Extracellular degradation of lipoprotein lipase in rat adipose tissue
© Wu et al; licensee BioMed Central Ltd. 2005
Received: 27 August 2004
Accepted: 25 January 2005
Published: 25 January 2005
Recent studies in vivo indicate that short-term regulation of lipoprotein lipase (LPL) in rat adipose tissue is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product. It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process.
When explants of rat epididymal adipose tissue were incubated, LPL mass and activity decreased rapidly. Mass and activity within adipocytes remained constant for at least six hours, demonstrating that it was the extracellular portion of the enzyme that decreased. Adipocytes isolated from the explants after three or six hours of incubation retained their ability to secrete LPL to the medium. Addition of a cocktail of protease inhibitors to the incubation medium slowed down the decrease of LPL mass. Chloroquine was without effect, indicating that the degradation was not lysosomal. 125I-labeled LPL added to the medium was degraded to acid soluble products, indicating that the degradation occurred extracellularly. Fragmentation of the labelled lipase occurred in conditioned medium and this process was virtually abolished by two MMP inhibitors.
The decrease of LPL mass and activity that occurs when explants of rat adipose tissue are incubated is due to proteolysis of extracellular LPL. The adipocytes continue to produce and secrete the enzyme. The effect of inhibitors indicates, but does not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue.
Lipoprotein lipase (LPL) hydrolyzes triglycerides in very low-density lipoproteins and chylomicrons . Tissue-specific regulation of LPL activity is a major mechanism to distribute lipids among tissues according to the physiological needs . Current information indicates that in adipose tissue, the regulation is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product . This information derives from in vivo experiments. To study the underlying mechanism an in vitro model is urgently needed. Experiments with isolated adipocytes do not seem to bring out the mechanism and the in vivo experiments indicate that it is the extracellular LPL that is the target for the regulation . We have therefore explored the possibility to use tissue explants and report our experiences in this paper. The results support the view that it is the extracellular enzyme that is being regulated. Unfortunately the preparation of tissue explants seems to trigger a proteolytic response in the tissue.
LPL activity and mass decreased when explants of rat adipose tissue were incubated
We tried variations in technique and several different incubation media in experiments such as those in Figure 1. There was some variation in the absolute values but the results were in principle the same, a rapid decline of LPL mass and activity. The rate at which LPL mass decreased was similar with tissue explants from fed and fasted rats, and LPL activity roughly followed LPL mass in tissues from fed rats.
Changes in LPL mass during incubation. Explants of rat adipose tissue were incubated for three hours as in Figure 1. The tissue explants and the medium were recovered and then the vessel was rinsed out with warm (~80°C) SDS solution. This was then suitably diluted with Triton X-100 to match the composition of the medium used for the ELISA. Mean ± SEM of five parallel incubations.
LPL mass (ng/g tissue)
1882 ± 13
665 ± 11*
26 ± 6
12 ± 3
1882 ± 13
702 ± 8 *
Is the loss of LPL mass an intra- or extracellular event?
Is the LPL protein degraded?
The objective for this study was to find an in vitro system to study the mechanism for down-regulation of LPL activity in rat adipose tissue that occurs on food deprivation. Isolated adipocytes have been tried in several laboratories [5–9], but the differences with nutritional state are rather small. This is true whether one measures LPL within the cells or the rates at which the cells secrete LPL to the medium. These observations are repeated here. The lack of difference within adipocytes indicates that other cell types are needed and may in fact be responsible for the pronounced down-regulation of extracellular LPL activity that occurs on food deprivation [3, 4]. We therefore tried to use tissue explants, which have proved valuable in other studies of adipose tissue . Our results show that degradation of the enzyme was a major process when explants were incubated. The degradation occurred extracellularly; LPL mass and activity in the adipocytes did not change during incubation for up to six hours. Added 125I-LPL was degraded and this was prevented by addition of MMP inhibitors to the medium.
There must be damage of cells when the tissue is cut into small pieces, and there is probably some degree of hypoxia during incubation of the pieces. Cultured explants have, however, been widely and successfully used to explore various aspects of adipose tissue biology ( and references therein). In preliminary experiments we found that the explants retained their ability to take up glucose, to synthesize proteins and to secrete leptin. Adipocytes isolated from the explants after several hours of incubation had the same LPL activity as adipocytes from fresh tissue (Figure 4). This indicates that the cells produced LPL at an essentially unchanged rate, since the LPL activity in adipocytes decreased rapidly when protein synthesis was inhibited by cycloheximide (Figure 2). Hence, the rapid decrease of LPL when explants were incubated was not due to a general loss of functionality, but reflected specific processes leading to degradation of extracellular LPL.
Our results are in line with observations made already in the sixties. There was evidence from chromatographic separations for at least two different forms of LPL in rat adipose tissue . Cunnigham and Robinson found that incubation of fat pads from fed rats resulted in a rapid loss of LPL activity until a low activity, stable to prolonged incubation, was attained . In contrast, the LPL activity of isolated fat cells was stable to prolonged incubation. The concept of stable and unstable forms of the lipase can now be interpreted as a reflection of extracellular lipase that is exposed to proteolysis, and intracellular lipase that is protected from the extracellular proteases.
The degradation of LPL in conditioned medium was almost completely abolished by the two non-specific MMP inhibitors that we tested. We have not characterized the proteolytic activity further but note that adipose tissue produces at least two MMPs, 2 and 9 . The loss of LPL mass during incubation of tissue explants was relatively slow during the first two hours and then accelerated. It is likely that the tissue trauma and/or the loss of blood circulation triggered an activation of the MMP system. It has been shown that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression . It is of interest to note that LPL activity does not decrease during perfusion of fat pads , whereas it does decrease when whole fat pads are cut out and incubated in vitro .
Two pathways for turnover of adipose tissue LPL have been demonstrated so far. One is dissociation of the lipase, perhaps after loss of catalytic activity, into the blood and degradation in the liver . Release of LPL into blood from adipose tissue has been directly demonstrated by measurement of arterio-venous difference in man . This pathway can, however, not operate in tissue explants. Another pathway, demonstrated with cultured fat cells, is endocytosis and degradation in lysosomes . This pathway did not seem to contribute significantly in the present system since the rate at which LPL mass decreased was not affected by chloroquine. The present findings suggest a third pathway, extracellular proteolysis in the tissue.
The rapid decrease of LPL that occurs when adipose tissue explants are incubated engages only the extracellular enzyme. The adipocytes continue to produce and secrete the enzyme and intracellular LPL remains essentially constant for at least six hours.
The decrease in extracellular LPL is due to proteolytic cleavage/degradation of both active and inactive forms of the enzyme.
The effects of inhibitors indicate, but do not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue.
Male Sprague-Dawley rats were from Möllegaard Breeding Center (Ejby, Denmark). Unless otherwise stated, the rats were 23 days old and weighed around 60 g. After transport to Umeå they were allowed to acclimatize for seven to ten days by which time they had reached a weight of approximately 120 g. The rats were kept in a well ventilated, temperature (21°C) and humidity (40–45%) controlled room with free access to a standard laboratory chow (Laktamin AB, Stockholm) and tap water. The light in the room was on between 6 a.m. and 6 p.m. In experiments where the rats were to be fasted, food was withdrawn from the cages at 6 a.m. and a grid was placed at the bottom of the cages to prevent coprophagia. The adipose depot used in all experiments was the periepididymal one. The rats were killed by decapitation. Animal experiments were approved by the animal ethics committee in Umeå.
Cycloheximide, bovine serum albumin (BSA), the MMP inhibitors GM6001 (Galardin) and Captopril, chloroquine and collagenase were from SIGMA (St. Louis, MO, USA). Protease inhibitor cocktail tablets "Complete Mini" were from Roche Diagnostics, Mannheim, Germany. Heparin was from Lövens (Malmö, Sweden). Substrate for the LPL activity assay was 3H-labelled triolein in Intralipid (10%) kindly prepared by Pharmacia-UpJohn (Stockholm, Sweden). Parker medium (Parker 199) was from SBL (Stockholm, Sweden). 125 I-LPL was prepared as before . All other reagents were of the highest commercial grade possible.
LPL was extracted from tissues by homogenization in a Tris-HCL buffer (pH 8.2) containing detergents and protease inhibitors as described . The homogenate was centrifuged for 15 min at 3000 rpm after which the intermediate phase (between the floating fat droplets and the pellet) was used for assay of LPL activity and mass. In most cases the extract was kept on ice and assayed within a few hours. Under these conditions LPL activity is stable. In some cases the extracts were frozen and kept at -70°C for later assay.
LPL activity was measured as described previously . Briefly, two μl of tissue homogenate (triplicate samples) was incubated for 60 min at 25°C with substrate in the presence of ten μl heat-inactivated serum from fasted rats (as source of apolipoprotein CII) and 6% BSA. The total volume was 200 μl. After termination of lipolysis by addition of organic solvents, the fatty acids were extracted and counted for radioactivity. One mU of lipase activity represents one nmol of fatty acids released per minute.
LPL mass was measured with an ELISA as described . The chicken antibodies used recognize both active and inactive forms of the lipase . Briefly, three different dilutions of tissue homogenate were incubated in microtiter plate wells previously coated with affinity-purified chicken anti-LPL IgG. Detection was mediated via the 5D2 monoclonal antibody (a kind gift by Dr John Brunzell, University of Washington, Seattle) followed by a peroxidase conjugated anti-mouse IgG antibody. Absorbance at 490 nm was measured in a Spectramax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).
DNA content was assayed using Labarca's method .
In vitro incubation of adipose tissue
Epididymal adipose tissue was dissected out from fed or 24 h fasted rats. The tissue was cut into small pieces (5 mg or less). A total of about maximal 100 mg tissue pieces were immediately put into culture plates. Each well contained 15 ml of Parker Medium 199 supplemented with 2% BSA, 0.5% casein hydrolysate, 10 mM glucose and adjusted to pH 7.4. Incubations were at 37°C and 5 % CO2: 95 % O2 with continuous gentle shaking motion in a Cellstar Incubator (Queue Systems, Asheville, Canada). After incubation, the tissues were prepared for measurement of LPL activity and mass as described above. In some experiments samples of the medium were taken for assay of LPL activity and/or mass.
In some experiments adipocytes were prepared after collagenase treatment of the tissue pieces as described . To measure heparin releasable LPL (HR-LPL), adipose tissue explants were incubated with heparin (final concentration was 50 IU/ml) for 45 min at 37°C.
Student's t-test was used for analysis of the data.
This study was funded by the Swedish Medical Research Council Projects no. 727, 12203 and 12663.
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