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INTRODUCTION |
Lipoprotein lipase
(LPL)1 is an enzyme that acts
at the vascular endothelium (1, 2). It hydrolyzes triglycerides in
lipoproteins and thereby makes the fatty acids available for cellular
metabolic reactions. The enzyme is synthesized in and secreted by
parenchymal cells in the tissue, e.g. adipocytes and
myocytes, and is transferred to the endothelium to which it becomes
anchored by interaction with heparan sulfate proteoglycans. It was
shown already in 1959 that the LPL activity in adipose tissue changes
rapidly in response to the nutritional state (3, 4). Studies by the
Robinson group then demonstrated that there is a strong correlation
between the LPL activity and the uptake of fatty acids from labeled
chylomicrons in rat adipose tissue (5). This suggests that LPL is an
important determinant for the distribution of lipids between tissues
(6). The mechanism for the regulation is, however, not clear.
The regulation appears to be post-transcriptional. LPL mRNA in
(rat) adipose tissue turns over with a half-life of 17 h, far too
slowly to account for the rapid drop of enzyme activity when food is
withdrawn (7). Several groups have found that LPL mRNA and/or
relative rate of LPL synthesis (8-12) as well as LPL mass ((9, 13-15)
remain essentially unchanged during short-term fasting.
There are two forms of the lipase in tissues (15). One form is
catalytically active and has high affinity for heparin. This is the
predominant form in adipose tissue of fed rats. The other form is
catalytically inactive, has low affinity to heparin and predominates in
adipose tissue during fasting. When fasted rats are given food, the LPL
activity returns to fed levels in 4-6 h. This increase of activity
requires synthesis of new LPL molecules; there is no evidence that the
inactive lipase protein can be recruited into the active form (7,
16).
Many studies have shown that the LPL activity that is released when
pieces of adipose tissue are incubated in heparin-containing medium (4,
12) or when adipose tissue is perfused with heparin (17)
("heparin-releasable LPL") changes more than total tissue LPL
activity. This indicates that the regulation is exerted mainly on the
extracellular LPL (18).
Short-term regulation of LPL in adipose tissue thus appears to involve
changes both in the activity state of the LPL protein and in the
distribution of LPL between intra- and extracellular locations in the
tissue, but it is not known how these two parameters are related to
each other. To resolve this, we have measured LPL activity and mass in
adipose tissue and in adipocytes isolated from the tissue. In some
experiments we have blocked synthesis of new enzyme to study the
turnover of the different pools of LPL, and we have studied which pools
of LPL (active/inactive, intra/extracellular) that are accessible to
heparin release. All results point to extracellular LPL as the target
for short-term nutritional regulation.
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EXPERIMENTAL PROCEDURES |
Animals--
Male Sprague-Dawley rats (23 days old) weighing
around 60 g were from Möllegaard Breeding Center (Ejby,
Denmark). They were allowed to acclimatize for 7-10 days by
which time they had reached a weight of ~120 g. The rats were kept in
a well ventilated, temperature (21 °C)- and humidity
(40-45%)-controlled room with free access to a standard laboratory
chow (Laktamin AB, Stockholm, Sweden) and tap water. The light in
the room was on between 6 a.m. and 6 p.m. In experiments
where the rats were to be fasted, food was withdrawn from the cages at
6 a.m., and a grid was placed at the bottom of the cages to
prevent coprophagia. The rats were killed by decapitation. The adipose
depot used in all experiments was the periepididymal one. In some
experiments the tissue was cut into small pieces and digested with
collagenase to isolate the adipocytes as described (19). The animal
ethics committee in Umeå approved the animal experiments.
Materials--
Cycloheximide, bovine serum albumin, and
collagenase were from Sigma, protease inhibitor mixture tablets
"Complete Mini" was from Roche Diagnostics, Mannheim, Germany.
Heparin was from Lövens (Malmö, Sweden).
125I-LPL was prepared as described (20). All other reagents
were of the highest commercial grade.
Extraction, Separation, and Assay of LPL Activity and
Mass--
LPL was extracted from tissues by homogenization in an
ammonia buffer (pH 8.2) containing detergents and protease inhibitors as described (15) but with the protease inhibitor mixture tablets Complete Mini (Roche Diagnostics) instead of the inhibitors
previously used. The homogenate was centrifuged for 15 min at 3000 rpm,
after which the intermediate phase (between the floating fat droplets and the pellet) was used for assay of LPL activity and mass. Most of
the data on activity or mass were calculated relative to the amount of
DNA in the sample, which was measured by fluorometry as described by
Labarca and Paigen (21). In some experiments active and inactive forms
of LPL in the extracts were separated on heparin-agarose as described
(15).
LPL activity was measured as described (15). The substrate was
3H-labeled triolein in Intralipid (10%) kindly prepared by
Pharmacia-UpJohn (Stockholm, Sweden). Briefly, 2 µl of tissue
homogenate (triplicate samples) was incubated for 60 min at 25 °C
with substrate in the presence of 10 µl of heat-inactivated serum
from fasted rats (as source of apolipoprotein CII) and 6% bovine serum
albumin. The total volume was 200 µl. After termination of lipolysis
by addition of organic solvents, the fatty acids were extracted and
counted for radioactivity. One milliunit of lipase activity
represents 1 nmol of fatty acids released per minute.
LPL mass was determined with a sandwich enzyme-linked immunoadsorbent
assay as described before (15) but with bovine LPL as standard.
Briefly, three different dilutions of tissue homogenate were incubated
in microtiter plate wells previously coated with affinity-purified
chicken anti-LPL IgG. Detection was mediated via the 5D2 monoclonal
antibody (a kind gift by Dr. John Brunzell, Seattle, WA) followed by a
peroxidase-conjugated anti-mouse IgG antibody. Absorbance at 490 nm was
measured in a Spectramax microplate spectrophotometer (Molecular
Devices, Sunnyvale, CA).
Statistics--
Data are presented as mean ± S.D.
Student's t test was used.
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RESULTS |
Active LPL Is Mainly Extracellular in Adipose Tissue of Fed
Rats--
As reported earlier (15) LPL mass was similar, but
LPL activity was higher in adipose tissue from fed compared with fasted rats (Table I). To study the distribution
of LPL within the tissue we used collagenase to digest the
extracellular matrix. The adipocytes were then isolated by floatation.
LPL activity was similar in adipocytes from fed and fasted rats (Table
I). Hence, the higher LPL activity in fed rats was due entirely to
higher extracellular LPL activity.
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Table I
Distribution of LPL in rat adipose tissue
Epididymal adipose tissue was collected from fed or 24-hour fasted
rats. One portion of the tissue was cut into pieces and incubated with
collagenase. Adipocytes were then isolated by centrifugation. LPL
activity and mass were determined in extracts from whole tissue and
adipocytes, respectively. Extracellular LPL was calculated as the
difference between whole tissue and adipocytes. In the left columns the
values are the amounts present in 1 g of tissue or in adipocytes
isolated from 1 g of tissue, respectively. In the right columns
the values are per µg of DNA in tissue or adipocytes, respectively.
This gives a better comparison since the loss of triglyceride weight on
fasting does not influence the values and since direct determination of
DNA in the isolated adipocytes adjusts for variations in recovery. No
value for extracellular LPL can be calculated from these values. All
values are mean ± S.D. for six rats in each group.
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When bovine or rat LPL was incubated with collagenase, LPL activity and
mass decreased rapidly. To study why this happened, 125I-labeled bovine LPL was added to the medium, which
after incubation for a few minutes was analyzed by SDS-PAGE (data not
shown). Radioautography showed that a number of low-molecular weight
bands had been formed, demonstrating that LPL is rapidly cleaved by
collagenase under the conditions used to isolate adipocytes. Hence, it
was not possible to recover and directly measure extracellular LPL. The
pellet of sedimented cells (often called the stromal-vascular cells) contained only 3% of tissue LPL activity and 4% of tissue LPL mass.
Hence, virtually all LPL was either in adipocytes or
extracellular. The amount of extracellular LPL could therefore
be estimated as the difference between tissue total and adipocytes.
This activity was 3.5-fold higher in fed compared with fasted rats
(Table I). In contrast, extracellular LPL mass did not differ
significantly between the nutritional states. This experiment was
repeated six times with some variations (age of rats, time of fasting).
The difference in tissue LPL activity between fed and fasted rats ranged from 2-fold (as in this experiment) to more than 4-fold. It was
a consistent finding that LPL activity within adipocytes was the same
irrespective of the nutritional state so that the difference in tissue
LPL activity was always due to a difference in extracellular activity.
LPL mass on the other hand did not differ significantly with
nutritional state, in tissue total, in adipocytes, or
extracellular (Table I).
Separation of Active and Inactive LPL--
Earlier studies have
shown that rat tissues contain at least two forms of LPL (15). The
catalytically active form has high heparin affinity and elutes at about
1 M NaCl from heparin-agarose. Inactive LPL elutes earlier
in the gradient. There is more active than inactive LPL in adipose
tissue from fed rats, whereas the reverse is true for fasted rats. We
obtained similar results in the present study (Fig.
1, upper panels). The peak
ratio (area of mass in the first peak divided by that in the second
peak) was 0.49 ± 0.06 and 2.26 ± 0.14 in tissue extracts
from fed and fasted rats, respectively. In contrast, there was no
difference in the distribution of LPL between the active and inactive
forms within the adipocytes (Fig. 1, lower panels). The peak
ratios were 2.05 ± 0.15 and 2.25 ± 0.04 in fed and fasted
rats, respectively. The specific activity for the enzyme that eluted in
peak two was similar (about 1 milliunit/ng) for all four separations,
i.e. for total tissue LPL as well as for adipocyte LPL in
fed and in fasted rats.

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Fig. 1.
Separation of active and inactive forms of
LPL in extracts from adipose tissue and from isolated adipocytes.
Epididymal adipose tissue was dissected from fed and 24-h fasted rats.
Adipocytes were isolated from one portion of the tissue. Extracts of
whole tissue (upper panels) or adipocytes (lower
panels) were prepared as detailed under "Experimental
Procedures" and applied on a heparin-agarose column. The amount of
material applied corresponded to the same amount (by weight) of adipose
tissue in all four cases. The column was eluted by a salt gradient, and
LPL activity (squares) and mass (circles) was
determined in the fractions.
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Turnover of LPL Activity and Mass after Inhibition of Protein
Synthesis--
The size of the pools of LPL are determined in part by
the turnover rates, which might change with the nutritional state. To
explore this, rats were injected with cycloheximide. In the experiment
in Fig. 2, LPL mass decreased by 65-75%
in 2 h, indicating a half-life in the order of 1 h. There was
no difference between fed and fasted rats (Fig. 2, lower
panel). This rules out the possibility that the relatively large
amount of inactive LPL in adipose tissue of fasted rats represents
dead-end material that turns over slowly. After the first 2 h, the
decrease of LPL became much slower, presumably because incomplete
inhibition of protein synthesis allowed some continued production of
the enzyme.

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Fig. 2.
The effect of cycloheximide on
LPL activity and LPL mass in rat adipose tissue. Cycloheximide (35 mg/kg of body weight) was injected intraperitoneally into fed or 24-h
fasted rats. Rats were killed before and 2, 4, or 6 h after the
injection. The upper panel shows LPL activity, the
lower shows LPL mass. Filled symbols are values
for fed rats, open symbols are values for fasted rats. The
inset in the upper panel shows the data
recalculated in percent of the initial activity; the data points for
fed (filled symbols) and fasted (open symbols)
rats fall almost on top of each other. Mean ± S.D. for five rats
at each time.
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LPL activity dropped rapidly after cycloheximide was given. This is in
accord with earlier studies in vivo (7, 22, 23) and with
cells (16, 24). In the fed rats, LPL activity dropped at a similar rate
as LPL mass (Fig. 2, upper panel). The much lower activity
in the fasted rats also dropped. To be able to compare the rates
between the nutritional states we recalculated the data as percent of
initial activity (Fig. 2, inset). When expressed in this
way, the LPL activities decreased at virtually the same rate in the two
nutritional states. The loss of LPL mass and activity during the
initial rapid phase (about 2 h) and the height of the subsequent
slow phase differed between experiments, presumably due to variation in
the extent to which protein synthesis was inhibited.
It was not feasible to isolate adipocytes from all the animals in the
experiment in Fig. 2. Therefore a separate experiment was carried out
at a single time point (Fig. 3). In this
experiment, LPL mass decreased by 45-55% in 90 min, compared with
about 75% in 2 h in the experiment in Fig. 2. The percentage loss
of LPL mass was similar for tissue total, for adipocytes, and for
calculated extracellular LPL mass (Fig. 3, lower panel). LPL
activity decreased by 53 and 47% in whole tissue and by 52 and 42% in
adipocytes from fed and fasted rats, respectively (Fig. 3, upper
panel). These differences were not statistically significant. In
any case, this experiment shows that all four pools of LPL,
active/inactive, intra/extracellular, turn over rapidly and at similar
fractional rates.

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Fig. 3.
The effect of cycloheximide on the
distribution of LPL activity and mass in the tissue. Same
conditions as in Fig. 2 but only a single time point (90 min) was
studied. Adipocytes were prepared from one part of the tissue. The
black portion of the bar shows activity or mass in
adipocytes, the shaded portion shows the extracellular
activity as the difference between whole tissue and adipocytes. Mean
for five rats in each group.
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Release of LPL Activity and Mass from Adipose Tissue after
Injection of Heparin in Vivo--
Many studies have shown that LPL is
released from its endothelial binding sites by heparin (1, 2). We
questioned how much and what fraction of the total tissue LPL could be
released by perfusion with heparin. For this we injected heparin and
measured tissue LPL mass and activity at a series of times (Fig.
4). There was a gradual decline of LPL
activity with time. This was more pronounced in tissues from fed rats,
from 1700-800 milliunits/g in 60 min compared with
700-600 milliunits/g in fasted rats. In contrast, there was no change
in the activity within adipocytes. Hence, heparin mobilized a large
fraction of the extracellular enzyme, but none of the intracellular.
The inset in Fig. 4 shows the percent change in
extracellular activity with time after heparin injection. When the data
were expressed in this way, the decline of activity followed the same
time course for fed and fasted rats, and the percent of the initial
extracellular activity that was lost over the 60 min studied was also
the same, about 60% in both fed and fasted rats.

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Fig. 4.
Effect of heparin in vivo on
intra- and extracellular LPL in adipose tissue. Heparin (500 IU/kg
of body weight) was injected intravenously. Rats were killed before and
2, 15, or 60 min after the injection, and epididymal adipose tissue was
dissected out. The upper panel shows LPL activity: ( )
whole tissue, fed rats; ( ) whole tissue, fasted rats; ( )
adipocytes, fed rats; ( ) adipocytes, fasted rats. The
inset in the upper panel shows calculated
extracellular LPL activity: ( ) fed rats, ( ) fasted rats. The
lower panel shows LPL mass: ( ) whole tissue, fed rats;
( ) whole tissue, fasted rats; ( ) adipocytes, fed rats; ( )
adipocytes, fasted rats. Mean ± S.D. for five rats at each
time.
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The changes in LPL mass (Fig. 4, lower panel) followed the
same trends as for LPL activity. Heparin extracted more LPL mass in the
fed compared with the fasted rats, and the changes occurred only in the
extracellular portion. Within the adipocytes, LPL mass remained constant.
The specific activity of tissue LPL was 0.93 milliunit/ng in the fed
rats and 0.39 milliunit/ng in the fasted rats. A calculation of the
specific activity for the enzyme that was lost from the tissue in 60 min after heparin injection returns values of 1.8 milliunits/ng in the
fed and 1.7 milliunits/ng in the fasted rats. These figures carry a
large uncertainty since they were calculated as the ratio of
differences between rather large values. Furthermore, they do not take
into account the LPL that was produced and released from the adipocytes
during the 1-h experiment. Nonetheless, the figures indicate that
heparin released only, or at least mainly, the active species of
LPL.
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DISCUSSION |
This study shows that most of the LPL protein in adipose tissue is
located extracellularly, irrespective of the nutritional state. Some
earlier studies had indicated that regulation of LPL activity is
exerted by changes in the proportion of newly synthesized enzyme that
is secreted from the cells (9, 12, 25). If so, the larger amount of
inactive LPL in adipose tissue of fasted rats would be expected to be
present within adipocytes, retained in the ER. The specific activity of
intracellular LPL would be lower in fasted compared with fed rats,
whereas the specific activity of the extracellular enzyme would be
similar in both nutritional states. In contrast to these predictions,
we found that the specific activity of intracellular LPL remained the
same. It was the specific activity of the extracellular enzyme that
changed. A confounding factor could have been that the turnover rates
for the different pools of LPL changed with the nutritional state. In
fasted rats an increased turnover rate for inactive LPL in adipocytes
might compensate so that mass remained relatively constant even though more enzyme was retained in the ER. Conversely, the turnover rate of
extracellular LPL would have to be slower in fasted rats so that mass
remained high even though secretion was reduced. We did not, however,
find any marked differences with nutritional state in the turnover
rates for any of the pools of LPL. Our data thus indicate that the
nutritional regulation of adipose LPL is exerted on the activity state
of the extracellular enzyme.
Ben-Zeev et al. recently studied the synthesis and
maturation of human LPL transfected into Chinese hamster ovary
cells (16). A large fraction of newly synthesized LPL was retained in
the ER, formed disulfide-linked aggregates, and was degraded through an
energy-dependent pathway. In concert with this, we found
that about 70% of LPL protein within the adipocytes was inactive. In a
pulse-chase study with guinea pig adipocytes, Semb and Olivecrona found
that 65% of newly synthesized LPL was degraded within 4 h (24).
Speake et al. (26) and Lee et al. (12) have also shown that LPL is degraded in rat adipose tissue. All these data are in
agreement with the hypothesis that a large fraction of newly
synthesized LPL protein does not fold correctly, is therefore rejected
by ER quality control mechanisms (27), aggregates, and is degraded
within the ER.
The active species made up about 30% of total LPL protein in the
adipocytes. From the pattern of glycosylation Been-Zeev et al. concluded that most of the catalytically active LPL molecules were still in the ER (16). Roh et al. separated different
intracellular structures from rat adipocytes by centrifugation (28).
They found that LPL was mainly in the ER with a small pool being
present in low-density membrane vesicles that probably represent a
secretory compartment in adipocytes. In pulse-chase experiments with
guinea pig adipocytes, Semb and Olivecrona found that about one-third of pulse-labeled LPL had appeared in the medium within 30 min of chase
(24). This figure rose to 49% by 1 h. After that there was no
further appearance of labeled LPL in the medium. These data suggest
that when the LPL molecules have been cleared by the quality control
system, they move rapidly to the cell surface.
Several laboratories have reported that short-term changes of LPL
activity in adipose tissue occur without corresponding changes in LPL
mRNA (9, 29), relative rates of LPL biosynthesis (8, 12), or LPL
mass ((9, 15). There are also reports that LPL activity in adipocytes
isolated from fed or fasted rats is about the same (30). In concert
with this, we found no difference in adipocyte LPL activity with
nutritional state. In addition, our data show that there was no
difference with nutritional state in LPL mass or in the distribution of
LPL protein between inactive and active forms within the adipocytes.
To gain information on where in the tissue the extracellular LPL is
localized we injected heparin. Tissue LPL activity decreased by only
about 10% during the first 2 min. This presumably represented the LPL
molecules that were at the endothelial surface (17, 31, 32). Over the
next 60 min an additional 40% of the LPL activity disappeared from the
tissue. That tissue LPL activity continued to decrease long after of
the endothelial-bound enzyme had been released supports the view that
there is a relatively large pool of LPL molecules that recirculate
between the luminal surface of the endothelium and extravascular sites
in the tissue (1, 2). Calculation of the specific activity indicated
that heparin extracted mainly, or perhaps only, the active form of the
enzyme. This is expected since only this form has high affinity for heparin/heparan sulfate (33). That heparin releases primarily the
active form of LPL is in concert with an earlier study on the specific
activity of the LPL released into blood after injection of heparin
(34).
One hour after injection of heparin, tissue LPL activity had decreased
by about 50%. This corresponds to more than two-thirds of the
extracellular LPL activity. In contrast, LPL activity within the
adipocytes did not change. This differs from what is usually seen in
experiments with cultured cells where heparin increases the release of
LPL activity to the medium at the expense of cell-associated LPL (16,
24, 35). The mechanism for this enhanced release is presumably that
heparin acts as a soluble ligand for the enzyme and prevents its
recycling into the cells (36, 37). One might have expected a similar
process in the tissue, i.e. that heparin would drive the
enzyme out from the cells toward the endothelium. That this did not
happen indicates that even without exogenous heparin there is an excess
of liganding sites in the tissue to receive the enzyme as it emerges
from the adipocytes.
Heparin released more than five times as much LPL activity from
the adipose tissue of fed compared with fasted rats. When we calculated
the release as fraction of the active, extracellular LPL in the tissue
(Fig. 4, inset), the time course and extent of release was
similar for the two nutritional states. This indicates that the active,
extracellular LPL was equally accessible to release by heparin in both
nutritional states. Hence, the larger release in the fed rats was a
reflection of the larger amount of active extracellular LPL rather than
due to some difference in how the enzyme was located in the tissue.
In a recent study we found that down-regulation of LPL activity in
adipose tissue during fasting requires that a gene, separate from the
lipase gene, is switched on (7). The mechanism by which the product of
this gene accomplishes the switch from predominantly active to
predominantly inactive form(s) of LPL is not clear. Here we show that
the switch primarily affects the extracellular LPL. In vivo
experiments, as used here, are too complex to unravel the mechanism.
Studies with adipocytes have shown relatively small or no differences
in LPL activity and secretion between cells from fasted compared with
fed rats (12, 24, 25, 35, 38, 39). The next step must therefore be to
find a suitable cell system and/or conditions where the putative
LPL-regulating gene can be switched on and off and the effects on LPL
maturation, trafficking, and stability can be studied in detail.