Fatty acids reduce heparin-releasable LPL activity in cultured
cardiomyocytes from rat heart
Lorraine G.
Anderson,
Rogayah
Carroll,
H. Stephen
Ewart,
Anjli
Acharya, and
David L.
Severson
Smooth Muscle Research Group, Faculty of Medicine, The
University of Calgary, Calgary, Alberta, Canada T2N 4N1
 |
ABSTRACT |
Varying glucose and fatty acid (FA)
concentrations in the medium of cultured cardiomyocytes from adult rat
hearts were tested for effects on lipoprotein lipase (LPL) activity.
Glucose (5.5, 11, and 25 mM in the culture medium for 18-22 h) had
no effect on either heparin-releasable LPL (HR-LPL) or on cellular LPL
(C-LPL) activities. When cardiomyocytes were cultured overnight with 60 µM oleate, HR-LPL activity was reduced to 20% of control, with no
change in C-LPL activity or total C-LPL mass. Similar results (HR-LPL
and C-LPL activities) were obtained with 60 µM concentrations of
palmitate and myristate; linoleate and eicosapentaenoate did reduce
C-LPL activity, but the decrease in HR-LPL activity was much greater.
Oxfenicine, an FA oxidation inhibitor, did not alter the inhibitory
effect of 60 µM oleate on HR-LPL. Short-term incubations (1 and 3 h)
of cultured cardiomyocytes with 60 µM oleate did not displace LPL
into the medium. Immunodetectable LPL on the cell surface of
oleate-treated cultured cardiomyocytes was increased compared with
control cells, but heparin treatment released the same amount of LPL
mass that had reduced catalytic activity.
myocardial cells; immunohistochemistry
 |
INTRODUCTION |
IN THE ADULT HEART lipoprotein lipase (LPL) is
synthesized and processed in cardiomyocytes and then is translocated to
binding sites on the luminal surface of endothelial cells in the
coronary vasculature (4, 6, 8, 20). The functional endothelium-bound enzyme catalyzes the hydrolysis of the triacylglycerol (TG) component of circulating lipoproteins (21); nonesterified fatty acids (FA)
produced by the action of LPL are then available for oxidation in
cardiomyocytes.
Hypertriglyceridemia is a characteristic feature of insulin-dependent
diabetes mellitus, principally because of reduced catabolism of TG-rich
lipoproteins (28). The degradation of TG-rich lipoproteins was
decreased in diabetic perfused hearts (22, 23) as a consequence of
reduced functional endothelium-bound LPL activity (7, 17, 23). In
addition, LPL activity in cardiomyocytes, the precursor of the
functional enzyme on the vascular endothelium, was decreased after the
acute induction of insulin-deficient diabetes (5, 7, 9, 17).
Administration of insulin in vivo to diabetic rats rapidly reversed the
diabetes-induced decrease in LPL activity in cardiomyocytes, but in
vitro incubations of cardiomyocytes from either control or diabetic rat
hearts with insulin had no direct stimulatory effect on LPL activity
(5, 7), despite the presence of functional insulin receptors in the
cardiomyocyte preparations. Therefore, the inhibitory effect of
diabetes on LPL activity in isolated cardiomyocytes may not be a direct
consequence of insulin deficiency but rather secondary to some other
metabolic factor(s) that is altered in an acute, streptozotocin-induced model of diabetes. Hyperglycemia and increased circulating FA concentrations as a result of unrestrained adipose tissue lipolysis are
common metabolic features of insulin-deficient diabetes. Several investigations have reported that LPL activity in cultured adipocytes was decreased when the glucose concentration was increased (15) or when
FA were added to the culture medium (2, 16). Therefore, the objective
of the present study was to determine the effect of glucose and FA on
LPL activity in cultured cardiomyocytes from rat heart. Heparin can
displace the portion of total cellular LPL activity that is bound to
heparan sulfate proteoglycan (HSPG) binding sites on the surface of
cells (6). This heparin-releasable LPL (HR-LPL) activity was
selectively reduced when cardiomyocytes were cultured in the presence
of FA.
 |
MATERIALS AND METHODS |
Isolation, culture, and incubation of
cardiomyocytes from rat hearts.
Adult rat ventricular cardiomyocytes were isolated essentially as
described previously (26), except that aseptic techniques were used
with a laminar flow hood. After collagenase treatment, freshly isolated
cells were suspended in culture medium [Joklik minimal essential
medium at pH 7.4, supplemented with 1 mM
CaCl2, 0.2% (wt/vol) essentially
FA-free albumin (30 µM), 1.2 mM
MgSO4, 1 mM
DL-carnitine, 100 IU/ml
penicillin, and 100 µg/ml streptomycin, which had been filtered
through a 0.22-µm filter] to a cell density of 150,000 viable
cells/ml. Cell number and viability were determined by adding a 30-µl
aliquot of resuspended cardiomyocytes to an equal volume of 0.4%
(wt/vol) trypan blue in 0.9% (wt/vol) NaCl. Cell number was determined
microscopically in duplicate by counting with a hemacytometer. A
myocyte was designated as viable if, on microscopic examination, it was
rod shaped with clear cross striations and excluded trypan blue.
Cardiomyocytes were cultured overnight on laminin-coated plates, using
the rapid attachment model of Jacobson and Piper (13). A 2-ml aliquot
of culture medium was added to each 35-mm well of laminin-coated
six-well tissue culture plates, followed by 1 ml of the freshly
isolated myocyte suspension (150,000 viable cells/well). Within 3 h, a
large percentage of the myocytes had attached to the laminin-coated
wells. At this time, unattached cells and debris were removed from each
well by gently aspirating the medium and replacing it with 3 ml of
fresh culture medium. The culture plates were incubated at 37°C
overnight (18-22 h) in the absence and in the presence of FA under
a humidified atmosphere of 95%
O2-5%
CO2. When FA were added to the
overnight culture medium, appropriate quantities of stock FA solutions
(100 mM in hexane) were dried under
N2 and resuspended in an
equivalent volume of 0.12 M KOH in ethanol. The ethanol was removed by
warming under N2 gas, and the FA
(K+ salt) were resuspended into
the culture medium to give the desired final concentration. The
presence of FA in the medium had no effect on the yield or viability
(72%) of cardiomyocytes after the overnight (18-22 h) culture.
Approximately 50,000 viable cells/well (33% yield) were still attached
to the laminin-coated wells after overnight culture. In some
experiments, as noted, FA were added with fresh medium after overnight
culture in control medium, and the incubation was continued for an
additional 1, 3, or 18 h. For measurements of LPL mass,
larger numbers of cardiomyocytes (750,000 cells) were cultured
overnight, using 100-mm laminin-coated tissue culture dishes.
To determine the fate of oleate added to the overnight culture medium,
cardiomyocytes were cultured with 60 µM
[14C]oleate (1 µCi/ml of medium). Triplicate aliquots (10 µl) were removed from
the culture medium (3-ml initial volume) at various time intervals
(0-4 h and 16-18 h), and the radioactivity in these medium
samples was measured by liquid scintillation spectrometry. At the end
of the overnight culture, the remaining medium was removed and 0.2 ml
of 5 N HCl was added to each well. Cells were removed by scraping, and
the solution was transferred to a test tube. The wells were rinsed with
0.6 ml H2O, which was added to the
scraped cells, and then 4 ml of chloroform-methanol (2:1) were added to
the tube. After vortexing and centrifugation, the upper phase was dried
under N2; extracted lipids were
dissolved in 50 µl chloroform-methanol (2:1), carrier lipids were
added, and lipid classes were separated by thin-layer chromatography (10) with the use of a solvent system consisting of heptane-diethyl ether-glacial acetic acid (25:75:1). Under these conditions,
phospholipids remain at the origin; other lipids migrate in the
following order from the bottom of the plate: monoacylglycerol,
diacylglycerol, FA, and TG (10). Bands corresponding to these lipid
classes were identified by I2
staining, and the content of radioactivity was measured by liquid
scintillation spectrometry.
Incubation experiments with heparin were performed with cultured
cardiomyocytes to measure LPL activity released into the medium
(HR-LPL) and residual cellular LPL (C-LPL) activity. After overnight
incubation, the culture medium was removed and replaced with 1 ml of
fresh medium containing 5 U/ml heparin (Hepalean) in each well, and the
culture plates were returned to the incubator for 40 min. Basal
(constitutive) release of LPL into the medium when cardiomyocytes were
incubated without heparin was very low. LPL activity in the medium of
cultured cardiomyocytes after a 40-min incubation in the absence or in
the presence of heparin was (mean ± SE) 18.9 ± 1.6 and 208 ± 18 nmol · h
1 · mg
1,
respectively (n = 60). Thus
heparin increased LPL activity in the incubation medium by 11-fold.
After incubations with heparin, the medium was removed and centrifuged
(15,000 g; Eppendorf microcentrifuge) to collect any dislodged cells. The supernatant (medium) was decanted and frozen for subsequent determination of HR-LPL activity. The cells
remaining on the plates were incubated for 20 min at 4°C with 1 ml
of a buffer (buffer
A) consisting of 0.25 M sucrose, 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 1 mM EDTA, and 1 mM dithiothreitol (pH 7.5) and then
scraped with a plastic cell scraper. These scraped cells were then
added to the cell pellet from the initial medium centrifugation,
recentrifuged, as above, and the supernatant was discarded. The cell
pellet was then frozen and stored at
80°C. For
determinations of C-LPL activity, the frozen cell pellets were
resuspended in 0.2 ml 50 mM ammonia buffer (pH 8.0) containing 0.05%
Triton X-100 and 0.8 ml of buffer A and then sonicated with a Braun
Sonic sonicator at 75 W for two bursts of 30 s at 4°C (9).
LPL assay.
LPL activity in the incubation medium (HR-LPL) and in sonicated cell
homogenates (C-LPL) from cultured cardiomyocytes was determined by
measuring the hydrolysis of a sonicated
[3H]triolein substrate
emulsion (26), except that the triolein concentration was reduced from
0.6 to 0.1 mM to increase substrate specific activity and the
sensitivity of the assay. The standard assay contained 0.1 mM
glycerol-[9,10-3H]trioleate
(6 mCi/mmol), 25 mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 7.5), 0.05% (wt/vol) essentially FA-free bovine serum albumin (BSA), 50 mM MgCl2, and
2% (vol/vol) heat-inactivated chicken serum as the LPL activator. A
100-µl aliquot of the incubation medium or 50 µl of the sonicated
cell homogenates was then assayed in a final volume of 400 µl. When
LPL activity was measured in sonicated cell homogenates, 2 U/ml heparin
was added to the assay tubes. The formation of
[3H]oleate was
measured by liquid-liquid extraction (26), after a 30-min incubation at
37°C. All assays were performed in duplicate, under conditions in
which the reaction rate was linear with respect to protein content. LPL
activity is expressed routinely as nanomoles of oleate released per
hour per milligram protein in the sonicated cell homogenates. Protein
concentrations were measured by a Coomassie blue spectrophotometric
assay (29), with BSA as the standard. Results are expressed as means ± SE; n refers to the number of wells for which LPL activity was measured. Lipase activity in sonicated
cell homogenates was stimulated 4.7-fold by serum (apolipoprotein CII) and was inhibited by >80% by 1 M NaCl, indicating
that other TG lipases that may be present in homogenates do not
contribute significantly to LPL activity measurements.
Purification of bovine milk LPL and isolation of
anti-LPL antibodies.
LPL was purified to homogeneity from fresh bovine milk as described by
Liu and Severson (18). Purified bovine milk LPL was coupled to
Affigel-10 beads (12) for affinity purification of LPL antibodies from
chicken eggs. Egg-laying hens were initially injected with 100 µg of
bovine LPL in complete Freund's adjuvant in multiple subcutaneous
spots along the back (12). Booster injections of 100 µg LPL in
incomplete Freund's adjuvant were given in the thighs and lower neck
region at weekly intervals. Five weeks after the initial injection, 15 ml of blood were drawn from the wing veins and eggs were collected.
Immunoglobulin Y (IgY) was isolated, using the water
dilution procedure of Akita and Nakai (1). Ten egg yolks from preimmune and immunized hens were diluted sixfold with acidified distilled water
(pH 5.2) and then left standing for 5-6 h or overnight. The fluffy
solution was then centrifuged for 1 h at 10,000 g at 4°C. The supernatant was
collected, and sodium sulfate was added to a final concentration of
19% (wt/vol). After centrifugation at 10,000 g at room temperature, the pellet was
resuspended and dialyzed against 10 mM tris(hydroxymethyl)aminomethane
(Tris) (pH 8) and 0.15 M NaCl [Tris-buffered saline
(TBS)]; one-third of this water-soluble fraction was
applied to a 3-ml LPL-Affigel-10 column. The column was washed with
TBS, followed by 10 mM acetate buffer (pH 4.5) and 1 M NaCl.
LPL-specific antibody was eluted with 0.2 M glycine-HCl buffer (pH 2.7)
and collected in an equal volume of 0.2 M Tris · HCl
(pH 8). Total amount of affinity-purified antibody obtained from 10 eggs was ~5 mg; a single band corresponding to chicken IgY was
observed after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). This polyclonal anti-LPL IgY reduces LPL
activity (immunoinhibition), and both intact LPL and degradation products are detected by immunoblotting after SDS-PAGE (unpublished results), indicating that the antibody recognizes both active and
inactive forms of the enzyme. Control IgY was isolated from preimmune
egg yolks.
For biotinylation, the affinity-purified anti-LPL polyclonal antibody
(157 µg/ml) in phosphate-buffered saline (PBS) was incubated with 0.1 ml 1 M carbonate buffer (pH 9) and 80 µg
N-hydroxysuccinimidyl 6-(biotinamido)hexanoate in dimethylformamide for 90 min at 37°C (12). Excess biotin was removed by dialysis against PBS; biotinylated antibody was stored at
80°C.
Enzyme-linked immunosorbent assay for LPL
mass.
For measurements of LPL mass, cell pellets were sonicated in 0.2 ml of
25 mM NH4Cl, 5 mM EDTA, 0.8%
(wt/vol) Triton X-100, 0.04% (wt/vol) SDS, 33 µg/ml heparin, and 10 µg/ml leupeptin (pH 8.2). Incubation media were lyophilized and
resuspended in 0.2 ml H2O. A
sandwich enzyme-linked immunosorbent assay (ELISA) for LPL mass was
developed, using affinity-purified polyclonal antibodies from
egg-laying hens. Polystyrene microtiter plate wells (Immulon 1) were
coated with 100 µl of anti-LPL antibody (15 µg/ml in 0.05 M
carbonate buffer, pH 9.6) overnight at 4°C. The plates were then
washed once with PBS and 0.05% (wt/vol) Tween 20 (buffer B) and blocked for 3 h at 25°C
with 300 µl PBS containing 3% (wt/vol) BSA and 0.1% (wt/vol)
sucrose. After four more washes with
buffer B, 100-µl aliquots of samples (cell
homogenates or incubation media) diluted in PBS containing 0.05%
(wt/vol) Tween 20, 1 mg/ml heparin, 0.4% (wt/vol) BSA, 1 mM
phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin, and 1 µg/ml
pepstatin A (buffer
C) were added to the wells. The
plates were then sealed and incubated overnight at 4°C. Purified
bovine milk LPL was dialyzed overnight in PBS, diluted in
buffer
C, and applied to the wells
(0.1-1.0 ng) as a standard for the ELISA. After extensive washing,
100 µl of affinity-purified biotin-labeled anti-LPL antibody in PBS
containing 1% (wt/vol) BSA were added. After a second overnight
incubation at 4°C and washes with
buffer
B, the wells were incubated with 100 µl of peroxidase-labeled streptavidin in PBS and 1%
(wt/vol) FA-free BSA for 2 h at 25°C. The plates were washed four
more times, and color reaction was achieved by adding 100 µl
o-phenylenediamine (0.8 mg/ml in 0.15 M citrate buffer, pH 5) and 0.003% (vol/vol) H2O2.
After development for 10-15 min, the absorbance at 495 nm was
determined with the use of a Bio-Rad microplate reader. LPL mass
measurements in cell extracts and in postheparin medium (ng/mg cell
protein) were used to calculate LPL specific activity as milliunits per
nanogram LPL protein (9), where 1 mU is defined as the amount of enzyme
catalyzing the release of 1 nmol oleate per minute. For LPL specific
activity calculations, catalytic activity and mass were measured in the
same preparations in which cardiomyocytes cultured overnight in the
presence of FA were paired with control cultured cells.
Immunohistochemistry.
Cultured cardiomyocytes were resuspended into PBS (pH 7.4) containing
1% (wt/vol) FA-free albumin at 37°C. After centrifugation, the
cell pellet was resuspended into cold PBS containing 0.625 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid and 2.5 mM NaOH for 5 min. Cells were then fixed with 2.5% Formalin in PBS and finally stored in PBS at a concentration of 106 cells/ml. Aliquots (25 µl)
of this fixed but nonpermeabilized cell suspension were then incubated
with 2 µg/ml preimmune IgY or affinity-purified anti-LPL IgY in PBS
containing 3% albumin and 0.05% Tween (dilution buffer) overnight at
4°C. After washing, rhodamine [tetramethylrhodamine
isothiocyanate (TRITC)]-conjugated rabbit anti-chicken IgY was
added to the cells at 1:50 dilution in dilution buffer and incubated
for 1 h at 4°C in the dark. The cells were then washed extensively
and resuspended in 10-20 µl PBS. A 6-µl aliquot was mounted on
a slide in 90% glycerol in PBS. The slides were viewed on an Olympus
BH2-RFCA fluorescent microscope, and black and white pictures were
recorded on Kodak T-max 400 film.
Heparin-Sepharose chromatography.
An aliquot (4.5 ml) of the postheparin medium from freshly isolated
cardiomyocytes (2 × 106
cells/ml incubated with 5 U/ml heparin for 1 h) was added to 1.5 ml
heparin-Sepharose (7). After a 30-min incubation at 4°C, the
mixture was poured into a column, washed with 20 ml of 20 mM HEPES (pH
7.4), 20% (vol/vol) glycerol, and 0.02% Triton X-100
(buffer
D), and the flow-through fractions
(2 ml each) were collected. The column was then washed with successive
additions of buffer
D containing increasing concentrations
of NaCl (0.05, 0.6, and 1.5 M NaCl). Fractions were assayed immediately
for LPL activity. Because of the presence of variable amounts of NaCl in the column fractions, 50 mM
MgCl2 was omitted from the assay, and the final NaCl concentration in the assay was kept constant at 0.28 M.
Materials.
Collagenase (Worthington type II) was obtained from Technicon Canada
(Richmond, BC, Canada), and heparin (Hepalean; 1,000 U/ml) was
purchased from Organon Teknika (Toronto, ON, Canada). Joklik minimal
essential medium, Dulbecco's modified Eagle's medium (DMEM) (high and
low glucose), and penicillin/streptomycin were purchased from GIBCO
Canada (Burlington, ON, Canada).
[3H]triolein (glycerol
[9,10-3H]trioleate)
and [1-14C]oleic acid
were purchased from Amersham (Oakville, ON, Canada). Tissue culture
plates (6 well; Falcon) were coated with 15 µg/ml laminin in Hanks'
balanced salt solution for 3 h at 37°C; the plates were then air
dried and stored at 4°C until use. Freund's adjuvants, 4%
Formalin, oxfenicine, and ultrapure mouse laminin were purchased from
Sigma Chemical (St. Louis, MO). Rhodamine (TRITC)-conjugated rabbit
anti-chicken IgY was obtained from Bio/Can Scientific (Mississauga, ON,
Canada), and peroxidase-labeled streptavidin was from Boehringer
Mannheim (Laval, PQ, Canada). FA were purchased from either Sigma or
Serdary Research Laboratories (London, ON, Canada). All other chemicals
were from either Sigma or VWR Scientific of Canada (Edmonton, AB,
Canada).
 |
RESULTS |
LPL activity in cultured myocytes.
Total C-LPL activity was unchanged after overnight culture, but HR-LPL
activity was increased by 3.4-fold in cultured cardiomyocytes compared
with freshly isolated cells. Presumably, this selective increase in
HR-LPL activity reflects the replenishment of LPL and/or LPL
binding sites on the cell surface after collagenase treatment.
Effect of glucose concentration on LPL activity in
cultured cardiomyocytes.
Overnight culture of cardiomyocytes with varying glucose concentrations
(5.6, 11, and 25 mM) resulted in no significant differences in either
HR-LPL or C-LPL activities (Fig. 1).

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Fig. 1.
Effect of glucose on lipoprotein lipase (LPL) activity in cultured
cardiomyocytes. Cardiomyocytes were cultured overnight with standard
Joklik medium (11 mM glucose) and with low-glucose (5.5 mM) or
high-glucose (25 mM) DMEM. After 18-22 h, medium was replaced with
fresh Joklik medium containing 5 U/ml heparin, and heparin-releasable
(HR)-LPL and cellular (C)-LPL activities were measured after a 40-min
incubation. Results are expressed as means ± SE for number of wells
indicated in parentheses.
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Effect of FA on LPL activity in cultured
cardiomyocytes.
In contrast to the preceding results with glucose, the addition of
oleate to the overnight culture medium produced a
concentration-dependent inhibition of HR-LPL activity (Fig.
2), with no significant change in C-LPL
activity. The heparin-containing medium from oleate-treated cells did
not reduce control HR-LPL activity in mixing experiments, indicating
that an inhibitor was not released into the medium when cardiomyocytes
cultured in the presence of oleate were incubated with heparin. Because
considerable inhibition of HR-LPL activity was observed with 60 µM
oleate (2:1 molar ratio to the albumin concentration in the culture
medium), this concentration was used in subsequent experiments.

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Fig. 2.
Effect of oleate on LPL activity in cultured cardiomyocytes.
Cardiomyocytes were cultured overnight in medium containing indicated
concentrations of oleate. Fresh medium containing 5 U/ml heparin was
added to each well, and HR-LPL ( ) and C-LPL ( ) activities
were measured. Results are means ± SE for number of wells given in
parentheses. * P < 0.05;
*** P < 0.001 (Student's
t-test).
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Total LPL mass in control cells was 38.4 ± 10.7 ng/mg protein
(n = 4 preparations of cultured
cardiomyocytes). LPL mass in the medium of cultured cells incubated in
the absence of heparin was undetectable, consistent with the low level
of LPL activity measured as an index of constitutive LPL release. In
the presence of heparin, LPL mass in the medium of cultured
cardiomyocytes was 8.4 ± 1.6 ng/mg cell protein
(n = 6). Thus heparin released ~20%
of total cellular mass into the medium, but C-LPL and HR-LPL activities
were very similar (Figs. 1 and 2), indicating that heparin released
~50% of total cellular enzyme activity. This apparent discrepancy is
due to the presence of a substantial quantity of inactive enzyme mass
in cultured cardiomyocytes; LPL specific activities in cell extracts
and heparin-treated medium were 0.075 ± 0.008 and 0.72 ± 0.15 mU/ng LPL protein, respectively. Thus heparin must preferentially
release active dimeric LPL into the medium of control cells.
Overnight culture with 60 µM oleate did not significantly alter LPL
mass in cells (42.1 ± 7.7 ng/mg cell protein;
n = 4) or in the medium after
incubation with heparin (5.6 ± 0.5 ng/mg protein; n = 6) compared with mass
determinations in control cultured cardiomyocytes. As a consequence,
the specific activity of HR-LPL (mU/ng LPL protein) was reduced from
0.72 ± 0.15 to 0.44 ± 0.06 (P < 0.05) by the overnight incubation with oleate.
The effect of 60 µM concentrations of different FA in the overnight
culture medium on LPL activity is shown in Fig.
3. Palmitate (16:0) and myristate (14:0),
like oleate (18:1), produced a marked reduction in HR-LPL activity
without any significant change in C-LPL activity. Although linoleate
(18:2) and eicosapentaenoate (20:5) did significantly reduce C-LPL
activity to 57 and 52% of control, respectively (Fig. 3), HR-LPL
activity was inhibited to a much greater extent (to 15 and 9% of
control, respectively). Thus all FA resulted in a selective inhibition
of HR-LPL activity.

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Fig. 3.
Effect of fatty acids (FA) on LPL activity in cultured cardiomyocytes.
Cardiomyocytes were cultured overnight in absence (open bars) and in
presence (solid bars) of the following FA (all at 60 µM): palmitate
(16:0), myristate (14:0), oleate (18:1), linoleate (18:2), and
eicosapentaenoate (20:5). Fresh culture medium containing 5 U/ml
heparin was then added to each well, and HR-LPL
(A) and C-LPL
(B) activities were measured.
Results are means ± SE for number of wells indicated in
parentheses. * P < 0.05;
** P < 0.01;
*** P < 0.001 (Student's
t-test).
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The fate of 60 µM oleate during the overnight culture was examined
next. When [14C]oleate
was added to the culture medium, there was a time-dependent decrease in
medium radioactivity to ~40-50% of control (zero time) after 18 h (Fig. 4). When cardiomyocytes were
extracted after overnight culture with
[14C]oleate, most of
the radioactivity incorporated into cellular lipids was recovered in TG
(50 ± 1% of total radioactivity in the lipid extract;
n = 3) and phospholipid (36 ± 4%). Oxfenicine (100 µM), an FA oxidation inhibitor (30), did not
alter the inhibition of HR-LPL activity in cultured cardiomyocytes by
60 µM oleate (Fig. 5).

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Fig. 4.
Utilization of
[14C]oleate by
cultured cardiomyocytes. Cardiomyocytes were cultured overnight with 60 µM [14C]oleate (1 µCi/ml culture medium). At indicated times, triplicate 10-µl
aliquots of medium were removed, and radioactivity was measured.
Results are from 2 separate preparations ( and ) of cultured
cardiomyocytes.
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Fig. 5.
Effect of oxfenicine on inhibition of HR-LPL activity in cultured
cardiomyocytes by oleate. Cardiomyocytes were cultured overnight in
presence of no additions, 100 µM oxfenicine, 60 µM oleate (18:1),
and oxfenicine plus oleate. Results are means ± SE for number of
wells indicated in parentheses.
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Even though LPL mass displaced into the medium by heparin was not
reduced by prior incubation with oleate, additional experiments were
conducted to determine whether FA (oleate) displaced LPL, bound to HSPG
on the cell surface of cardiomyocytes (6, 7, 21), into the medium
during the overnight culture, thus reducing HR-LPL activity measured in
the subsequent 40-min incubation with heparin. LPL activity in the
medium after overnight culture in the absence and in the presence of 60 µM oleate was 15 ± 3 (n = 12)
and 3 ± 1 (n = 12)
nmol · h
1 · mg
1,
respectively. However, any LPL displaced by oleate likely would have
been inactivated during the long (18-22 h) incubation time at
37°C. Therefore, the effect of oleate added to cardiomyocytes after
overnight culture on LPL activity in the medium and on HR-LPL activity
was determined (Table 1). Incubation of
cultured cardiomyocytes with 60 µM oleate for 1 h did not alter
either medium LPL activity or HR-LPL activity. In contrast, the
addition of heparin to the 1-h incubation displaced LPL into the medium
and consequently reduced HR-LPL activity in the subsequent incubation
with heparin (Table 1). Increasing the incubation time with 60 µM
oleate to 3 h still produced no increase in medium LPL activity, even
though HR-LPL activity was reduced significantly. Incubation of control overnight-cultured cardiomyocytes with 60 µM oleate for an additional 18 h reduced HR-LPL activity to 8 nmol · h
1 · mg
1
(Table 1), compared with control activity of 120 ± 19 nmol · h
1 · mg
1
when cells were cultured for another 18 h without oleate. This inhibitory effect was similar to the reduction in HR-LPL activity observed when oleate was present in the initial overnight culture medium (Figs. 2 and 3), but LPL activity in the medium was still not
increased (Table 1).
When LPL activity was measured in the medium of cultured cardiomyocytes
after incubation with 60 µM oleate for 1 or 3 h (Table 1), the final
assay incubation contained 15 µM oleate (100 µl of medium assayed
in a total volume of 400 µl; see MATERIALS AND METHODS). The direct addition of 15 µM oleate to
assays with control medium from heparin-treated cardiomyocytes did not
reduce HR-LPL activity (119 and 125 nmol · h
1 · mg
1
in the absence and presence of 15 µM oleate, respectively).
Therefore, LPL activity in the medium of oleate-treated cells is not
masked by interference of FA introduced into the LPL assay.
LPL activity released into the medium of cultured cells by heparin is
somewhat unstable at 37°C; therefore, the possibility that oleate
increases the rate of LPL inactivation in the medium was examined. As
shown in Fig.
6A,
incubation of the medium of heparin-treated cultured cardiomyocytes for
up to 60 min at 37°C resulted in a time-dependent decrease in
HR-LPL activity. However, this inactivation was reduced, not enhanced,
when 60 µM oleate was present (Fig.
6B).

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Fig. 6.
Stability of HR-LPL activity. Cardiomyocytes were cultured overnight
under control conditions. Fresh medium containing 5 U/ml heparin was
added, and after a 40-min incubation, medium was collected for
determination of HR-LPL activity. A:
heparin-treated medium was incubated at 37°C for indicated times,
and HR-LPL activity was measured. Results are means of 2 experiments
with different cultured cardiomyocyte preparations.
B: HR-LPL activity was measured in
medium at zero time and after 40-min incubation at 37°C with either
no additions (open bars) or with 60 µM oleate added to medium (solid
bars). Results are means ± SE
(n = 7 incubations).
|
|
The reversibility of the inhibitory effect of oleate on HR-LPL activity
was also examined. Cardiomyocytes were first cultured overnight with
oleate to reduce HR-LPL activity (to 31 ± 5 nmol · h
1 · mg
1;
n = 9). The cells were then cultured
for an additional 18 h in fresh medium containing no oleate; HR-LPL
activity increased to 60 ± 6 nmol · h
1 · mg
1
(n = 15), relative to 120 ± 19 nmol · h
1 · mg
1
in control cardiomyocytes cultured continuously in the absence of
oleate (Table 1). Thus the inhibitory effect of oleate on HR-LPL is at
least partially reversible in this time frame.
The possibility that the binding of FA to LPL (11) might alter the
ability of the enzyme to bind to heparin was tested by subjecting
postheparin medium (HR-LPL) to heparin-Sepharose chromatography (Fig.
7). LPL binds tightly to heparin-Sepharose
so that high ionic strength is required to displace the enzyme (7). The presence of 60 µM oleate in the postheparin medium had no effect on
the binding of LPL to the column or on its elution with 1.5 M NaCl
(Fig. 7).

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Fig. 7.
Heparin-Sepharose chromatography. LPL in postheparin medium (~1,055
nmol/h) was bound to heparin-Sepharose in absence ( ) and in presence
( ) of 60 µM oleate. After flow-through fractions were collected,
column was washed with buffer containing indicated concentrations of
NaCl, and LPL activity was determined in column fractions. Recovery of
LPL activity eluted with 1.5 M NaCl was 68% ( oleate) and 75%
(+oleate).
|
|
Histochemical detection of LPL on the cell surface
of cultured cardiomyocytes.
The detection of LPL on the surface of nonpermeabilized cultured
cardiomyocytes by immunofluorescence, using an affinity-purified chicken polyclonal antibody to LPL, was examined next. A typical pattern of immunostaining with control cultured cardiomyocytes is shown
in Fig.
8A; the
most intense fluorescent labeling was observed at the edges of cells.
Surprisingly, cardiomyocytes after overnight culture with 60 µM
oleate consistently exhibited more immunodetectable LPL on the cell
surface (Fig. 8B) compared with control cultured cardiomyocytes.

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Fig. 8.
Immunohistochemical detection of cell surface LPL on cultured
cardiomyocytes. Cardiomyocytes were cultured overnight in absence
(A) and in presence
(B) of 60 µM oleate. After
fixation, cells were incubated with an affinity-purified chicken
polyclonal antibody to bovine milk LPL and resulting immunofluorescence
was detected. Single cells shown in A
and B are representative of many cells
examined microscopically from 4 experiments, in which control and
oleate-treated cardiomyocytes were compared. Cells were photographed
and developed at same exposure time. No immunofluorescence was detected
if control preimmune immunoglobulin Y (IgY) replaced anti-LPL IgY.
|
|
 |
DISCUSSION |
Kern et al. (15) reported that increasing the glucose concentration in
the culture medium of isolated human adipocytes from 5.5 to 25 mM
reduced the basal constitutive release of LPL activity by 50%. The
basal or constitutive release of LPL into the medium of cultured
cardiomyocytes is very low and was unaffected at different glucose
concentrations (results not shown). Variation of the glucose concentration in the culture medium (5.5, 11, and 25 mM) also had no
significant effect on either HR-LPL or C-LPL activities in cultured
cardiomyocytes, suggesting that the hyperglycemia that accompanies
diabetes does not regulate myocardial LPL activity.
A number of investigations have shown that functional endothelium-bound
LPL activity is regulated acutely by FA to prevent the excessive
delivery of lipolytic products to tissue cells. First, FA inhibit LPL
activity directly by product inhibition and by reducing the activation
by apolipoprotein CII (3); LPL has four to six FA binding sites (11).
Second, FA can displace LPL from binding sites on the surface of
cultured endothelial cells (27). Thus this latter mechanism can account
for the observation that infusion of a TG emulsion to humans (24) or
administration of an oral fat load (14) increased plasma LPL activity.
These acute mechanisms for regulating endothelium-bound LPL may be
inadequate under certain circumstances, such as the chronic increases
in plasma FA that occur in diabetes, so it is reasonable to anticipate that FA may also regulate the activity of LPL in parenchymal cells (e.g., cardiomyocytes and adipocytes).
Addition of a low concentration of oleate (60 µM) at a physiological
FA-to-albumin ratio (2:1) to the overnight culture medium produced a
profound decrease in HR-LPL activity to ~20% of control but no
significant change in residual C-LPL activity (Fig. 2) or in C-LPL
mass. This selective inhibitory effect on HR-LPL activity was also seen
with saturated FA, palmitate, and myristate. Although linoleate and
eicosapentaenoate did result in a significant decrease in C-LPL
activity, the reduction in HR-LPL activity was much greater (Fig. 3).
The selective inhibition of HR-LPL activity in cultured cardiomyocytes
by FA is similar to results obtained by Amri et al. (2) with cultured
Ob 1771 adipocytes, in which FA produced a greater decrease in HR-LPL
activity compared with C-LPL activity, with no change in LPL mass
measured by immunoblotting. On the other hand, different results were
obtained with cultured rat adipocyte precursors (16) and cultured
chicken adipocytes (19), in which FA in the culture medium decreased
LPL activity in cell extracts by a transcriptional mechanism, with
reductions in LPL mRNA and LPL synthesis/mass (16, 19). Therefore, the
mechanism of FA inhibitory effects on LPL activity can vary markedly,
depending on the cell system.
The inhibitory effect of oleate on HR-LPL activity in cultured
cardiomyocytes was not altered by oxfenicine (Fig. 5), an inhibitor of
FA oxidation (30). This is perhaps not surprising, since cultured
cardiomyocytes are quiescent and have low rates of FA oxidation (25);
esterification to form endogenous TG is the principal metabolic fate
for FA added to the medium of cultured cardiomyocytes. Amri et al. (2)
observed that nonmetabolized FA analogs were capable of reducing LPL
activity in cultured Ob 1771 adipocytes.
LPL bound to HSPG on the cell surface of cultured cardiomyocytes could
have been displaced into the culture medium by FA, as shown with
cultured endothelial cells (27). This FA-induced displacement could
then produce the observed selective reduction in HR-LPL activity. This
potential mechanism seems unlikely, however, for the following reasons.
First, displacement of cell surface LPL by FA should be rapid; the
addition of oleate to cultured endothelial cells released 80% of bound
LPL into the medium during a 1-h incubation at 37°C (27). By
comparison, a 1-h incubation of cultured cardiomyocytes with oleate
resulted in no increase in medium LPL activity (Table 1). LPL activity
potentially present in the medium was not masked by the transfer of
inhibitory amounts of oleate into the LPL assay, and oleate did not
accelerate the inactivation of LPL in the culture medium. In addition,
any displacement of LPL into the culture medium during the 1-h
incubation, even if not detected by direct assay of enzyme activity,
should also reduce HR-LPL activity as shown with heparin (Table 1), but
the incubation with oleate for 1 h did not reduce HR-LPL activity. A
small reduction in HR-LPL activity was seen after the 3-h incubation with oleate, but no increase in medium LPL could be detected. Therefore, the time course for the inhibitory effect of oleate on
HR-LPL (3-18 h) is inconsistent with a displacement mechanism. Furthermore, the mass of LPL displaced into the medium by heparin was
not reduced after overnight culture with oleate, and more immunodetectable LPL (rather than less) was evident on the cell surface
of oleate-treated cardiomyocytes compared with control cultured cells
(Fig. 8). These results with cultured cardiomyocytes are consistent
with a previous investigation from our laboratory that reported that FA
did not release LPL from binding sites on the coronary vasculature of
perfused hearts or from the surface of freshly isolated cardiomyocytes
(26).
Other mechanisms therefore must account for the selective reduction in
HR-LPL activity when FA are added to the medium of cultured
cardiomyocytes. For example, the processing of LPL (7) could be
affected by FA so that more inactive monomeric but immunodetectable LPL
is present on the cell surface. Because the overnight culture with FA
did not change total C-LPL mass measured by ELISA, FA must stimulate
the transport of LPL from intracellular compartments to the cell
membrane of cultured cardiomyocytes. As a result, the relative
proportion of inactive to active forms of LPL on the cell surface may
be increased by FA treatment to account for the observation that
heparin released the same total mass of LPL (active and inactive
enzyme) into the medium of oleate-treated cells, even though HR-LPL
activity was reduced substantially. The acute presence of FA in
postheparin medium did not alter binding of LPL to heparin-Sepharose
(Fig. 7). Nevertheless, cell surface binding sites (HSPG) for LPL may
be altered by chronic exposure of cardiomyocytes to FA to impair LPL
displacement by heparin, since oleate treatment did not change LPL mass
displaced into the medium by heparin, even though the total
immunodetectable LPL mass on the cell surface was increased
considerably (Fig. 8). Diabetes alters sulfation of HSPG in hepatocytes
(31); FA may produce similar changes in HSPG composition in cultured
cardiomyocytes and thus influence LPL binding. The constitutive (basal)
release of LPL activity is extremely low, so FA are unlikely to produce the increase in cell surface immunodetectable LPL by somehow
stabilizing binding of LPL to reduce constitutive secretion into the
medium.
In summary, FA selectively reduced HR-LPL activity in cultured
cardiomyocytes, despite an increase in immunodetectable LPL on the cell
surface. The FA-induced impairment of the heparin-induced release of
LPL into the medium may be due to alterations in the intracellular
processing of LPL and the subsequent movement of the enzyme to the cell
membrane, together with changes in cell surface binding sites. More
investigations are required to determine if this inhibitory effect of
FA on HR-LPL activity could contribute, at least partially, to the
diabetes-induced reduction in LPL activity in cardiomyocytes.
 |
ACKNOWLEDGEMENTS |
The expert administrative assistance of L. Youngberg is gratefully
acknowledged.
 |
FOOTNOTES |
This work was supported by an operating grant from the Medical Research
Council of Canada.
Address for reprint requests: D. L. Severson, The University of
Calgary, Faculty of Medicine, Department of Pharmacology and
Therapeutics, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1.
Received 20 March 1997; accepted in final form 25 June 1997.
 |
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