From the Departments of Medicine,
§ Pathology and Immunology, and ¶ Cell Biology and
Physiology and the
Center for Cardiovascular Research,
Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, November 28, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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Lipoprotein lipase (LpL) provides tissues
with triglyceride-derived fatty acids. Fatty acids affect Lipoprotein lipase
(LpL)1 catalyzes the
rate-limiting step for clearance of circulating triglycerides. The
hydrolysis of lipoprotein-associated triglycerides in the capillary
beds of peripheral tissues such as muscle and adipose tissue produces
free fatty acids that are available for local uptake (1). LpL is the
major physiological determinant of fatty acid delivery to peripheral
tissues in vivo. The enzyme is highly glycosylated and
functions as a head-to-tail dimer (2, 3) under well-defined conditions
(4-6). Functional LpL binds to proteoglycans at the cell surface and
capillary endothelium and is displaced from these sites by heparin
(7-9). LpL is synthesized by a variety of cells (adipocytes, myocytes,
and monocyte/macrophages) where its expression is differentially
regulated by nutritional, hormonal, and developmental signals (10-14).
LpL mRNA was recently identified in human and mouse islets and the
rat insulinoma cell line INS-1 (15).
LpL regulation is complex and varies between cells. In adipocytes, LpL
is increased by activation of protein kinase C ( Glucose is known to regulate LpL in adipocytes (21, 22). Incubation of
rat adipocytes in the absence of glucose produces a 49-kDa form of LpL
that is catalytically inactive and not secreted (21). In the same
cells, incubation in the presence of glucose produces a 55-kDa
catalytically active form of LpL that is secreted. Glucose is essential
for glycosylation, which is required for LpL enzymatic activity (23),
and increases the rate of LpL translational and post-translational
events. Glucose regulation of LpL expression in macrophages differs
from adipocytes. Sartippour et al. (24) demonstrated that
glucose increases LpL expression in J774 macrophages in a protein
kinase C- and AP1-dependent manner. LpL regulation in pancreatic islets has not been studied.
LpL mRNA was recently identified in human and mouse islets as well
as insulinoma cells (15). LpL expression was shown to be inversely
related to insulin secretion, indicating that LpL may be a
physiologically relevant provider of fatty acids to Materials--
Male C57BL/6 mice were obtained from
Harlan Sprague-Dawley (Indianapolis, IN). CMRL-1066 and RPMI 1640 tissue culture media, penicillin, streptomycin, Hanks' balanced salt
solution, and L-glutamine were obtained from Life
Technologies. Fetal bovine serum was from HyClone (Logan, UT).
Collagenase type P was obtained from Roche Molecular Biochemicals.
Ficoll, D-mannoheptulose, diazoxide, purified LpL, and low
molecular weight (Mr 3000-5000) heparin were
from Sigma. Rat sera, a source of apolipoprotein C-II, was obtained from Sigma Chemical Co. and Chemicon. Glycerol
tri-[14C]oleate was obtained from Amersham Pharmacia
Biotech. For Western blot analysis, the primary antibody was
chicken-anti-bovine milk LpL (25) and the secondary antibody was
peroxidase-conjugated donkey anti-chicken IgG (Jackson
ImmunoResearch). For immunohistochemical studies, the primary
antibodies were rabbit-anti-human LpL (26, 27) and guinea
pig-anti-human insulin (Linco). The secondary antibodies were
FITC-conjugated donkey anti-rabbit IgG and Cy3-conjugated donkey-anti-guinea pig IgG (Jackson ImmunoResearch). CellTiter 96 AQueous One Solution containing MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inert salt reagent) was purchased from Promega (Madison, WI).
Pancreatic Islet Isolation and Culture--
Islets were isolated
from 6- to 8-week-old male C57BL/6 mice by collagenase digestion as
previously reported (28). Islets were cultured overnight in an
atmosphere of 95% air, 5% CO2 in CMRL-1066 tissue culture
medium (CMRL) containing 5.5 mM glucose, 2 mM
L-glutamine, 10% (v/v) heat-inactivated fetal bovine
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Mouse
islets (200 per experimental condition) were cultured in 35-mm sterile dishes.
Cell Culture--
All experiments were performed in triplicate.
INS-1 and RINm5F cells were maintained under conditions previously
described (15, 29) in 12-well tissue culture plates (3 × 106 cells/well). INS-1 cells, established by Asfari
et al. (30), were a kind gift from Chris Newgard (Dallas, TX).
Isolated pancreatic mouse islets (200) were seeded into a 35-mm dish
containing 1 ml of culture media. The islets were washed with CMRL
(without fetal calf serum, containing 0.1% bovine serum albumin and 3 mM glucose) and incubated for 30 min prior to acute exposure (4 h) to 3 or 20 mM glucose. For studies using
diazoxide or mannoheptulose, these agents were present during the
preincubation and glucose stimulation period. For determining the
effect of room temperature, preincubation and glucose stimulation
protocols were performed in a gassed, humidified chamber at 25 °C.
Glucose stimulation was performed by replacing preincubation media with stimulation media (containing 0.1% bovine serum albumin and 3 or 20 mM glucose).
After glucose stimulation, islets were collected and media replaced
with 125 µl of CMRL containing 100 µg/ml heparin and incubated for
30 min. The heparin-containing media were then collected, a detergent
solution was added to the plates, and the islets were incubated until
visually dissolved. The heparin-containing media and the detergent
extracts were assayed for LpL activity (described below). The
stimulation culture supernatant was assayed for insulin by radioimmunoassay.
INS-1 cells (3 × 106) were seeded in 12-well culture
plates 48 h prior to glucose stimulation. Preincubation and acute
glucose stimulation of INS-1 cells were performed as described with
mouse islets, except cells were cultured in RPMI media, and the absence of glucose was used for the basal conditions. The effect of prolonged insulin exposure (24 h) was performed by adding exogenous porcine insulin (0.25, 0.5, or 1 µM) to the incubation media.
After acute exposure to an elevated glucose concentration or prolonged
exposure to exogenous insulin, heparin was added to the incubation
media to a final concentration of 100 µg/ml and incubated for 30 min. The culture media were then collected, a detergent solution was added,
and cells were incubated until visually dissolved. The heparin-containing culture supernatant and the detergent extract were
assayed for LpL activity as described below. The culture supernatant
was assayed for insulin.
LpL Enzyme Activity Assay--
Lipase activity was measured by
an in vitro assay in which radiolabeled fatty acids
esterified to glycerol are cleaved and recovered after a
chloroform/methanol/heptane-based extraction (17). The units of
activity are reported as moles of free fatty acid released per specific
number of islets or cells per unit time. LpL activity is
distinguishable from other lipase activities by its sensitivity to high
molar salt concentration (4). "Heparin-releasable" LpL activity is
the amount of activity in the supernatant of heparin-treated islets or
cells. "Detergent-extractable" is the amount of activity after
detergent solubilization of remaining cells or islet pellets following
heparin treatment. Detergent solubilization involves incubating
Cellular Metabolism Assay--
Metabolism was determined using
an assay based on the ability of NADH or NADPH generated by substrate
flux to reduce a tetrazolium substrate and produce a stable colored
product. INS-1 or RINm5F cells were cultured in 96-well culture plates
(2 × 105 cells/well). At the start of the experiment,
the cells were washed with culture media and stimulated with glucose as
described above. Subsequently, 20 µl/well tetrazolium reagent was
added to the media and the optical density at 540 nm was determined
after 4 h on a Molecular Devices Thermomax plate reader
(Sunnyvale, CA).
Immunohistochemistry--
Dispersed islets were obtained by
dispase digestion of freshly isolated mouse islets and cultured for
24 h in 5.5 mM glucose. Duplicated aliquots of
dispersed islet cells were treated with or without heparin (100 µg/ml) for 30 min prior to centrifugation onto glass slides and
fixation in Bouin's solution. INS-1 cells (30,000) were seeded into
8-well collagen I-coated culture slides (Becton Dickinson, Beford, MA)
and cultured for 48 h in the absence of glucose. Paired wells
containing INS-1 cells were treated with or without heparin (100 µg/ml) for 30 min prior to fixation in Bouin's solution. Fixed cells
were assayed for insulin and LpL localization using antibodies as
described above followed by FITC-conjugated donkey anti-primary
antibodies that allowed fluorescence detection. Visualization of
localization of LpL and insulin in cells was performed with a Sarastro
2000 dual-laser Zeiss Axioskop confocal microscope.
Acute Glucose Exposure Stimulates LpL Activity but Not Total LpL
Mass--
To determine whether glucose acutely stimulates LpL activity
in islets, we first measured total LpL activity in mouse islets. In
preliminary experiments, LpL enzyme activity (defined by salt inhibition and apoCII dependence) was detected in islets and shown to
be linearly dependent on the number of islets assayed (data not shown).
Glucose caused a time-dependent stimulation of mouse islet
total LpL activity (Fig. 1). LpL enzyme
activity was 91% higher in the presence of 20 mM glucose
in comparison to basal glucose (3 mM) following a 4-h
incubation period (p < 0.01). The same effect was seen
at 8 h. Glucose-stimulated insulin secretion from these islets at
1, 4, and 8 h increased over basal secretion by 12-, 20-, and
2.5-fold, respectively (data not shown).
Western blotting (Fig. 2) of mouse islets
(lanes 2 and 3), and the Acute Glucose Exposure Stimulates LpL Translocation to the Plasma
Membrane--
In adipocytes, physiologically relevant LpL is thought
to reside in a cellular compartment at or close to the cell surface. LpL at this site is released to the media by heparin. To determine whether glucose affects the proportion of LpL that is accessible to
heparin, we examined LpL enzyme activity and LpL protein mass from
glucose-stimulated islets and INS-1 cells after treatment with 100 µg/ml heparin. Heparin-releasable LpL activity from mouse islets
(Fig. 3A) and INS-1 cells
(Fig. 3B) was significantly higher after 4-h exposure to 20 mM glucose than with basal glucose treatment. Heparin-releasable LpL protein mass also increased in INS-1 cells treated with 20 mM glucose for 4 h (Fig.
4, lanes 3 versus 5). These data suggest that glucose
acutely promotes the translocation of active LpL to the surface of
Effects of Insulin Secretion and Cellular Metabolism on
Glucose-stimulated LpL Activity--
Because insulin has been shown to
regulate LpL activity and expression in adipocytes (17), we studied the
relationship between glucose-stimulated insulin secretion and LpL
activity in islets and
In 3T3-L1 adipocytes, prolonged exposure to exogenous insulin
(10 LpL and Insulin Reside in Separate Vesicles in Pancreatic
Fatty acids affect islet function, but the origin of islet-associated
fat is poorly understood. Cells can obtain fatty acids through three
mechanisms: de novo lipogenesis, importing circulating free
fatty acids, or hydrolyzing lipoprotein-associated triglycerides through the action of LpL. The first two are unlikely to be utilized by
islets. The first, de novo lipogenesis, is extremely energy inefficient (44). The second, importing free fatty acids from the
plasma, is not a major source of fat for most tissues. The bulk of
circulating free fatty acids are derived from the hydrolysis of
triglycerides in adipose tissue, transported to the liver, converted
back to triglycerides, and secreted in the form of lipoproteins (45).
LpL, by hydrolyzing lipoprotein-associated triglycerides in the
capillaries of peripheral tissues, is probably the major provider of
fatty acids to skeletal muscle, heart, and adipose tissue (46). We
recently provided an initial description of LpL expression in islet
cells and showed that manipulating LpL expression in these cells can
affect insulin secretion (15). These data suggest that In the current study, we demonstrate that Like glucose-stimulated insulin secretion, glucose-stimulated LpL
activation required glucose metabolism. Incubating We also provide visual evidence that LpL and insulin exist in discrete
secretory organelles. Our immunohistochemical studies show that LpL
protein is associated with Exposure of Based on the current results, our working model for how islet LpL may
contribute to -cell
function, and LpL overexpression decreases insulin secretion in cell
lines, but whether LpL is regulated in
-cells is unknown. To test
the hypothesis that glucose and insulin regulate LpL activity in
-cells, we studied pancreatic islets and INS-1 cells. Acute exposure
of
-cells to physiological concentrations of glucose stimulated both
total cellular LpL activity and heparin-releasable LpL activity.
Glucose had no effect on total LpL protein mass but instead promoted
the appearance of LpL protein in a heparin-releasable fraction,
suggesting that glucose stimulates the translocation of LpL from
intracellular to extracellular sites in
-cells. The induction of
heparin-releasable LpL activity was unaffected by treatment with
diazoxide, an inhibitor of insulin exocytosis that does not alter
glucose metabolism but was blocked by conditions that inhibit glucose
metabolism. In vitro hyperinsulinemia had no effect on LpL
activity in the presence of low concentrations of glucose but increased
LpL activity in the presence of 20 mM glucose. Using
dual-laser confocal microscopy, we detected intracellular LpL in
vesicles distinct from those containing insulin. LpL was also detected
at the cell surface and was displaced from this site by heparin in
dispersed islets and INS-1 cells. These results show that glucose
metabolism controls the trafficking of LpL activity in
-cells
independent of insulin secretion. They suggest that hyperglycemia and
hyperinsulinemia associated with insulin resistance may contribute to
progressive
-cell dysfunction by increasing LpL-mediated delivery of
lipid to islets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
,
, and
isoforms) and inhibition of protein kinase C decreases LpL synthesis
(16). Insulin increases adipocyte LpL activity by both
post-transcriptional and post-translational mechanisms (17). This
effect is attenuated by rapamycin, an inhibitor of the mTOR (mammalian
target of rapamycin) signaling pathway (18). In cardiac muscle cells,
the stimulatory effect of insulin on LpL requires dexamethasone and
appears to exert its effects by altering the cytoskeleton (19, 20).
However, insulin does not regulate LpL in murine peritoneal macrophages
and macrophage cell lines (7).
-cells. In this
study, we address the regulation of LpL in
-cells. Our results show
that glucose metabolism controls the intracellular trafficking of
LpL-containing vesicles through mechanisms that function independent of
the exocytosis of insulin. Hyperglycemia-induced translocation of LpL
to the cell surface, a process amplified by hyperinsulinemia,
represents a potential mechanism underlying the progressive
-cell
lipotoxicity associated with insulin resistance.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cells or islet pellets with a detergent solution containing 2.0 g/liter deoxycholate for 30 min at 37 °C. "Total LpL activity"
is the amount of activity after detergent solubilization of cells or
islet pellets that have not been exposed to heparin-treatment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Acute exposure to an elevated glucose
concentration stimulates LpL activity in pancreatic islets. Two
triplicate groups containing 200 pancreatic islets/group were
preincubated in basal (3 mM) glucose for 30 min.
Subsequently, one triplicate group was maintained under basal glucose
and one triplicate group was exposed to elevated (20 mM)
glucose for the indicated time periods. Following the incubation
periods, the islets were dissolved in detergent extract buffer and the
extract was assayed for LpL activity as previously described. Identical
results were seen in three independent experiments. Data are expressed
as mean ± S.E. *, p < 0.05 versus
1 h.
-cell lines INS-1
(lanes 4 and 5), and RINm5F (lanes 6 and 7) showed that these cell types produce an LpL protein
with the same molecular weight as LpL from differentiated 3T3-L1
adipocytes (lane 1). However, total LpL mass was not
increased in any of these cell types after incubation in 20 mM glucose for 4 h (compare lanes 2 versus 3, 4 versus
5, and 6 versus 7). These
results suggest that post-translational mechanisms contribute to the
acute induction of LpL enzyme activity in
-cells.
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Fig. 2.
Acute exposure to an elevated glucose
concentration does not increase total LpL mass in pancreatic islets
or -cell lines. Triplicate groups/wells
containing 200 pancreatic islets (lanes 2 and 3)
or 3 × 106 cells (INS-1, lanes 4 and
5, or RIN-m5F, lanes 6 and 7)/well
were incubated in basal (3 mM for islets; none for INS-1 or
RIN-m5F cells, even-numbered lanes) or elevated (20 mM, odd-numbered lanes) glucose for 4 h.
Islets or cells were dissolved in SDS extraction buffer, the pooled
extract was separated by 10% SDS-polyacrylamide gel electrophoresis,
then transferred to nitrocellulose. Western blotting was performed
using a chicken anti-LPL-specific antibody and an horseradish
peroxidase-conjugated donkey anti-chicken antibody followed by
detection with chemiluminescence. Data shown are representative of
three experiments.
-cells.
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Fig. 3.
Acute exposure to elevated glucose
concentration increases heparin-releasable LpL activity in islets (A)
and INS-1 cells (B). Triplicate groups/wells of islets or 3 × 106 INS-1 cells/well were incubated in basal (3 mM for islets; none for INS-1 cells) or elevated (20 mM) glucose for 4 h. Following the incubation
periods, the islets or cells were exposed to 100 µg/ml low molecular
weight heparin for 30 or 45 min. The culture supernatant was collected,
the remaining islets or cells were dissolved in detergent extract
buffer, and the heparin supernatant and extract were assayed for LpL
activity. The same results were seen in more than three experiments.
Data are presented as mean ± S.E. *, p < 0.01 versus basal.
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Fig. 4.
Acute exposure to elevated glucose
concentration increases heparin-releasable LpL mass in INS-1
cells. Triplicate wells containing 3 × 106 INS-1
cells/well were incubated in the absence (lanes 5 and
6) or with elevated (20 mM, lanes 3 and 4) glucose for 4 h. Following the incubation
periods, the cells were incubated for 45 min with 100 µg/ml low
molecular weight heparin. The culture supernatants were collected,
pooled, and concentrated using heparin-treated Amicon filters 10,000 nominal molecular weight limit (hep.). The remaining
cells were dissolved in SDS extraction buffer (det.).
The heparin supernatant and cell extract were then subjected to Western
blot detection for LpL. The same results were seen in two independent
experiments.
-cells. Diazoxide, an agent known to
hyperpolarize
-cells and inhibit insulin secretion, blocked
glucose-stimulated insulin secretion (Fig.
5A) but did not affect
glucose-stimulated LpL activity (Fig. 5B) or glucose
metabolism (data not shown). Inhibition of glucose metabolism by
incubation at 25 °C or in the presence of mannoheptulose (10 mM) prevented the stimulation of LpL activity by glucose
(Fig. 5B) as well as glucose-stimulated insulin secretion (Fig. 5A). Incubation at 25 °C or in the presence of
mannoheptulose decreased glucose metabolism by 50% and 100%,
respectively (data not shown).
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Fig. 5.
Effect of room temperature incubation,
mannoheptulose, or diazoxide on glucose-stimulated insulin secretion
(A) and LpL activity (B) in
islets. Mouse islets were incubated in basal (3 mM) or
elevated (20 mM) glucose for 4 h in the presence or
absence of 10 mM mannoheptulose, 250 µM
diazoxide, or at room temperature. Following the incubation periods,
the islets were exposed to 100 µg/ml low molecular weight heparin for
30 min, then the culture supernatant was collected and assayed for
insulin content (A) and heparin-releasable LpL activity
(B). Cells were also assayed for cellular metabolism using
MTS and measuring A540 for each well.
Identical results were seen in three experiments. Data are presented as
mean ± S.E. *, p < 0.01 versus 20 mM glucose. These data were generated using islets. The
same results were obtained using INS-1 cells (not shown).
12 to 10
6 M) increases
heparin-releasable LpL activity (17). Treatment of INS-1 cells with
0.25-1.0 µM exogenous insulin caused a
dose-dependent increase in heparin-releasable LpL activity
above that produced by 20 mM glucose alone (Fig.
6). This effect required elevated glucose
concentrations, because exogenous insulin at 1.0 µM did not stimulate LpL activity at low glucose concentrations (1 mM).
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Fig. 6.
Long-term exposure to exogenous insulin
stimulates LpL activity in INS-1 cells. Triplicate wells
containing 3 × 106 INS-1 cells/well were incubated in
basal (1 mM) or elevated (20 mM) glucose for
24 h in the presence or absence of 1 µM insulin.
Following the incubation period, the cells were exposed to 100 µg/ml
low molecular weight heparin for 45 min. The culture supernatant was
collected, the remaining cells were dissolved in detergent extract
buffer, and the heparin supernatant and extract were assayed for LpL
activity. The same results were seen in more than three independent
experiments. Data are presented as mean ± S.E. **,
p < 0.05; *, p < 0.01 versus 20 mM glucose.
-Cells--
Diazoxide treatment blocks glucose-stimulated insulin
secretion but not glucose-stimulated induction of LpL activity,
implying that LpL and insulin reside in separate and distinct vesicles. To address this issue directly, we used fluorescent
immunohistochemistry to localize these proteins in
-cells.
Dual-laser confocal fluorescent microscopic images of islets (Fig.
7) showed that LpL (red) and insulin (green) reside in separate vesicles within
insulin-expressing
-cells. Membrane-associated LpL present on
-cells (Fig. 7A, white arrows) was not observed in
heparin-treated cells (Fig. 7B). Similar results were seen
in INS-1 cells (Fig. 8). Serial confocal
microscopy sections showed that both control (panels A-C)
and heparin-treated (panels D-F) INS-1 cells contained
insulin (green) and LpL (red) residing in
separate vesicles. However, INS-1 cells were polarized with respect to
LpL expression with the heparin-releasable fraction localized to the
apical region. Panels A and D of Fig. 8 represent
the apical region of cells, panels B and E
intermediate levels, and panels C and F basal
regions. Apical-associated LpL (indicated by white arrows in
A) was not observed after heparin-treatment (D).
For both dispersed islet cells and INS-1 cells, heparin displacement of
membrane-associated LpL was seen in the majority of cells.
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Fig. 7.
LpL is located in insulin-expressing cells in
pancreatic islets. Dispersed islet cells were isolated and
cultured for 24 h. Control, untreated (A) or
heparin-treated (B) dispersed islet cells were centrifuged
onto glass slides and fixed with Bouin's solution. Insulin (FITC,
green) and LpL (Cy3, red) expression were
detected using fluorescence immunohistochemistry observed using a
dual-laser confocal microscope. The acquired images demonstrate
separate and distinct areas of localization for insulin and LpL.
Heparin treatment (B) results in the removal of
membrane-associated LpL (white arrows, A). The
same results were seen using two independent islet cell preparations,
and the images shown are representative of the appearance of cells on
multiple different slides. In other experiments (not shown), LpL
protein was also shown to be associated with -cells in intact
islets.
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Fig. 8.
LpL is located in INS-1 cells. Control,
untreated (A-C) and heparin-treated (D-F)
adherent, cultured INS-1 cells were fixed with Bouin's solution.
Insulin (FITC, green) and LpL (Cy3, red)
expression were detected using fluorescence immunohistochemistry
observed using a dual-laser confocal microscope. Serial section images
(0.5 µm in thickness and separated by 1.5 µm) demonstrate separate
and distinct areas of localization for insulin and LpL. Heparin
treatment (D) results in the removal of apical
membrane-associated LpL from adherent, cultured INS-1 cells
(white arrows, A). Images shown are
representative of the appearance of cells on multiple different
slides.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Cell lipid metabolism is critical for the normal regulation of
insulin secretion and may be involved in the obesity-related
-cell
failure that leads to type 2 diabetes. Fatty acids are required for
glucose-stimulated insulin secretion in some systems (31). In islets
and rodents, short term exposure to elevated fatty acid concentrations
enhances glucose-stimulated insulin secretion (32-34). Long term
exposure decreases glucose-stimulated insulin secretion (35, 36). Basal
insulin secretion is stimulated by chronic exposure of islets to fatty
acids (37-39).
-Cell failure in Zucker diabetic fatty rats is
preceded by accumulation of islet triglyceride (40), an effect that can
be reversed by therapies associated with a decrease in islet
triglycerides (41). Cytosolic acyl-CoA may serve as an effector
molecule in the control of glucose-stimulated insulin secretion,
perhaps through the activation of protein kinase C isoforms (42). Fatty
acids stimulate islet ceramide synthesis, a process that may lead to
apoptosis in islets (43).
-cells, like
parenchymal cells in other tissues, derive physiologically relevant
stores of fatty acids through the action of LpL.
-cells from pancreatic
islets and
-cell lines express functional LpL activity with
properties similar to that described in adipocytes. LpL activity was
inhibited by salt, dependent on a source of apolipoprotein C-II for
activity, and heparin-releasable. LpL in detergent extracts and a
heparin-releasable fraction from mouse islets and two
-cell lines,
INS-1 and RINm5F, had the same apparent molecular weight as LpL from
differentiated 3T3-L1 adipocytes. Like adipocytes,
-cells showed an
increase in LpL enzyme activity after exposure to glucose. Total and
heparin-releasable LpL activities of islets were increased 2-fold in
the presence of elevated glucose (20 mM) compared with
basal glucose levels. Glucose treatment had no effect on total LpL
protein content but caused an increase in the heparin-releasable
fraction of LpL protein. These results imply that glucose acutely
induces the translocation of active LpL from intracellular stores to
the cell surface of islets.
-cells either in
the presence of mannoheptulose or at room temperature, interventions
that decrease glucose metabolism, prevented the induction of LpL
activity by glucose. Glucose metabolism appears to be tightly linked to
the regulation of LpL activity, because the 50% reduction in glucose
metabolism associated with incubation at room temperature completely
inhibited LpL activity. Unlike glucose-stimulated insulin secretion,
glucose-stimulated LpL activation was unaffected by treatment with
diazoxide, an agent that opens islet KATP channels and
prevents insulin secretion. These results provide physiological
evidence that functional LpL is not located in insulin secretory granules.
-cells in intact islets as well as in
dispersed primary
-cells and INS-1 cells. However, intracellular LpL
is localized to secretory granules that are distinct from those
containing insulin. Unlike insulin, LpL is also found at the cell
surface where it can be released by treatment with heparin.
-cells to elevated levels of exogenous insulin for
24 h stimulated heparin-releasable LpL activity by ~25% above that of glucose. This effect of insulin required the presence of
elevated concentrations of glucose, because insulin had no effect at
basal glucose concentrations. In 3T3-L1 adipocytes, insulin increases
heparin-releasable LpL activity without affecting LpL transcription,
mRNA levels, or protein synthetic rate, consistent with
post-translational activation of LpL (17). The current data suggest
that hyperinsulinemia may have similar effects on
-cell LpL activity
in the setting of hyperglycemia.
-cell dysfunction is shown in Fig.
9. Under basal conditions (left
panel), monomeric islet LpL dimerizes and translocates to the
capillary endothelium. At this site, LpL acts on circulating
lipoproteins to yield free fatty acids. These are transported to the
-cell by poorly understood mechanisms that may involve fatty acid
transporters (47). Intracellular fatty acids are then re-esterified to
yield triglyceride, the neutral lipid that accumulates in the
-cells
of diabetic animals. Fatty acids can also be released from
intracellular triglyceride stores through the activity of
hormone-sensitive lipase, known to be expressed and active in
-cells
(48). In the setting of hyperglycemia and hyperinsulinemia associated
with peripheral insulin resistance (right panel),
translocation of active LpL to the capillary endothelium is increased,
and more triglyceride-derived fatty acids are delivered to the
-cell. Because fatty acids and glucose compete as respiratory
substrates in many tissues (49), chronically increasing
-cell fatty
acids would decrease glucose metabolism leading to decreased insulin
secretion. Increasing fatty acids may also directly impair
-cell
function and promote apoptosis (40, 50-51). Consistent with
this notion of chronic lipotoxicity, islets from mice with LpL
deficiency secrete more insulin than islets from animals with normal
LpL activity. These LpL-deficient mice have higher fasting insulin
levels than wild-type mice in the setting of lower blood sugars and no
evidence of abnormal insulin responsiveness (15).
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Fig. 9.
Working model for LpL-mediated delivery
of fatty acids to -cells. LpL molecules
are represented by symbols within a circular secretory
granule in a
-cell. The depiction of monomers inside the cell
and dimers at the capillary endothelium is an oversimplification.
In summary, -cell LpL is regulated by glucose and insulin. The
promotion of LpL activity requires glucose metabolism and involves
altered trafficking of secretory granules that are distinct from those
containing insulin. This demonstration of a regulated, physiologically
relevant pathway for delivering lipid to
-cells provides a framework
for clarifying the mechanisms underlying the insulin secretory failure
that characterizes type 2 diabetes.
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ACKNOWLEDGEMENTS |
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We thank Richard Hresko for supplying the 3T3-L1 adipocytes and Joan Fink for technical assistance.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grants DK06181, HL58427, DK53198, and T32 DK07296, by Washington University Clinical Nutrition Research Unit Grant DK56341, by Washington University Diabetes Research and Training Center Grant DK20579, and by an American Diabetes Association Mentor-based Fellowship.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Clay F. Semenkovich, Division of Atherosclerosis, Nutrition, and Lipid Research, Washington University School of Medicine, Campus Box 8046, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-4454; Fax: 314-747-4477; E-mail: semenkov@im.wustl.edu.
Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010707200
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ABBREVIATIONS |
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The abbreviations used are: LpL, lipoprotein lipase; FITC, fluorescein isothiocyanate; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inert salt reagent; CMRL, CMRL-1066 tissue culture medium.
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