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Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Medical School, Houston, Texas 77225
Address all correspondence and requests for reprints to: Victoria P. Knutson, Ph.D., Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Health Science Center, Medical School, 6431 Fannin, Houston, Texas 77225. E-mail: vknutson{at}farmr1.med.uth.tmc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As might be expected for an enzyme that is rate limiting for serum triglyceride hydrolysis, the physiological impact of LPL deficiency or overexpression is dramatic. Familial LPL deficiency is an autosomal recessive disorder characterized by fasting hypertriglyceridemia, accumulation of chylomicrons due to their delayed clearance, and elevated cholesterol levels (4, 5). Patients with this deficiency present in childhood with severe abdominal pain, pancreatitis, and failure to thrive. The lipid and lipoprotein profile of these patients, especially their total cholesterol/HDL ratio, predicts an increased risk for coronary artery disease (4). The targeted disruption of the LPL gene in mice leads to severe hypertriglyceridemia, with death occurring in neonates (6). Muscle-specific (6) or liver-specific (7) replacement of LPL in the knockout animals partially normalized their lipid profiles, but physiological consequences were still apparent. Overexpression of LPL in obese rodents prevented diet-induced obesity (8). Thus, the regulation of LPL expression is critically important for normal lipid-lipoprotein homeostasis.
The cell biology of LPL is complex and unique. Although the enzyme is catalytically active and physiologically relevant while tethered to the glycocalyx on the luminal membrane of endothelial cells, the enzyme is synthesized in the underlying parenchymal cells, especially adipose and muscle cells (9, 10). No messenger RNA for LPL has been detected in endothelial cells (10). Therefore, LPL must be synthesized in and released from the adipocyte/myocyte, move across the interstitial fluid, associate with the basal surface of the endothelial cell, and subsequently move across the endothelial monolayer for ultimate expression on the luminal surface of the endothelial cell. The regulation of these processes is incompletely understood.
LPL synthesis in adipocytes has been shown to be regulated by insulin (11, 12, 13), but with the complex intra- and intercellular itinerary of LPL, there is the potential for the insulin-mediated regulation of this important enzyme at multiple steps of its routing from the adipocyte to the luminal domain of the endothelial cell. The intent of this study was to focus on early steps in the intercellular routing of LPL and determine whether cultured endothelial cells modulate the release of LPL from cultured 3T3-L1 adipocytes in an insulin-regulated manner. The results demonstrate that endothelial cells isolated from adipose tissue are highly insulin responsive, and insulin stimulation of the endothelial cells results in the release from the endothelial cells of a factor that mobilizes LPL from the adipocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human adipose endothelial (HAE) cells were isolated from abdominal adipose tissue removed by liposuction, as previously described (16, 17). The endothelial cells were further purified from contaminating fibroblasts by incubation in medium 199 formulated with D-valine instead of L-valine (Sigma), as we have previously described for the isolated of bovine aorta endothelial cells (18). For optimal growth, the HAE cells were seeded onto 100-mm plastic dishes (Falcon, Becton Dickinson and Co.) that had been precoated overnight at 37 C with 2.5 ml 5 µg/ml fibronectin (Sigma) in medium 199. The cells were split at a 1:3 ratio and were grown in medium 199 containing 10% FBS and supplemented with 4 mM glutamine, 100 U/ml heparin (Sigma), 12.5 µg/ml endothelial cell growth factor (Collaborative Research, Waltham, MA), 0.1% fungizone (Life Technologies, Inc./BRL, Gaithersburg, MD), 0.1 mM penicillin G and 41 µM streptomycin sulfate. The HAE cells could be serially passaged up to 20 times and maintain endothelial cell characteristics such as lectin binding, vonWillibrand factor production, and the binding and degradation of acetylated LDL (18) (data not shown). All cell monolayers were fully confluent at the time of use.
Bovine aorta endothelial (BAE) cells were isolated and maintained as we have previously described (18). These cells were serially passaged from primary cultures of BAE cells, and the cells used in these experiments were from early passages (passages 8–10) derived from the primary cultures.
LPL assay
LPL activity was quantitated as previously described (19) using
rat serum (Pelfreeze Biologicals, Rogers, AR) as a source
of the LPL cofactor apolipoprotein CII and
[3H]triolein (glycerol
tri-[9,10(N)-3H]oleate, NEN Life Science Products, Boston, MA) as substrate in a glycerol emulsion. To
each tube, 100 µl cell culture medium from the adipocytes were added
to 100 µl substrate emulsion, and after mixing, the samples were
incubated at 37 C for 1 h. The reaction was terminated, and the
liberated free fatty acids were retained in the aqueous phase after
extraction into organic solvents. The aqueous phase was subjected to
scintillation counting and quantitated by the method of internal
standards. The rate of production of free fatty acids was linear for at
least 90 min under these assay conditions. One milliunit of activity is
represented by the release of 1 nmol fatty acid/min.
LPL activity associated with the 3T3-L1 adipocytes was quantitated as insulin-releasable activity, heparin-releasable activity, or the residual cell-associated activity. Insulin-releasable activity was determined by incubating 3T3-L1 adipocytes with the indicated concentration of insulin for the indicated periods of time in DMEM-1% charcoal-stripped-FBS. The culture medium was then removed from the monolayers and immediately assayed for LPL activity, or the samples were quick-frozen in a dry ice-acetone slurry and stored at -80 C. Immediately after LPL release by insulin, heparin-releasable LPL activity was determined by replacing fresh medium (DMEM-1% charcoal-stripped FBS) on the cells containing the indicated concentration of heparin for the indicated period of time. The culture medium was then removed from the cells and immediately assayed for LPL activity, or the medium was quick-frozen and stored at -80 C. Residual cell-associated LPL activity was assessed after maximal release of cell surface LPL by insulin and heparin treatment. After heparin release, the monolayers were rapidly washed in PBS, and the adipocytes were delipidated by extraction in acetone, as previously described (12). The resultant dried acetone powders were resuspended in 50 mM NH4Cl (pH 8.1), 0.4% Triton X-100, and 0.04% SDS with a motorized Teflon pestle in a Dounce homogenizer (Kontes Co., Vineland, NJ), and assayed for LPL activity within 30 min of resuspension.
Each experimental point was obtained from two 100-mm dishes of adipocytes, assayed in duplicate. The data are adjusted to represent the LPL activity obtained per 100-mm dish of confluent cells.
LPL release activity from HAE cells
Twenty-four hours before an experiment, the HAE cell monolayers
were treated to remove the supplements to the cell culture medium and
remove any serum-derived hormones. To achieve this, the cells were
washed three times with sterile PBS, and the medium replaced on the
cells was DMEM containing 1% charcoal-stripped FBS. Immediately before
use, the HAE cells were again washed with PBS before the addition of
DMEM and 1% charcoal-stripped FBS in the presence or absence of the
indicated concentration of insulin.
Statistical analyses
All data are presented as the average ± the variance of
the data. Statistical significance was determined by two-tailed
t tests with comparisons between the insulin-treated and
insulin-free samples. P < 0.05 is considered
significant and is denoted by an asterisk in the
figures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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demonstrate that maximal release of
LPL activity from the adipocytes required 45–60 min. These results are
consistent with data presented by others (20), in which maximal release
was found between 30–60 min of stimulation. To determine the insulin
concentration dependence of this release, the adipocytes were incubated
with variable insulin concentrations for 60 min before harvesting the
medium and quantitating LPL activity released from the adipocytes. As
shown in Fig. 1B, insulin induced a 4.4-fold increase in LPL release
from the adipocytes, with an EC50 of
approximately 0.25 nM and with maximal stimulation apparent
at 1 nM. Further increasing the insulin concentration to
100 nM resulted in the same level of LPL release as that
demonstrated with 10 nM insulin (data not shown). Other
investigators have reported comparable EC50
values for insulin-induced release of LPL from 3T3-L1 adipocytes and
other cultured adipose cells (11, 20).
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. Using a heparin
concentration of 100 µg/ml, the time course of LPL release from the
adipocyte was rapid (Fig. 2A), with maximal release occurring by 15
min, the earliest time point examined. The rapid release of LPL by
heparin shown here is consistent with the short periods of heparin
release used by other investigators (13). The heparin concentration
dependence of LPL release from the adipocytes (Fig. 2B) demonstrates
that maximal LPL release was achieved with a heparin concentration of
100 µg/ml. These high heparin concentrations were also used by others
(13) for maximal release of LPL activity from the adipocytes. In all
subsequent experiments, maximal heparin release was achieved with 100
µg/ml heparin for 20 min.
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. In the basal cells, in the absence of
insulin stimulation, only 1.6% of the total cellular LPL activity
(defined as insulin-releasable plus heparin-releasable plus residual
cell-associated LPL activity) was released from the cells into the
medium over a 1-h period. The majority (65.7%) of the total cellular
activity could be released from the cell by heparin. Upon stimulation
of the cells with insulin for 1 h, total cellular LPL activity
increased by 1.44-fold, from 76.1 to 109.4 mU/dish. Although insulin
increased the release of LPL into the cell culture medium by 6-fold
(from 1.2 to 7.1 mU/dish), a large, 1.7-fold increase in
heparin-releasable LPL activity was induced by insulin (from 50 mU/dish
in the basal cells to 86.6 mU/dish in the stimulated cells). Insulin
treatment did not significantly modify the level of intracellular LPL
activity.
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In summary, a 1-h insulin treatment of the adipocytes led to a 1.8-fold increase in the LPL activity available at the plasma membrane of the adipocyte (insulin-releasable plus heparin-releasable), with no further sizable change upon insulin challenge for 18 h.
Under physiological conditions, insulin released from the pancreas into
the blood first encounters the endothelial cells of the
microvasculature before moving across the vessel wall and binding to
the insulin receptor on the adipocytes. Therefore, it was of interest
to ascertain whether insulin stimulation of endothelial cells isolated
from adipose tissue would result in the release of a factor from the
endothelial cells that would liberate LPL activity from the 3T3-L1
adipocytes. Confluent cultures of HAE cells on a plastic substrate were
stimulated in the presence or absence of 10 nM insulin for
up to 90 min. This HAE cell-conditioned culture medium (which, for the
insulin-treated cells, contained 10 nM insulin) was removed
from the HAE cells and placed upon 3T3-L1 adipocytes for 1 h.
Medium was then removed from the adipocytes and assayed for LPL
activity. The results of this experiment are shown in Fig. 4. For the zero time point, the
adipocytes were incubated for 1 h with fresh medium in the
presence or absence of 10 nM insulin. Thus, the difference
in the zero time values is due solely to insulin-induced release of LPL
from the adipocytes, with no contribution in the zero time LPL values
from HAE cell-derived factors. In the absence of insulin stimulation,
the HAE cells released an activity that caused a low level of release
of LPL activity from the adipocytes. Maximal release of this activity
was attained after 30 min of incubation with the HAE cells, and
thereafter was maintained for up to 90 min. Treatment of the HAE cells
with 10 nM insulin resulted in the rapid production of a
factor that caused high levels of release of LPL from the adipocytes.
Maximal LPL release activity was obtained from the HAE cells at 5–20
min of stimulation with insulin. By 30 min of insulin stimulation, this
activity had declined to near-basal levels. As insulin was added to the
culture medium bathing the HAE cells, and because it is unlikely that
all of the added insulin was degraded over the short incubation periods
with the HAE cells, this insulin was carried over into the subsequent
incubations with the adipocytes. This direct effect of insulin on the
adipocytes led to the release of LPL from the adipocytes and
contributed a uniform 8–10 mU/dish of LPL activity to each "+Ins"
data point shown in Fig. 4. From these data, it is apparent that
insulin stimulation of the HAE cells results in the rapid, but
transient, release of a factor that induces the release of LPL activity
from the 3T3-L1 adipocytes.
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). Therefore, to determine the amount of LPL activity
remaining on the adipocytes after stimulation with the HAE cell release
factor, HAE cells were incubated with 10 nM insulin for 15
min, and the conditioned cell culture medium was collected from the HAE
cells and added to the 3T3-L1 adipocytes for 1 h. The 3T3-L1 cells
were then immediately incubated with 100 µg/ml heparin for 30 min,
and the collected samples were assayed for LPL activity. It was
consistently found that less than 10 mU/dish of LPL activity could be
released by heparin from the adipocytes after the release of LPL by the
HAE cell-derived factor (data not shown). Therefore, the
insulin-induced factor derived from the HAE cells releases most, if not
all, of the LPL activity present on the surface of the adipocytes.
To assess the specificity of the release of the insulin-induced LPL
release factor, endothelial cells isolated from macrovessels, BAE
cells, were incubated in the presence or absence of 100 nM
insulin for various periods of time, and the cell culture medium was
collected and subsequently added to 3T3-L1 adipocytes for 1 h. The
culture medium from the adipocytes was then harvested, and LPL activity
released into the medium was quantitated. The results of this
experiment are shown in Fig. 5. In the
absence of insulin stimulation, conditioned media derived from the BAE
cells contained no activity that stimulated the release of LPL from the
adipocytes. In the presence of insulin, no increase in LPL release
activity, above that induced by insulin alone, could be detected for
the first 20 min of stimulation. By 45 min, the release of LPL from the
adipocytes was stimulated 1.4-fold above that seen with insulin alone.
With time, the BAE cells may release greater levels of a LPL release
factor, but over the period when HAE cells are highly responsive to
insulin, BAE cells are not. Thus, there is a degree of specificity in
the expression and/or insulin sensitivity of the release of endothelial
cell-derived LPL release factor. The microvessel-derived HAE cells are
insulin responsive, whereas the macrovessel-derived BAE cells are
not.
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The HAE cells were incubated with the indicated concentration of
insulin for 20 min, and the conditioned medium was collected from the
cell monolayers. Conditioned medium was then added to 3T3-L1 adipocytes
for 60 min, and medium was collected and assayed for LPL activity. The
insulin concentration that produced half-maximal LPL release was
approximately 0.5 nM, and maximal release factor was
generated at 5 nM insulin. The open circle
demonstrates the release of LPL from the adipocytes incubated with 10
nM insulin in the absence of any endothelial
cell-derived factors.
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. The HAE cells were incubated
with 10 nM insulin for 20 min, and the conditioned medium
was collected. The conditioned medium was then added to 3T3-L1
adipocytes for the indicated periods of time, and the medium was
collected and assayed for LPL activity. The release of LPL from the
adipocytes by the HAE cell-derived factor was rapid, attaining maximal
release in 30 min, with a half-time of approximately 15 min.
The effect of repeated challenges with insulin on the release of the
factor from the HAE cells was then determined (Fig. 7). The HAE cells were treated with 10
nM insulin over a 30-min time course, and medium was
collected and assayed by the release of LPL from the adipocytes
(challenge 1). The cells were then washed, and insulin-free cell
culture medium was replaced on the cells for 2.5 h. The cells were
stimulated a second time with 10 nM insulin over a 30-min
time course, and the medium was collected and again assayed by the
release of LPL from the adipocytes (challenge 2). A separate set of HAE
cells was treated with 100 nM insulin for 18 h,
conditions that desensitize 3T3-L1 adipocytes to subsequent insulin
stimulation. These desensitized HAE cells were washed extensively to
remove the insulin and then restimulated with 10 nM insulin
over a 30-min time course, and the medium was collected and assayed by
release of LPL from the naive adipocytes (desensitized). The results
from this experiment are shown in Fig. 7. After a rest period of
2.5 h, the HAE cells responded to a second challenge with insulin
in a manner as robust as the initial challenge; the amplitude of the
response and the time course of the response are as extensive in the
second insulin challenge as in the first. However, if the HAE cells
were chronically stimulated with insulin, the maximal level of
LPL-releasing factor generated by the HAE cells in response to a
subsequent acute insulin challenge was decreased by a factor of 4 when
comparing the 15-min time point and correcting for zero time levels of
LPL release. These data demonstrate that the release of LPL from the
adipocytes is mediated through the response of the endothelial cells to
insulin.
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were basal,
nonstimulated cells. An identical experiment was performed in which the
adipocytes were chronically pretreated with 100 nM insulin
for 18 h, washed to remove the insulin, and then incubated with
medium conditioned by the HAE cells, as described in Fig. 7. With the
desensitized adipocytes, results identical to those shown in Fig. 7
were obtained. Conditioned medium derived from acutely
insulin-challenged HAE cells induced the release of LPL from the
desensitized adipocytes, but conditioned medium obtained from
desensitized HAE cells induced a blunted release of LPL from the
desensitized adipocytes (data not shown). These data further
substantiate the conclusion that the regulation of release of LPL from
the 3T3-L1 adipocytes is in part mediated through the response of the
endothelial cells to insulin. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism by which insulin mediates the release of LPL may be through the induction of a phosphatidylinositol phospholipase C activity. It has been proposed that LPL is anchored to the plasma membrane of the cell by means of a glycosyl-phosphatidylinositol anchor and that insulin treatment of the cells induces a phosphatidylinositol phospholipase C activity that cleaves this anchor and releases LPL activity from the cell (25). However, it has also been demonstrated that insulin treatment induces the release of phosphatidylinositol-anchored HSP (25), and adipose cells have been found to express high levels of HSP (22, 23). Thus, insulin treatment of the adipocytes may result in the direct release LPL activity, or, in an indirect manner, insulin treatment may release the HSP to which LPL is bound. By either mechanism, the amount of LPL released by insulin is a small fraction (8.2%) of the amount of LPL released by heparin, and the time course for maximal release by insulin is approximately 4 times longer than the time required for heparin release.
Insulin treatment of 3T3-L1 adipocytes has been documented to increase the level of cellular and heparin-releasable LPL activity through posttranscriptional and posttranslational mechanisms (13). These early studies used extended (2-day) incubations to demonstrate these effects. Our data demonstrate that within 1 h of stimulation of the adipocytes with physiological concentrations of insulin, the pool of readily available, heparin-releasable LPL activity nearly doubles. This increase in LPL activity occurs without any significant decrease in the remaining cell-associated LPL activity. The source of this LPL may be newly synthesized LPL, as the biosynthetic half-time for LPL in 3T3-L1 adipocytes has been found to be 1 h (26). Alternately, as it has been shown that there is a large intracellular pool of inactive, probably monomeric, LPL (26), it is possible that this inactive pool of LPL is readily activated and recruited to the heparin-releasable pool. Studies to define the source of this LPL activity are currently in progress.
Insulin treatment of microvessel endothelial cells isolated from adipose tissue results in the production of a factor that greatly facilitates the release of LPL from the 3T3-L1 adipocytes. No comparable effect can be observed with endothelial cells derived from macrovessels. To our knowledge, this is the first demonstration of such cross-talk between adipocytes and endothelial cells. This endothelial cell-derived factor, or LPL release factor, releases nearly the entire pool of heparin-releasable LPL from the adipocytes. The time course for the release of LPL from the adipocytes with the LPL release factor is significantly slower (half-time, 15 min) than the release of LPL from adipocytes mediated by heparin (maximal release in 15 min). The slower LPL release time course for the factor makes it unlikely that the factor is itself heparin derived from the endothelial cells. We are currently pursuing the identity of this LPL release factor.
A peak of LPL release factor is demonstrated within 15 min of insulin stimulation of the HAE cells, and this maximal activity returns to near-basal levels within the ensuing 5–10 min. This rapid decline in LPL release factor activity does not appear to be due to the degradation or thermodynamic instability of the factor. The factor can be harvested from the HAE cells after 15 min of insulin stimulation, and it retains full activity after storage at 4 C for 24 h or when subjected to a round of freeze-thawing (data not shown). The loss of activity appears to be a function of the factor’s continued interaction with the HAE cells. Whether this is due to the release of an inactivating factor from the HAE cells or is due to the reassociation of the LPL release factor with the HAE cells is currently under investigation.
The expression of the LPL release factor from the HAE cells into the culture medium is highly insulin responsive. The maximal production of this factor in the cell culture medium bathing the HAE cells occurs within 15–20 min of insulin stimulation, with an EC50 of 500 pM. Significant levels of the factor are generated upon stimulation of the cells with 100 pM insulin. Therefore, its production occurs rapidly, and with physiological concentrations of insulin. The production of this release factor may be highly relevant to the release of LPL from the adipocyte in a physiological context.
Also relevant from a physiological context is the ability of the HAE cells to respond to multiple challenges with insulin. After a recovery period of 2 h, a second round of stimulation of the HAE cells with insulin resulted in the release of the LPL release factor from the HAE cells with a profile as robust as the initial insulin challenge. In a normal human feeding pattern, with many hours separating meals and the corresponding peaks (postprandial) and troughs (preprandial) in the blood levels of insulin, VLDL, and chylomicrons, it would be anticipated that the endothelial cells should be able to repeatedly respond to these waves of hormones and LPL substrates. This flexibility would optimize the availability of LPL, with maximal hydrolysis of the triglycerides associated with the lipoprotein particles.
Further extending the analysis of this in situ data to the physiological context, it appears that the site of regulation of bioavailable LPL is the endothelial cell. The effect of insulin on the adipocyte is to increase the pool of readily available LPL. However, this population of LPL for the most part remains attached to the adipocyte and is not available to be routed to the luminal domain of the endothelial cell. It is insulin’s effect on the endothelial cell, mobilizing the LPL-releasing factor, that releases the LPL from the adipocyte for ultimate transit to and across the endothelial cell monolayer.
The role of the endothelial cell in regulating LPL is further underscored in the hyperinsulinemic conditions that we have shown to mimic the insulin-resistant state (21). Chronic treatment of 3T3-L1 adipocytes with insulin results in a cellular distribution of LPL within the adipocyte that is very similar to what is seen in the adipocyte that has been stimulated with insulin for only 1 h. Upon chronic insulin stimulation, LPL found in the cell culture medium is elevated 5-fold above basal levels, and heparin-releasable LPL in the chronically stimulated cells is increased 1.3-fold above basal cells. So, although chronic insulin stimulation of 3T3-L1 adipocytes leads to a desensitized state with respect to insulin-induced lipogenesis and glycogen synthesis (21), in the desensitized adipocyte the readily available pool of LPL is elevated above the basal cell and is very similar to that in the acutely stimulated adipocyte. Chronic insulin stimulation of HAE cells, on the other hand, significantly blunts the release of LPL release factor from endothelial cells. Hence, under insulin-resistant conditions, despite the availability of LPL at or near the surface of the adipocyte, LPL cannot be extensively mobilized from the fat cell because the HAE cell is unable to respond to insulin. It appears, therefore, that the endothelial cell acts as the primary locus for the regulation of LPL mobilization from the adipocyte.
Previous investigators have demonstrated the important role played by the endothelial cell in the movement of LPL from parenchymal cells to its site of action, the luminal side of the endothelial cell monolayer. Goldberg’s laboratory has demonstrated that a heparanase-like activity is released from bovine aorta macrovessel endothelial cells after 16 h of incubation of the cells with lysophosphatidylcholine (a product of LPL hydrolysis). This heparanase-like activity causes the release of LPL from adipocytes (27). The relevance of these findings to a normal physiological setting is unknown. It is difficult to know whether, under the normal conditions of blood flow, sufficient lysophosphatidylcholine would be able to accumulate and regulate the endothelial cells. In addition, lysophosphatidylcholine treatment for 16 h was required to accumulate the indicated levels of heparanase activity. In contrast, insulin acts upon the endothelial cell by binding to its high affinity receptor, which we (28) and others (29, 30) have demonstrated to be present at high levels on endothelial cells. In addition, the rapid and robust response of the endothelial cells to low concentrations of insulin suggest that this process is physiologically significant. Nevertheless, these recent studies from the Goldberg laboratory indicate that endothelial cells play a role in regulating LPL release from adipocytes. Whether the LPL-releasing factor that we have identified here also maintains a heparanase-like activity is currently under investigation. Others have identified oligosaccharides derived from endothelial cell HSP with high affinity for LPL (31). The binding of these oligosaccharides to LPL stabilizes the activity of the enzyme and facilitates both its binding to the apical surface of the endothelial cell and its transcytosis across the endothelial cell monolayer (27). It is not known whether insulin induces binding and transcytosis of LPL to macrovessel (bovine aorta) endothelial cells, nor is it known whether these same processes are promoted by insulin in microvessel (adipose) endothelial cells. Studies to address these questions are currently underway.
These data indicate that at least three loci exist through which insulin regulates LPL. 1) Acting directly upon the adipocyte, insulin increases the release of LPL from the adipocyte. The magnitude of this response is small, and the insulin signaling pathway mediating this effect is unknown. 2) Acting directly on the adipocyte, insulin induces an increase in the population of LPL at the surface of the adipocyte (heparin-releasable LPL). The mechanism by which this large increase in LPL activity is acquired at the cell surface is unknown. 3) Insulin, acting on the microvessel endothelial cell, induces the release of a factor that, when incubated with the adipocyte, quantitatively releases the cell surface LPL activity from the adipocyte. The identity of this factor derived from the insulin-stimulated endothelial cells is unknown, and the mechanism by which insulin regulates its release is unknown.
The identity of the microvessel endothelial cell-derived LPL release factor and the mechanism by which it is regulated by insulin are under active investigation.
Received August 27, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or presence (•) of 100
nM insulin for the indicated periods of time. The cell
culture medium was then collected and assayed for LPL activity. B,
Insulin concentration response for the release of LPL from the
adipocytes. The adipocytes were incubated with the indicated
concentration of insulin for 60 min. The cell culture medium bathing
the cells was then collected and assayed for LPL activity.
). A parallel set of endothelial cells was incubated in the absence
of insulin for the indicated periods of time, and after incubation of
this conditioned medium with the adipocytes for 60 min, the resultant
medium was assayed for LPL activity (
).