimulated adipocytes (perilipins, like HSL, are phosphorylated by PKA as a
consequence of adipocyte activation, causing them to redistribute away from
the lipid droplet, thus permitting HSL to bind to the droplet surface;
Østerlund, 2001) and
(2) the adipocyte lipid binding protein (ALBP) specifically interacts with HSL
and most likely removes the fatty acid product of lipolysis
(Holm et al., 2000
).
Activated HSL displays a preference (34-fold) for hydrolysis of the
1- or 3-ester bond of its acylglycerol substrate compared with the 2-ester
linkage (Holm et al., 2000).
Interestingly, the T. newnesi enzyme displays a slight preference for
DAG compared with TAG and for the sn-1,2 over the sn-1,3
isomer of DAG (Fig. 5), implying that initial cleavage of the sn-3 linkage increases the
likelihood of the resulting sn-1,2 species being converted to a
2-monoglyceride. Thus, sn-2 monoacylglycerols are expected to be the
primary substrates for monoacylglycerol lipase.
An interesting regulatory aspect of lipid mobilization from adipose tissue
of Antarctic fish is how this storage depot is managed to meet the potentially
differing and conflicting demands of buoyancy regulation and as an energy
source to fuel aerobic metabolism. Our results do offer some possible insight
into this process. Our present data suggest that saturated species of TAG
(especially short-chain species such as tri-14:0) are poorly hydrolyzed by
HSL. Previous results likewise show that especially 14:0 accounts for a
disproportionate fraction of stored neutral lipid in these species
(Lund and Sidell, 1992) and,
consequently, may be among the primary species of fatty acid involved in
buoyancy regulation. Evidence has also been accumulating that the corporeal
stores of lipid in Antarctic fishes increase with increasing body size,
conferring greater buoyant advantage as the animals become larger
(Friedrich and Hagen, 1994
;
Hagen et al., 2000
;
Near et al., 2003
). In
combination, these observations make it possible to envision that the spectrum
of dietary lipid in excess of immediate needs is readily partitioned into
storage by lipogenic machinery but that subsequent hormone-activated
mobilization of these stores favors the breakdown of TAGs substituted with
more unsaturated fatty acids, leaving a gradually increasing component of TAGs
substituted with saturated fatty acids, especially 14:0, over time. Thus, a
pool of relatively metabolically inert neutral lipids would be accumulated and
contribute to whole animal buoyancy.
In summary, our results indicate that adipose tissue HSL of T. newnesi releases fatty acids from a TAG emulsion in a nonrandom fashion. PUFAs are preferentially released, followed by monoenes; saturated species of TAG are hydrolyzed most slowly. Degree of fatty unsaturation, more than chain length, is the primary determinant of hydrolytic rates. These data indicate that HSL is a potentially important determinant of the specific fatty acids released from the storage depots of Antarctic fish. In addition, there is a reasonable correspondence between the fatty acids released due to the action of adipose HSL and the patterns of peripheral fatty acid catabolism by liver and muscle in these fish and an equivalent correspondence between those most poorly released and the composition of accumulated storage TAG in adipose tissues.
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Acknowledgments |
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