Download Endothelial cells respond to hyperglycemia by increasing the LPL

Am J Physiol Endocrinol Metab 306: E1274–E1283, 2014.
First published April 15, 2014; doi:10.1152/ajpendo.00007.2014.
Endothelial cells respond to hyperglycemia by increasing
the LPL transporter GPIHBP1
Amy Pei-Ling Chiu,1 Fulong Wang,1 Nathaniel Lal,1 Ying Wang,1 Dahai Zhang,1 Bahira Hussein,1
Andrea Wan,1 Israel Vlodavsky,2 and Brian Rodrigues1
1
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada; and 2Cancer
and Vascular Biology Research Center, Rappaport Faculty of Medicine, Technion, Haifa, Israel
Submitted 3 January 2014; accepted in final form 9 April 2014
heparanase; lipoprotein lipase; platelet-derived growth factor; glycosylphosphatidylinositol-anchored high-density lipoprotein binding
protein 1
WITH UNINTERRUPTED CONTRACTION being a unique feature of the
heart, cardiac muscle has a high demand for energy. This organ
demonstrates substrate promiscuity, enabling it to utilize multiple sources of energy such as fatty acids (FA), carbohydrates,
amino acids, and ketones (2). Among these, carbohydrate and
FA are the major participants from which the heart derives
most of its energy. Accordingly, in a basal setting, glucose and
lactate account for ⬃30% of energy, whereas 70% of ATP
generation is derived from FA oxidation (25). Concerning FA,
the heart has a limited capacity to synthesize this substrate and
thus relies on 1) release of FA from adipose and transport to the
Address for reprint requests and other correspondence: B. Rodrigues,
Faculty of Pharmaceutical Sciences, Univ. of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 (e-mail: rodrigue@mail.
ubc.ca).
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heart after complexing with albumin (24), 2) breakdown of
endogenous cardiac triglyceride (TG) (14), and 3) lipolysis of
circulating TG-rich lipoproteins to FA by lipoprotein lipase
(LPL)positioned at the endothelial cell (EC) surface of the
coronary lumen (26).
In diabetes, because glucose uptake and oxidation are impaired, the heart is coerced to use FA almost exclusively for
ATP generation (33). Multiple adaptive mechanisms, either
whole body or intrinsic to the heart, operate to make this
achievable. These include augmented adipose tissue lipolysis,
where breakdown of stored TG increases circulating FA that
are transported to the heart. If delivered to the liver, these FA
can raise circulating lipoprotein concentrations, as hepatic FA
availability is a rheostat for VLDL synthesis. In so doing,
VLDL-TG is an additional and major resource to increase FA
delivery to the heart for oxidation (31). Innate to the cardiac
muscle, the uptake of albumin-bound FA is driven by plasma
membrane FA transporters (for example, CD36), which increase following diabetes (21). Diabetes also enhances adipose
TG lipase to mobilize the storage pool of TG within cardiomyocytes (43). Finally, the utilization of VLDL-TG as a FA
source by the diabetic heart is influenced not only by elevated
plasma VLDL concentrations but also the vascular content of
LPL, a rate-limiting enzyme in circulating TG clearance (2).
We were the first to report higher luminal LPL activity following diabetes (32).
The increase in cardiac LPL with diabetes was immediate
and unrelated to gene expression and involved exaggerated
processing to dimeric, catalytically active enzymes, an obligatory step for ensuing secretion (39). Transfer to the coronary
lumen requires movement of LPL to the cardiomyocyte plasma
membrane by AMP-activated protein kinase, protein kinase D,
and p38 MAPK (18). Activation of these kinases following
diabetes facilitated LPL vesicle formation in addition to promoting cytoskeleton rearrangement for secretion onto surface
heparan sulfate proteoglycans (HSPG) (10). For its onward
movement across the interstitial space to the basolateral side of
vascular ECs, detachment of LPL from the myocyte surface is
a prerequisite and is likely mediated by high-glucose-induced
secretion of EC heparanase (38). This endoglycosidase, exceptional in its ability to degrade heparan sulfate (HS), instigates
LPL release from cardiomyocyte HSPG (38). From here, LPL
traverses the interstitial space, and glycosylphosphatidylinositolanchored high-density lipoprotein-binding protein 1 (GPIHBP1)
is suggested to deliver it across ECs onto the luminal binding
sites of these cells, where the enzyme is functional (42). Thus,
GPIHBP1, a protein expressed abundantly in the heart but only
on capillary ECs, operates as a transporter, collecting LPL
0193-1849/14 Copyright © 2014 the American Physiological Society
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Chiu AP, Wang F, Lal N, Wang Y, Zhang D, Hussein B, Wan
A, Vlodavsky I, Rodrigues B. Endothelial cells respond to hyperglycemia by increasing the LPL transporter GPIHBP1. Am J Physiol
Endocrinol Metab 306: E1274 –E1283, 2014. First published April
15, 2014; doi:10.1152/ajpendo.00007.2014.—In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use
fatty acid (FA) almost exclusively for ATP. The vascular content of
lipoprotein lipase (LPL), the rate-limiting enzyme that determines
circulating triglyceride clearance, is largely responsible for this FA
delivery and increases following diabetes. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein [GPIHBP1; a
protein expressed abundantly in the heart in endothelial cells (EC)]
collects LPL from the interstitial space and transfers it across ECs
onto the luminal binding sites of these cells, where the enzyme is
functional. We tested whether ECs respond to hyperglycemia by
increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL
activity and GPIHBP1 gene and protein expression. The increased
LPL and GPIHBP1 were located at the capillary lumen. In vitro,
passaging EC caused a loss of GPIHBP1, which could be induced on
exposure to increasing concentrations of glucose. The high-glucoseinduced GPIHBP1 increased LPL shuttling across EC monolayers.
GPIHBP1 expression was linked to the EC content of heparanase.
Moreover, active heparanase increased GPIHBP1 gene and protein
expression. Both ECs and myocyte heparan sulfate proteoglycanbound platelet-derived growth factor (PDGF) released by heparanase
caused augmentation of GPIHBP1. Overall, our data suggest that this
protein “ensemble” (heparanase-PDGF-GPIHBP1) cooperates in the
diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to
restore metabolic equilibrium, curb lipotoxicity, and help prevent or
delay heart dysfunction that is characteristic of diabetes.
HIGH-GLUCOSE REGULATION OF GPIHBP1
from the interstitial space and transferring it across ECs (7,
9, 15).
Given that GPIHBP1 mRNA levels change rapidly (22), we
tested whether this protein has a part to play in the transfer of
LPL to the vascular lumen after diabetes. Our data suggest a
novel mechanism in which high-glucose-induced secretion of
heparanase not only liberates LPL from the myocyte cell
surface but can also induce GPIHBP1 expression, thus promoting LPL shuttling to the apical side of ECs.
MATERIALS AND METHODS
nase, protein was concentrated with an Amicon centrifuge filter
(Millipore), followed by the procedures listed above.
RNA isolation and real-time PCR. Total RNA was isolated from rat
hearts and RAOECs using Trizol, followed by chloroform and isopropanol extraction, washing by ethanol, and dissolving in DEPCH2O. RNA was reverse transcribed into cDNA with a mixture of
random primers, oligo-dT, and SuperScript III. cDNA were amplified
by TaqMan probes (GPIHBP1 and ␤-actin) in triplicate, using a
7900HT Fast Real-Time PCR system. Gene expression was calculated
with the comparative cycle threshold method.
EC culture. RAOECs (Cell Applications) and bovine coronary
artery ECs (BCAECs; Clonetics) were cultured at 37°C in a 5% CO2
humidified incubator to 80 –90% confluence (38). Cells were detached
following trypsin digestion, and aliquots were used for seeding of a
new culture. Cells from passages 5–10 were used for detection of
GPIHBP1 and heparanase.
EC and cardiomyocyte coculture. Only RAOECs and BCAECs
from passages 8 –10 were used for this experiment. ECs were seeded
on Transwell inserts (24-mm diameter, 1.0-␮m pore size; Falcon). On
reaching 80 –90% confluence, these inserts were placed in a six-well
plate that had cardiomyocytes attached to the bottom. Cardiomyocytes
were plated at a density of 200,000 cells/well (40).
LPL transport assay. To study basolateral to apical transport of
LPL, RAOECs were seeded on Transwell inserts until the cells
formed a tight monolayer. The inserts were then placed in six-well
plates. Subsequently, LPL (5,000 U/10 ␮g/ml) was added to the
bottom chamber. After 1 h, LPL that translocated to the apical surface
of ECs was collected by heparin displacement and LPL activity
determined.
In vitro lipolysis of VLDL. Isolation of VLDL (without chylomicrons) by density gradient ultracentrifugation was achieved using
serum from 16-h-fasted rats (6 PM to 10 AM). Fasting of rats for this
duration led to the production of more VLDL and minimized the
contribution from chylomicrons. In vitro lipolysis of VLDL-TG was
carried out on the basis of previously described methods (36). Briefly,
various concentrations (0 – 0.8 mM) of VLDL-TG were incubated
with heparin-releasable LPL at 37°C for 30 min. At the end of
incubation the reaction was stopped by the addition of precooled 0.3
M Na2PO4 (pH 6.9), and the tubes were immediately immersed in ice.
From this reaction mixture, 50 ␮l was pipetted in triplicate to measure
free fatty acids released by hydrolysis of VLDL-TG with a NEFA C
kit (Wako Chemicals).
Cytokine array. Media were collected from RAOECs (EC culture
medium) incubated with DMEM containing either 5.5 or 25 mM
glucose for 12 h. Myocytes (myocyte culture medium) incubated with
DMEM or DMEM containing 500 ng/ml purified heparanase (HPA)
were also collected. Media were centrifuged at 2,000 rpm for 5 min.
Array membranes were incubated with blocking buffer at room
temperature (RT), followed by a 1-h incubation with EC culture
medium or myocyte culture medium at RT for 2 h. Membranes were
washed (3 times for 5 min) and incubated with working primary
antibody and anti-horseradish peroxidase, respectively, at RT for 2 h.
Array membrane was detected with enhanced chemiluminesence
(ECL) and Hyperfilm ECL Film (GE Healthcare).
Reagents and antibodies. Heparin (Hepalean, 1,000 U/ml) was
from Organon. [3H]triolein was purchased from Amersham Canada.
Anti-HPA antibody monoclonal antibody 130 was from InSight (Rehovot, Israel). Anti-GPIHBP1 antibody was from Novus Biologicals.
Anti-PECAM (CD31) was from Chemicon (Millipore). Anti-␤-actin
(C4), goat anti-mouse antibody, and goat anti-rabbit antibody were
from Santa Cruz Biotechnology. To measure free fatty acid released
from VLDL-TG breakdown, a NEFA C assay kit was purchased from
Wako. Purified active and latent heparanase were a kind gift from Dr.
Israel Vlodavsky, Hadassah University Hospital, Jerusalem, Israel.
Phosphatidylinositol-specific phospholipase C (PI-PLC), Alexa
555-labeled anti-mouse IgG, Alexa 488-labeled anti-rabbit IgG,
Trizol, SuperScript III RT, GPIHBP1 (Rn01503971_g1) and actin
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Experimental animals. This investigation adhered to the Guide for
the Care and Use of Laboratory Animals published by the National
Institutes of Health and was approved by the Animal Care Committee
of the University of British Columbia (certificate no. A13-0098).
Adult male Wistar rats (250 –320 g) were injected with streptozotocin
(STZ), a ␤-cell-specific toxin. STZ (55 mg/kg) was administered
intravenously as a single dose to generate a model of poorly controlled
type 1 diabetes (19, 20). Hyperglycemia was confirmed in blood
samples from the tail vein using a glucometer (AccuSoft) and glucose
test strips (Accu-Chek Advantage; Roche). STZ animals were kept for
4 days before hearts were removed for both LPL and GPIHBP1
measurement.
Heart perfusion and isolation of cardiomyocytes. Rats were anesthetized with 65 mg/kg ip pentobarbital sodium, the thoracic cavities
were opened, and hearts were carefully excised. After cannulation of
the aorta, the heart was secured by tying below the innominate artery
and perfused retrogradely with Krebs-Henseleit buffer (37). Ventricular calcium-tolerant myocytes were prepared by way of a previously
described procedure (34). Briefly, myocytes were made calcium
tolerant by successive exposure to increasing concentrations of calcium. Cardiomyocytes were plated at a density of 200,000 cells/well
on laminin-coated six-well culture plates. Cells were maintained using
Medium-199 and incubated at 37°C in a 5% CO2 humidified incubator.
LPL activity. To measure coronary LPL, Krebs buffer containing
heparin (8 U/ml) was used, perfusion effluent was collected over 5
min, and LPL activity was determined. To release EC surface-bound
LPL, cells were incubated with heparin (8 U/ml) for 3 min. LPL
activity was determined by measuring in vitro hydrolysis of [3H]triolein substrate (18). Radiolabeled FA products were extracted and
estimated by liquid scintillation counting.
Immunofluorescence. To visualize LPL and GPIHBP1 localization,
rat hearts were perfused with cold PBS and fixed in 10% paraformaldehyde overnight, and 8- to 10-␮m parafilm-embedded sections were
prepared. Slides were incubated in a 56°C oven for 30 min and
dehydrated by xylene (twice for 10 min) and ethanol (35–95%, 5 min)
followed by PBS. Sections were permeabilized with 0.2% Triton
X-100 in PBS for 30 min and incubated with blocking buffer containing 10% donkey serum and 0.2% Triton X-100 in PBS for 30 min
at room temperature. Slides were then incubated with primary antibodies at 4°C overnight: 1:100 for anti-CD31, anti-GPIHBP1 (8), and
anti-LPL. Secondary antibodies were used at a dilution of 1:200 and
incubated at room temperature for 1 h. For immunofluorescence
detection of proteins in rat aortic endothelial cells(RAOECs), cells
were seeded on slides and grown until they were 80 –90% confluent.
Following treatment with or without high glucose (25 mM), cells were
washed with cold PBS and fixed with methanol at 4°C for 1 h, and the
above procedures (permeabilization, blocking, primary/secondary antibodies) were repeated.
Western blot. To measure protein expression, 30 ␮g of protein was
loaded. Membranes were incubated with primary antibodies (1:1,000
for anti-GPIHBP1, 1:2,000 for anti-␤-actin, and 1:500 for anti-heparanase) at 4°C overnight and subsequently with secondary antibodies
(1:1,000) at room temperature for 1 h. To measure medium hepara-
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HIGH-GLUCOSE REGULATION OF GPIHBP1
(Rn00667869_m1) probe, and TaqMan fast advanced master mix
were obtained from Invitrogen. Protein array kit (Rat Cytokine Array
C2) was purchased from RayBiotech.
Statistical analysis. Values are means ⫾ SE. Wherever appropriate,
one-way ANOVA followed by the Bonferroni test was used to
determine differences between group mean values. The level of
statistical significance was set at P ⬍ 0.05.
RESULTS
Fig. 1. Streptozotocin (STZ) diabetes increases cardiac lipoprotein lipase (LPL) activity and glycosylphosphatidylinositol-anchored
high-density lipoprotein-binding protein 1
(GPIHBP1) expression. Rats were made hyperglycemic by iv injection of 55 mg/kg
STZ. After 4 days of hyperglycemia, animals were euthanized; hearts were removed
and either perfused with heparin (8 U/ml) to
release coronary LPL activity (A, left and top
right) or used for immunofluorescent detection of LPL (A, bottom right) and protein
measurement of GPIHBP1 (B). Coronary
LPL activity was determined at the indicated
times by measuring the in vitro hydrolysis of
[3H]triolein substrate. LPL immunofluorescence was detected using an anti-rabbit LPL
antibody. Scale bar, 20 ␮m. *P ⬍ 0.05 compared with control (CON); n ⫽ 3.
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Cardiac LPL and GPIHBP1 after STZ diabetes. Following
injection of STZ and maintenance of animals for 4 days, there
was a pronounced increase in blood glucose (Fig. 1A, right).
Because utilization of glucose is impaired following diabetes,
the heart rapidly adapts to use FA exclusively, an effect
determined largely by LPL (18). Indeed, perfusion of hearts
with heparin indicated that LPL activity increased following
hyperglycemia (Fig. 1A, top right). Interestingly, immunofluorescence detection of LPL revealed that most of the
increased enzyme in diabetic hearts was located at the
coronary lumen (Fig. 1A, bottom right). In the heart, LPL
synthesized in cardiomyocytes is shuttled across ECs with the
help of GPIHBP1 (12, 42). To determine whether the increased
vascular content of LPL following diabetes is associated with
GPIHBP1, its protein (Fig. 1B) and mRNA (Fig. 2B) expression were examined and determined to be augmented. Because
the increased GPIHBP1 had an exclusive endothelial location
(Fig. 2A), our data suggest that GPIHBP1 is important for
myocyte to EC transfer of LPL to increase FA delivery to the
diabetic heart.
Endothelial GPIHBP1 following cell passage and influence
of high glucose. GPIHBP1 is expressed exclusively in endothelial cells (42). We determined the level of this glycoprotein
in RAOECs and BCAECs and unexpectedly identified loss of
this glycoprotein following passaging of these cells (Fig. 3A).
In an attempt to mimic in vivo hyperglycemia, RAOECs were
exposed to increasing concentrations of glucose for 12 h.
Interestingly, in cells from later passages (in which GPIHBP1
expression had been suppressed), although concentrations of
glucose between 10 and 15 mM increased GPIHBP1 mRNA
(Fig. 3B, right) and protein (Fig. 3C, right), the increases were
more robust with the higher concentration of glucose. Choosing 25 mM glucose and different times, high glucose increased
GPIHBP1 mRNA expression, an effect that was pronounced
after 12 h (Fig. 3B). Measurement of GPIHBP1 protein under
these conditions uncovered an even more rapid increase within
4 h of incubation with high glucose (Fig. 3C). This increased
EC GPIHBP1 was predominantly membrane bound (Fig. 4A).
It should be noted that RAOECs and BCAECs from the earlier
passages (e.g., passages 5–7) also responded to high glucose by
increasing GPIHBP1 expression (data not shown). Overall,
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HIGH-GLUCOSE REGULATION OF GPIHBP1
these data imply that by sensing the impending loss of glucose
transport, ECs respond by increasing GPIHBP1 to shuttle LPL.
GPIHBP1-associated transfer of LPL across the endothelial
monolayer. To test the functional relevance of the high-glucose-induced augmentation of GPIHBP1, LPL transport from
the basolateral to the apical side of RAOEC monolayers was
determined. As expected, there was an increase in heparinreleasable LPL activity at the apical side of ECs exposed to
high glucose (Fig. 4B), an effect that was blunted by PI-PLC
(Fig. 4B, inset). The osmotic control mannitol had no influence
on LPL transport (Fig. 4B). The LPL that had been shuttled
following high glucose was functionally active, as exposure of
this apical heparin-releasable enzyme to lipoprotein triglyceride was capable of increasing its hydrolysis to FA (Fig. 4C).
Since prior exposure of cells to PI-PLC [which is expected
cleave GPIHBP1 (15) and prevent LPL shuttling] abolished
this TG breakdown, our data indicate that in ECs exposed to
high glucose, enhancement of GPIHBP1 is an effective stimulus for increasing delivery of FA to cardiomyocytes to support
energy requirements.
GPIHBP1 expression is linked to endothelial content of
HPA. HPA, an EC endoglycosidase, can cleave HS side chains
on HSPGs in the extracellular matrix and on the cell surface of
cardiomyocytes to release LPL for transfer across the interstitial space to reach ECs (29). As described previously, exposure
of RAOECs to high glucose rapidly increased the secretion of
both active (Fig. 5A) and latent (data not shown) HPA. Intriguingly, in addition to its ability to rapidly liberate cardiomyocyte
HSPG-sequestered LPL, exposure of ECs to active but not
latent (data not shown) HPA for 12 h produced a robust
increase in GPIHBP1 mRNA and protein (Fig. 5B). Because
passaging of RAOECs progressively decreased HPA expression (Fig. 5C) similarly to loss of GPIHBP1, our data have
uncovered a novel autocrine role (either direct or indirect) for
active HPA to affect GPIHBP1 expression.
Cardiomyocytes induce EC GPIHBP1. Although GPIHBP1
is highly expressed in ECs in vivo, ECs in culture lose
expression of this glycoprotein (1). Because ECs in the heart
are closely appositioned to cardiomyocytes, we reasoned that a
paracrine influence from cardiomyocytes may be responsible
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Fig. 2. Increased GPIHBP1 is located at the
capillary lumen in diabetic hearts and is associated with augmented gene expression. After
4 days, both CON and STZ hearts were collected for immunofluorescence microscopy
and mRNA measurement. Paraffin-embedded
sections were used and permeabilized with
0.2% Triton X in PBS. A: primary anti-mouse
CD31 and anti-rabbit GPIHBP1 antibodies
were used to stain the capillary lumen. Scale
bar, 20 ␮m. B: GPIHBP1 mRNA level was
determined using TaqMan (ABI) following
RNA isolation and reverse transcription. *P ⬍
0.05 compared with CON; n ⫽ 3.
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HIGH-GLUCOSE REGULATION OF GPIHBP1
for EC expression of GPIHBP1. Using a coculture system to
mimic the intact heart, we determined that in ECs in which
GPIHBP1 had been silenced (passages 8 –10), a simple introduction of cardiomyocytes into the vicinity restored GPIHBP1
expression to levels observed with early cell passages (Fig. 6,
A and B). These results point toward paracrine factors from
cardiomyocytes influencing EC GPIHBP1 expression. Assuming that the stimulus for the release of signaling mediators from
myocytes could originate from ECs (likely HPA), we exposed
the coculture to high glucose. Noteworthy, a further expression
of EC GPIHBP1 was evident in this condition (Fig. 6C),
suggesting that high-glucose-induced HPA released from ECs
can affect GPIHBP1 by both autocrine and paracrine signaling.
HPA-released platelet-derived growth factor from myocytes
and ECs can induce GPIHBP1 expression. ECs and myocyte
HSPG-anchored proteins can be liberated by HPA (38). Using a
protein array and ECs exposed to high glucose or myocytes
treated with active HPA, a number of proteins were increased in
the culture medium. These included monocyte chemotactic protein-1, prolactin, receptor for advanced glycation end products,
thymidylate kinase TMP-1 (data not shown), and platelet-derived
growth factor (PDGF) (Fig. 7A). Given the acknowledged role of
PDGF in FA metabolism (35), we treated EC with recombinant
PDGF and discovered an induction of GPIHBP1 from passage 7
(which still express GPIHBP1) and passage 10 (in which there is
a loss of GPIHBP1) (Fig. 7B). Nevertheless, the increase was
more robust in cells from passage 10, as these cells had an
extremely low initial expression of GPIHBP1. Our data suggest a
mediator role (either autocrine or paracrine) for this growth factor
in FA delivery to the diabetic myocyte.
DISCUSSION
Underutilization of glucose and overreliance on FA are hallmarks
of the diabetic heart (33). Although albumin-bound circulating FA is
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Fig. 3. Passaging of endothelial cells causes loss of GPIHBP1, which can be induced on exposure to high glucose. Rat aortic endothelial cells (RAOECs) and bovine coronary
artery endothelial cells (BCAECs) were cultured to 80–90% confluence. A: cells were detached following trypsin digestion, and aliquots were used either for detection of
GPIHBP1 or seeding of a new culture. B and C: RAOECs from passages 7–10 were treated with increasing concentrations of glucose (B and C, right) for different times, and
GPIHBP1 mRNA (B) and protein (C) were determined using TaqMan and Western blotting, respectively. *P ⬍ 0.05 compared with 0 h or 5.5 mM glucose; n ⫽ 3–4.
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HIGH-GLUCOSE REGULATION OF GPIHBP1
an important source of this substrate, its molar concentration is
⬃10-fold less than that of lipoprotein TG (26). As such, the hydrolysis of circulating TG is suggested to be the predominant source of
FA for cardiac utilization during diabetes (3). Lipoprotein-TG breakdown occurs at the coronary lumen, with the assistance of LPL
positioned at apical surface of ECs. Following diabetes, in the absence of changes in LPL synthesis, coronary LPL activity is aug-
mented by posttranslational modifications. These include an
increase in LPL dimerization (39), vesicle transport (18), and
shuttling of the enzyme from the myocyte surface to the
basolateral side of ECs (6). Data from the current study present
the novel idea that in response to hyperglycemia, transport of
LPL to the apical side of ECs to assist in TG breakdown is
achievable through induction of GPIHBP1.
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Fig. 4. GPIHBP1 induced by high glucose
increases LPL shuttling across endothelial cell
monolayers. A: RAOECs seeded on slides
were placed in a 6-well plate and treated with
5.5 (CON) or 25 mM glucose (HG) for 12 h.
Slides were then washed with ice-cold PBS,
fixed with methanol, and permeabilized using
0.2% Triton X in PBS. Anti-rabbit GPIHBP1
(1:100) and anti-mouse CD31 antibodies (1:
100) were used for immunofluorescence staining. Scale bar, 20 ␮m. B: in a separate experiment, RAOECs were seeded on Transwell
inserts and grown until they formed a tight
monolayer. Cells were then treated with CON
or HG for 4 and 12 h, respectively. In some
experiments, this was followed by treatment
with or without phosphatidylinositol-specific
phospholipase C [PI-PLC; 1 U/ml for 1 h
(inset)]; 25 mM mannitol (Mnt) for 12 h was
used as an osmotic control. Following the
indicated times, these Transwell inserts were
removed and placed in a different 6-well plate
with DMEM on the basolateral side containing
10 ␮g/ml purified LPL. After incubation for 1
h and washing (3 times) with PBS, medium
containing heparin (8 U/ml) was used for 3
min to release apical surface-bound LPL. LPL
activity was determined by measuring the in
vitro hydrolysis of [3H]triolein substrate. C:
using the above protocol (with incubation of
HG for 12 h), the heparin-releasable LPL medium was incubated with increasing concentrations of very-low density lipoprotein-triglyceride (VLDL-TG; 0 – 0.8 mM) at 37°C for
30 min and the concentration of released free
fatty acid (FA) determined. One group of cells
was treated with 1 U/ml PI-PLC prior to incubation with high glucose. *P ⬍ 0.05; n ⫽ 3.
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Fig. 5. Endothelial heparanase (HPA) can increase GPIHBP1 gene and protein
expression. A: RAOECs from passages 6 –10 were used for determination of
latent (L-HPA) and active heparanase (A-HPA). Cells from passages 7–10
were treated with HG (25 mM) for 12 h, and medium was collected. B: the
medium was concentrated using an Amicon column at 4°C and subsequently
centrifuged for 15 min at 14,000 g. HPA in this concentrated medium was
determined by Western blot; n ⫽ 4. C: recombinant HPA was used to treat
RAOECs (passages 7–10) for 12 h, cell lysates were collected, and GPIHBP1
mRNA (top) and protein (bottom) were determined using TaqMan and Western blot, respectively; n ⫽ 3– 4. *P ⬍ 0.05.
Fig. 6. Loss of endothelial GPIHBP1 expression can be restored on coculture
with myocytes (Myo). A and B: RAOECs (A) or BCAECs (B) (passages 8 –10)
were seeded on Transwell inserts and cultured until 80 –90% confluence. The
Transwell insert was then transfered to a 6-well plate containing attached Myo
and cocultured for 12 h. Isolation of Myo was achieved by collagenase
digestion of rat hearts, and cells were attached to laminin-coated 6-well plates
in M199 containing 0.1% BSA and kept overnight. After coculture with
myocytes, endothelial cell protein was collected and used for Western blot of
GPIHBP1. C: in some experiments, endothelial cells (with or without coculture
with Myo) were exposed to normal (5.5 mM) or high glucose (HG; 25 mM) for
12 h, and GPIHBP1 was determined; n ⫽ 3– 4. *P ⬍ 0.05 compared with
control; #P ⬍ 0.05 compared with normal glucose.
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HIGH-GLUCOSE REGULATION OF GPIHBP1
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Following a single injection of a moderate dose (55 mg/kg)
of STZ, there is an induction of hypoinsulinemia and hyperglycemia. Increasing the dose to 100 mg/kg also creates an
environment of hyperlipidemia (39). In the former situation,
coronary LPL activity is augmented, whereas with the latter
setting and the presence of higher circulating FA, LPL is
turned off (32). With D55 hearts, in the absence of any change
in protein synthesis, the increase in LPL activity principally at
the vascular lumen could be explained largely by posttranslational modifications that increased transfer of myocyte enzyme
to the ECs (30). Not yet determined is whether the concluding step, moving LPL across EC, is also increased following diabetes. GPIHBP1, a glycosylphosphatidylinositol-an-
chored protein expressed exclusively in ECs (15), is a recent
addition to the mechanism that transfers LPL across ECs (42).
GPIHBP1 has a strong binding affinity for LPL, accepting it
from the interstitial space and moving it to the coronary lumen
(41). Intriguingly, the mRNA for GPIHBP1 changes much
more rapidly than most mRNAs in mammalian cells (22). Our
results present the novel observation that to guarantee FA
supply to the diabetic heart, the LPL transporter GPIHBP1
increases rapidly in ECs.
In an attempt to elucidate the mechanism by which diabetes
influences GPIHBP1, we exposed two different EC lines to
glucose, a substrate whose concentrations increase rapidly
following STZ. However, given that under standard cell culture
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Fig. 7. Endothelial cells and myocyte heparan sulfate
proteoglycan (HSPG)-bound platelet-derived growth factor (PDGF) can augment GPIHBP1. A: RAOECs were
cultured with or without HG (25 mM) for 12 h, whereas
myocytes were incubated with or without A-HPA also for
12 h. With both cell types, the culture medium was
collected and tested using a cytokine array. Of the proteins detected, PDGF was chosen. B: purified recombinant PDGF (200 ng/ml) was used to treat RAOECs
[passage 7 (left) or 10 (bottom right)] for 12 h, and
GPIHBP1 was determined by Western blot. *P ⬍ 0.05;
n ⫽ 2– 4. C: summarization of how the endothelial cell
increases its GPIHBP1 in response to hyperglycemia.
Briefly, HG induces secretion of endothelial HPA, which
has multiple functions. These include 1) stimulation of
myocyte HSPG-bound LPL release, 2) autocrine and
paracrine actions to provoke liberation of PDGF, and 3)
reuptake into endothelial cells to influence gene transcription. The latter 2 effects contribute toward augmentation
of GPIHBP1, the transporter that moves LPL across EC
to its apical side. Out here, LPL is responsible for
VLDL-TG hydrolysis to FA, the preferred substrate for
the diabetic heart.
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HIGH-GLUCOSE REGULATION OF GPIHBP1
HSPG are ubiquitously present in every tissue compartment,
particularly the extracellular matrix, cell surface, intracellular
granules, and nucleus (17). They consist of a core protein to
which several linear HS side chains are covalently linked and
function not only as structural proteins but also as anchors due
to the high content of charged groups in HS (16). The latter
property is implicitly used to electrostatically bind a number of
different proteins (chemokines, coagulation factors, enzymes
like LPL, and growth factors such as vascular endothelial
growth factor) (23). Attachment of these bioactive proteins is a
clever arrangement, providing the cell with a rapidly accessible
reservoir, precluding the need for de novo synthesis when the
requirement for a protein is increased (4). Heparanase is an
endoglycosidase, exceptional in its ability to degrade HS,
thereby instigating ligand release. Using a cytokine array and
incubation of cardiomyocytes with active heparanase, a number of cytokines were increased in the myocyte culture medium. We focused on PDGF, as this protein can regulate
angiogenesis but also play a significant role in increasing the
metabolic reliance of smooth muscle on FA (35). Favorably,
PDGF increased EC GPIHBP1. Because exposure of ECs to
high glucose also increased PDGF in the medium, likely
through an autocrine stimulus of heparanase secretion with the
release of PDGF bound to EC HSPG, our data suggest that this
protein “ensemble” (heparanase-PDGF-GPIHBP1) cooperates
in the diabetic heart to regulate FA delivery and utilization by
the cardiomyocytes.
Overall, through their release of heparanase, ECs, which are
the first responders to hyperglycemia, are responsible for
regulating LPL-derived FA delivery to the cardiomyocytes.
Although this mechanism serves to guarantee FA supply and
consumption when glucose utilization is compromised, it unintentionally provides a surfeit of FA to the diabetic heart,
sponsoring a setting where FA uptake exceeds the mitochondrial oxidative capacity. Chronically, the resulting increase
in the conversion of FA to potentially toxic FA metabolites,
including ceramides, diacylglycerols, and acylcarnitines,
paired with increased formation of reactive oxygen species
secondary to elevated FA oxidation, can provoke cardiac cell
death (lipotoxicity).
GRANTS
The present study is supported by an operating grant from the Canadian
Diabetes Association. Y. Wang and D. Zhang are the recipients of Doctoral
Student Research Awards from the Canadian Diabetes Association.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
A.P.-L.C. and Y.W. conception and design of research; A.P.-L.C., N.L., and
B.H. performed experiments; A.P.-L.C. analyzed data; A.P.-L.C., F.W., and
I.V. interpreted results of experiments; A.P.-L.C. prepared figures; A.P.-L.C.
drafted manuscript; A.P.-L.C., D.Z., A.W., I.V., and B.R. edited and revised
manuscript; A.P.-L.C. and B.R. approved final version of manuscript.
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