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INTRODUCTION |
Lipoprotein lipase
(LPL)1 is a central enzyme in
lipid metabolism that is expressed primarily in adipose tissue and
muscle (1). The regulation of lipoprotein lipase is complex and
regulation may occur at the transcriptional, translational, and
posttranslational levels (2). LPL activity is decreased in the adipose
tissue of patients with diabetes. After treatment to control
hyperglycemia in both type I and type II diabetes, there is an increase
in LPL activity (3, 4), along with an increase in LPL synthetic rate
with no change in LPL mRNA, suggesting regulation at the level of
translation (5). Similar observations have been made in experimental
models of insulin-deficient diabetic rats. Whereas short term insulin
treatment of insulin-deficient rats increased LPL protein more than LPL
mRNA, prolonged insulin treatment increased both LPL protein and
LPL mRNA levels (6).
Glucose and insulin modulate protein kinase C (PKC) activity in rat
adipocytes (7). Hyperglycemia is known to increase cellular
diacylglycerol (DAG), which in turn is the natural activator of PKC.
Elevated DAG, resulting in PKC activation has been identified in
insulin-deficient diabetic adipose tissue (8-10). The role of
hyperinsulinemia and hyperglycemia in the activation of PKC isoforms
and involvement in insulin resistance has been studied in various
animal models of diabetes (11). PKC is present in the soluble
cytoplasmic fraction in cells prior to stimulation, and the treatment
of cells with the phorbol ester 12-O-tetradecanoyl phorbol
13-acetate (TPA) resulted in activation and translocation of PKC to the
membrane. TPA activates PKC by interacting with the DAG binding site
(12), although the degree of stimulation of PKC by TPA is much greater
than the induction by DAG. However, prolonged treatment with phorbol
ester can down-regulate PKC activity and PKC protein by depleting
cellular PKC protein (13, 14).
The eleven related isoforms of PKC have been described and can be
classified into three subgroups, depending on the requirement for
diacylglycerol, phospholipid, and calcium for activation. These
isoenzymes are characterized by differences in their four domains, the
regulatory domains, C1 and C2, and the catalytic domains, C3 and C4
(15). Each PKC isoform has a distinct tissue distribution and
physiological function. Recent studies indicate that PKC isoforms
and
are activated preferentially in the vasculature of diabetic
animals. However, other PKC isoforms are also increased in the
glomeruli and retinal tissues isolated from diabetic animals and in
cells cultured in the presence of high glucose (16, 17).
In this study, the effect of PKC activation and depletion by TPA on LPL
activity was examined in adipocytes. Although stimulation of PKC did
not increase LPL, depletion or inhibition of PKC resulted in a decrease
in LPL translation. These data suggest that PKC plays a vital role in
the expression of LPL activity in adipocytes.
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MATERIALS AND METHODS |
Cell Culture and Differentiation--
3T3-F442A cells were
obtained from Dr. Howard Green (Harvard Medical School, Boston, MA).
The cells were maintained in Dulbecco's modified Eagle's medium,
supplemented with 10% calf serum (Life Technologies, Inc.). For
experiments, the cells were grown to confluence in 12-well cluster
plates, and differentiated by incubation in medium with 10% fetal calf
serum containing 100 nM insulin for 5-7 days.
Differentiated adipocytes were treated with the indicated concentration
of TPA dissolved in 1 µl of Me2SO in 1 ml of culture medium. Control cells received the same amount of Me2SO.
PKC inhibitors staurosporine and calphostin C were purchased from
Calbiochem. Staurosporine and calphostin C stock solutions were made in
Me2SO at 1,000× concentration. Calphostin C was activated
after addition to the culture medium by exposure to light for 10 min as
recommended by the manufacturer.
Measurement of LPL Activity--
Heparin releasable and
extractable LPL were prepared as described previously (18). To measure
heparin releasable LPL, the medium was aspirated, and cells were
treated with 10 units/ml of heparin in Dulbecco's modified Eagle's
medium for 45 min at 37 °C. After collecting this fraction, the cell
layer was extracted in a detergent-containing buffer (19). LPL
catalytic activity was measured as described previously (20). Samples
were incubated with emulsified substrate containing
[3H]triolein and human serum as a source of apo C-II for
45 min at 37°. LPL activity has been expressed as nanomoles of free
fatty acid (FFA) released/hr/mg of cell protein.
LPL Synthetic Rate--
The synthetic rate of LPL was measured
in control and TPA-treated adipocytes by pulse labeling the cells for
the specified time intervals with [35S]methionine (100 µCi/ml). The unincorporated label was aspirated, and the total
cellular proteins were extracted in lysis buffer containing 50 mM phosphate buffer, pH 7.4, 2% deoxycholate, 1% SDS, 20 mM phenylmethylsulfonyl fluoride, 2 mM
leupeptin, and 2 mM EDTA. Labeled LPL was
immunoprecipitated using LPL specific polyclonal antibodies described
earlier (21). Immunoprecipitated samples were analyzed on 10% SDS-PAGE
followed by autoradiography as described earlier (22).
RNA Extraction and Northern Blotting--
RNA was extracted from
adipocytes using the method of Chomczynski and Sacchi (23). Equal
amounts of total RNA from the various treatment groups were analyzed
using 2.2 M formaldehyde, 1% agarose gels. Northern blots
were probed with the 32P-labeled hLPL cDNA (24) and
glyceraldehyde-3-phosphate dehydrogenase cDNA probe as described by
us previously (22, 25). Previous studies involving murine LPL,
including 3T3-F442A adipocytes have yielded two RNA species at 3.2 and
3.6 kilobases because of the two alternative sites of polyadenylation
(26, 27).
Western Blot Analysis--
The cell layer was rinsed in ice-cold
phosphate buffered saline and total protein was extracted using the
cell lysis buffer described above. The lysates were centrifuged at
1,500 × g for 15 min to separate the insoluble debris.
Proteins were fractionated using 10% SDS-PAGE. Samples containing 15 µg of total protein were electrophoresed for Western blot analysis
and transferred onto nitrocellulose membranes using 300 mA current for
2-3 h. Membranes were treated with 20 mM Tris/HCl, pH 7.6, 150 mM NaCl, 0.2% Tween 20, and 5% nonfat dry milk
overnight at 4° C.
Detection of LPL Protein--
LPL polyclonal antibody (rabbit)
was a generous gift from Dr. Ira Goldberg (Columbia University, New
York, NY). Anti-rabbit horseradish peroxidase conjugate was obtained
from Sigma Chemical Company. The primary antibody was used at 1:2,000
dilution and the secondary at 1:5,000. Membranes were washed
extensively with Tris buffer containing 0.1% Tween 20 to remove the
excess of primary or secondary antibody. The LPL protein was detected
using chemoluminescence reagents (Amersham Pharmacia Biotech).
Detection of PKC Isoforms--
The isoform specific primary and
secondary antibodies were purchased from Transduction Laboratories,
Lexington, KY. The primary antibodies were used at 1:1,000 dilution and
the secondary antibody at 1:5,000 dilution. Membranes were washed, and
the PKC protein bands were detected using chemoluminescence.
Data Analysis--
All experiments were performed in triplicate
with triplicate wells in each, and data are expressed as mean ± S.E.
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RESULTS |
Effect of TPA Treatment on LPL Activity--
3T3-F442A adipocytes
were treated with 100 nM TPA for varying time intervals. As
shown in Fig. 1, heparin releasable LPL
activity was inhibited by TPA treatment. Although there was no
significant decrease in activity during the first 60 min of treatment
with 100 nM TPA, LPL activity began to diminish after
3 h of treatment and was inhibited by 80% after 6 h of
treatment with TPA (control, 1.06 ± 0.1; TPA treated, 0.158 ± 0.025 µmol of FFA/hr/mg of protein). Overnight treatment with TPA
had no additional inhibitory effect on LPL activity. More than 75% of
the total LPL activity was found in the heparin releasable fraction of
3T3-F442A adipocytes. Total extractable LPL activity was also inhibited
82% in TPA-treated cells (control, 1.6 ± 0.1; TPA treated,
0.280 ± 0.08 µmol of FFA/hr/mg of protein). In addition to
studying 3T3-F442A adipocytes, primary cultures of rat adipocytes were
examined, and TPA inhibited heparin releasable LPL activity, but to a
lesser degree than in 3T3-F442A cells. After overnight treatment of rat
adipocytes with 100 nM TPA, control and TPA-treated LPL
activity was 44.6 ± 5.1 and 31.1 ± 2.9 nmol of
FFA/hr/106 cells, respectively.

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Fig. 1.
Effect of TPA on LPL activity in 3T3-F442A
adipocytes. A, cells were grown to confluence in
12-well culture dishes and differentiated for 5-6 days. They were
treated with 100 nM TPA for the specified time intervals.
Heparin releasable LPL activity was measured as described under
"Materials and Methods." LPL activity in control adipocytes was
1.06 ± 0.1 µmol of FFA/hr/mg of protein. B, dose
response to TPA on LPL activity in 3T3-F442A adipocytes. Differentiated
cells were treated with varying doses of TPA ranging from 3 to 200 nM for 6 h, and heparin releasable LPL activity was
measured. LPL activity in control adipocytes was 0.63 ± 0.1 µmol of FFA/hr/mg of protein. All data represent the results of three
experiments performed in quadruplicate.
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To study the dose response of TPA, cells were treated with increasing
concentrations of TPA. As indicated in Fig. 1B the
inhibition of LPL activity was dose-dependent and 6 nM TPA inhibited LPL activity by 30%. The inhibition
increased with increasing concentration of TPA and was maximal at 100 nM TPA.
To determine whether the decrease in LPL activity in response to TPA
treatment resulted from a decrease in LPL mRNA levels, cells were
treated with 100 nM TPA for varying time intervals, followed by RNA extraction and Northern blot analysis. The addition of
TPA had no inhibitory effect on LPL mRNA levels as shown in Fig.
2. LPL mRNA remained the same at all
the time points studied. The same blot was also probed for
glyceraldehyde-3-phosphate dehydrogenase mRNA as a control for
equal loading of RNA. The expression of glyceraldehyde-3-phosphate
dehydrogenase mRNA did not change with TPA treatment.

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Fig. 2.
Effect of TPA treatment on LPL mRNA in
3T3-F442A adipocytes. The cells were treated with 100 nM TPA for the specified time interval. Total RNA was
extracted and analyzed using Northern blot analysis. Densitometric
analysis of the autoradiograms indicates no change in the intensity of
LPL and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
bands. The data shown represent one of two experiments.
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Because TPA treatment did not decrease LPL mRNA, the decrease in
LPL activity following TPA treatment could result from either a
decrease in synthetic rate of LPL or a change in LPL posttranslational processing. To determine whether TPA treatment decreased LPL synthetic rate, control and TPA-treated adipocytes were pulse labeled with [35S]methionine for 30 min, and LPL was
immunoprecipitated. As shown in Fig.
3A, LPL synthetic rate was
decreased by 75-80% with 6 h of TPA treatment as compared with
the untreated control. To assess total LPL immunoreactive mass, total
cell lysates from control, and TPA-treated cells were analyzed for LPL
protein by Western blot analysis. As shown in Fig. 3B, LPL
mass was decreased in both 6- and 16-h TPA-treated lysates.

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Fig. 3.
Effect of TPA on LPL synthesis in 3T3-F442A
adipocytes. A, the cells were treated with TPA (100 nM) for 6 h and pulse labeled with
[35S]methionine. Equal trichloroacetic acid precipitable
counts were immunoprecipitated using anti-LPL antibodies and analyzed
on 10% SDS-PAGE. The data shown represents one of three experiments.
B, Western blots were performed after TPA treatment on total
cell lysates. The data shown represent one of three experiments.
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The decreased [35S]methionine incorporation described
above could have been due to a decrease in LPL translation, or to an
increase in LPL degradation. To determine whether there were any
effects of LPL degradation, we performed [35S]methionine
pulse labeling for 10, 20, 30, and 40 min, followed by
immunoprecipitation of LPL. As shown in Fig.
4, [35S]methionine
incorporation into LPL was linear in both control and TPA-treated
cells, and there was a 50-60% decrease in LPL synthesis in the
TPA-treated cells at all time intervals. Thus, the decrease in LPL
synthesis in the TPA-treated cells at the shorter (10 and 20 min) pulse
labeling times, and the linear incorporation into both control and
TPA-treated cells, indicate that the decrease in LPL activity resulted
from a decrease in LPL translation, and not from an increase in LPL
degradation.

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Fig. 4.
Effect of TPA on LPL synthetic rate in
3T3-F442A adipocytes. The cells were treated with TPA (100 nM) for 16 h and pulse labeled with
[35S]methionine for the indicated time intervals. Equal
TCA precipitable counts were immunoprecipitated using anti-LPL
antibodies and analyzed on 10% SDS-PAGE. A, autoradiograph
of the immunoprecipitated LPL protein; B, densitometric
analysis of the image.
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To study the effect of prolonged TPA treatment on the expression of PKC
isoforms, 3T3-F442A adipocytes were treated with TPA, and total cell
lysates were analyzed using Western blots. As shown in Fig.
5, TPA treatment decreased the expression
of PKC
,
,
, and
within 6 h of treatment, and
overnight treatment resulted in a further decrease, such that isoforms
,
, and
were undetectable. PKC isoforms µ,
, and
remained unchanged with TPA treatment. Isoforms
and
were not
detectable in 3T3-F442A adipocytes.

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Fig. 5.
Depletion of PKC isoforms in response to
prolonged TPA treatment. Cells were treated with 100 nM TPA for 6 or 16 h. 20 µg of total cell protein
was analyzed using Western blot analysis for the presence of various
PKC isoforms using specific antibodies (see "Materials and
Methods").
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To examine earlier times after addition of TPA, 3T3-F442A adipocytes
were treated with 100 nM TPA, and total cell lysates were
analyzed using Western blots. As shown in Fig.
6, there were small decreases in all PKC
isoforms at 2 h and much greater decreases at 4 and 6 h of
TPA treatment. In addition, we observed an increase in the expression
of isoforms
and
after 30 min of TPA treatment (data not shown).
As described in Fig. 1, there was no significant change in LPL activity
until 2.5-3 h after TPA treatment. Thus, the decrease in PKC isoforms,
as demonstrated by Western blotting, occurred at the same time as the
decrease in LPL activity.

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Fig. 6.
Depletion of PKC isoforms in response to TPA
treatment. Cells were treated with 100 nM TPA for 2, 4, 6, and 16 h. 20 µg of total cell protein were analyzed using
Western blotting for the presence of PKC isoforms using specific
antibodies.
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Because prolonged TPA treatment of cells is known to deplete PKC (28),
we examined the effect of directly inhibiting PKC activity using PKC
inhibitors. The addition of staurosporine (1 µM)
inhibited LPL activity by 50%, compared with control cells. In
contrast to TPA, which required several hours to inhibit LPL, the
inhibitory effect of staurosporine was observed within 60 min of
addition (data not shown). To further demonstrate the effect of PKC
inhibition on LPL, calphostin C, which is a more specific inhibitor of
PKC, was added to 3T3-F442A adipocytes. As shown in Fig.
7A, LPL activity was inhibited
by calphostin C, and this inhibition occurred quickly. Following the
addition of 1 µM calphostin C, LPL activity was inhibited
by 30% in 30 min and 75% in 2 h. Inhibition of LPL activity by
calphostin C increased with increasing concentrations with maximal
inhibition of LPL by 2 µM calphostin C (Fig.
7B).

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Fig. 7.
Effect of PKC inhibitors on LPL
activity. A, the cells were treated with increasing
concentrations of calphostin C for 4 h and heparin releasable LPL
activity was measured. Control LPL activity was 3.15 ± 0.23 µmol of FFA/hr/mg of protein. B, the cells were treated
with 1 µM calphostin C for varying time intervals, and
heparin releasable LPL activity was measured as described under
"Materials and Methods." Control LPL activity was 2.57 ± 0.17 µmol of FFA/hr/mg of protein. All experiments were performed in
triplicate and expressed as the mean ± S.E.
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DISCUSSION |
LPL hydrolyzes the triglyceride core of lipoproteins and is
subject to regulation by a number of different hormones (2). The
physiologic regulators of LPL include catecholamines, which inhibit
adipose lipid accumulation during periods of active lipolysis (e.g. fasting), and insulin, which stimulates LPL in concert
with an inhibition of lipolysis (e.g. after a meal). A
number of studies have demonstrated that the regulation of LPL is
complex. Under some conditions, levels of LPL mRNA are regulated,
whereas other times there is regulation of LPL translation, or
posttranslational processing (2). Regulation of LPL translation has
been demonstrated in response to several conditions, including glucose
(29), thyroid hormone (30, 31), epinephrine (32-34), and in response
to improved diabetes control (5, 6). In all of these instances, there was no change in LPL mRNA expression, but the synthetic rate of LPL
was altered, resulting in decreased LPL activity. In this study we
report that expression of LPL activity in adipocytes is decreased by
depletion of PKC, and that this inhibition of LPL takes place at the
level of translation.
To determine whether PKC was involved in the regulation of LPL
activity in adipocytes, we studied the effect of TPA, which can mimic
in part the effect of DAG, the natural ligand for PKC (35). The
activation of PKC is an immediate response to TPA, and occurs within 30 min (36, 37). We observed that TPA treatment caused no significant
change in LPL activity during this time interval. The inhibitory effect
of TPA on LPL activity, however, was evident after longer treatment
with TPA. Long term treatment with TPA is known to decrease PKC in
several cell types (38). Phorbol ester-induced down-regulation of PKC
has been documented in several studies and is mainly due to increased
rate of degradation (39, 40). For example, Shea et al. (41)
demonstrated that with prolonged TPA treatment of human neuroblastoma
cells, the rate of degradation of PKC-
is faster than the rate of
synthesis, which resulted in the depletion of PKC protein. To
demonstrate that the inhibitory effects of TPA on LPL were caused by
PKC depletion, we directly inhibited PKC with specific inhibitors. This
resulted in inhibition of LPL activity. We also demonstrated the
depletion of several PKC isoforms by Western blotting. Thus, depletion
of PKC or direct inhibition using PKC inhibitors inhibited LPL
activity, indicating that physiological levels of active PKC play a
vital role in the regulation of LPL activity. Because stimulation of PKC did not stimulate LPL, our data suggest that PKC must play a
constitutive role to maintain LPL synthesis.
In our previous studies (32, 33), we have demonstrated that LPL
translation is regulated by an RNA-binding protein that is stimulated
by catecholamines, and binds to the 3'-untranslated region of the LPL
mRNA. Other examples of regulatory RNA-binding proteins have
involved phosphoproteins (42), and thus it is possible that PKC may
alter LPL translation through the phosphorylation of a protein
intermediate. Phorbol esters trigger phosphorylation and activation of
RAF1, which is a 75-kDa phosphoprotein with intrinsic kinase activity
and is an important physiological substrate for PKC
(43). Activated
RAF functions as a kinase kinase and triggers signaling proteins
including cytosolic enzymes like S6 kinase.
The role of PKC in the phosphorylation and regulation of neuromodulin
and neurogranin, and other nuclear RNA-binding proteins which regulate
translational initiation, splicing and ribosomal assembly has been
described recently (44). PKC phosphorylation of these proteins inhibits
their binding to their target RNA in vitro. It is possible
that PKC may play a similar role in the regulation of LPL activity. PKC
could be involved in the phosphorylation of regulatory proteins that
are involved in the translation of LPL. Depletion of PKC may
dephosphorylate the binding protein and inhibit LPL activity.
PKC plays a vital role in other aspects of gene expression and insulin
signaling in 3T3-L1 adipocytes (45). Depletion of PKC by prolonged
treatment of adipocytes with TPA resulted in an activation of
GTPase-activating protein and inhibition p21ras GTP loading
(substitution of GTP for GDP on p21ras), which results in an
alteration of insulin action. Another mechanism by which PKC alters
insulin action may involve serine phosphorylation on the C-terminal
domain of the insulin receptor, which results in a decrease in insulin
receptor tyrosine kinase activity, and a decrease in insulin signaling
(46).
In this study, we demonstrated that PKC depletion results in a decrease
in LPL translation. PKC is an important regulatory protein involved in
catalyzing specific substrate phosphorylation in eukaryotic cells (35),
and may be relevant to the changes in LPL that occur with diabetes. LPL
activity is decreased in the adipose tissue of patients and animals
with both insulin-deficient and insulin-resistant diabetes, and
improved diabetes control increased LPL activity (4-6, 47). Elevated
blood glucose levels stimulates the production of DAG in many cells,
including adipocytes (36, 48). One would expect that glucose-mediated
increases in DAG would activate PKC, and hence maintain LPL expression. Perhaps such is the case under normal fasting/feeding conditions where
elevations in blood glucose are modest and transient. With diabetes,
however, where blood glucose is chronically elevated, it is possible
that the elevated DAG, or perhaps some related mechanism, results in a
down-regulation of PKC and hence an inhibition of LPL expression.
In summary, we have demonstrated that the depletion of PKC in
adipocytes resulted in a decrease in LPL translation, although the
stimulation of PKC did not affect LPL. These data suggest that PKC is
necessary for the normal constitutive expression of LPL and may provide
an important link to the signal transduction events that regulate this
important enzyme.