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The Distribution of Lipoprotein Lipase in Rat Adipose Tissue

2003; Elsevier BV; Volume: 278; Issue: 14 Linguagem: Inglês

10.1074/jbc.m212736200

1083-351X

Gengshu Wu, Gunilla Olivecrona, Thomas Olivecrona,

Diabetes, Cardiovascular Risks, and Lipoproteins

Lipoprotein lipase (LPL) acts at the vascular endothelium. Earlier studies have shown that down-regulation of adipose tissue LPL during fasting is post-translational and involves a shift from active to inactive forms of the lipase. Studies in cell systems had indicated that during fasting LPL might be retained in the endoplasmic reticulum. We have now explored the relation between active/inactive and intra/extracellular forms of the lipase. Within adipocytes, neither LPL mass nor the distribution of LPL between active and inactive forms changed on fasting. Extracellular LPL mass also did not change significantly, but shifted from predominantly active to predominantly inactive. To explore if changes in secretion were compensated by changes in turnover, synthesis of new protein was blocked by cycloheximide. The rates at which intra- and extracellular LPL mass and activity decreased did not change on fasting. To further explore how LPL is distributed in the tissue, heparin (which detaches the enzyme from the endothelial surface) was injected. Tissue LPL activity decreased by about 107 in 2 min and by 507 in 1 h. Heparin released mainly the active form of the lipase. There was no change of LPL activity or mass within adipocytes. The fraction of extracellular LPL that heparin released and the time course were the same in fed and fasted rats, indicating that active, extracellular LPL was distributed in a similar way in the two nutritional states. This study suggests that the nutritional regulation of LPL in adipose tissue determines the activity state of extracellular LPL. Lipoprotein lipase (LPL) acts at the vascular endothelium. Earlier studies have shown that down-regulation of adipose tissue LPL during fasting is post-translational and involves a shift from active to inactive forms of the lipase. Studies in cell systems had indicated that during fasting LPL might be retained in the endoplasmic reticulum. We have now explored the relation between active/inactive and intra/extracellular forms of the lipase. Within adipocytes, neither LPL mass nor the distribution of LPL between active and inactive forms changed on fasting. Extracellular LPL mass also did not change significantly, but shifted from predominantly active to predominantly inactive. To explore if changes in secretion were compensated by changes in turnover, synthesis of new protein was blocked by cycloheximide. The rates at which intra- and extracellular LPL mass and activity decreased did not change on fasting. To further explore how LPL is distributed in the tissue, heparin (which detaches the enzyme from the endothelial surface) was injected. Tissue LPL activity decreased by about 107 in 2 min and by 507 in 1 h. Heparin released mainly the active form of the lipase. There was no change of LPL activity or mass within adipocytes. The fraction of extracellular LPL that heparin released and the time course were the same in fed and fasted rats, indicating that active, extracellular LPL was distributed in a similar way in the two nutritional states. This study suggests that the nutritional regulation of LPL in adipose tissue determines the activity state of extracellular LPL. lipoprotein lipase endoplasmic reticulum Lipoprotein lipase (LPL)1 is an enzyme that acts at the vascular endothelium (1Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Google Scholar, 2Olivecrona T. Olivecrona G. Betteridge D.J. Illingworth D.R. Shepherd J. Lipoproteins in Health and Disease. Arnold, London1999: 223-246Google Scholar). It hydrolyzes triglycerides in lipoproteins and thereby makes the fatty acids available for cellular metabolic reactions. The enzyme is synthesized in and secreted by parenchymal cells in the tissue, e.g. adipocytes and myocytes, and is transferred to the endothelium to which it becomes anchored by interaction with heparan sulfate proteoglycans. It was shown already in 1959 that the LPL activity in adipose tissue changes rapidly in response to the nutritional state (3Hollenberg C.H. Am. J. Physiol. 1959; 197: 667-670Google Scholar, 4Eckel R.H. Borensztajn J. Lipoprotein Lipase. Evener Publishers, Chicago1987: 79-132Google Scholar). Studies by the Robinson group then demonstrated that there is a strong correlation between the LPL activity and the uptake of fatty acids from labeled chylomicrons in rat adipose tissue (5Cryer A. Riley S.E. Williams E.R. Robinson D.S. Clin. Sci. Mol. Med. 1976; 50: 213-221Google Scholar). This suggests that LPL is an important determinant for the distribution of lipids between tissues (6Preiss-Landl K. Zimmermann R. Hammerle G. Zechner R. Curr. Opin. Lipidol. 2002; 13: 471-481Google Scholar). The mechanism for the regulation is, however, not clear. The regulation appears to be post-transcriptional. LPL mRNA in (rat) adipose tissue turns over with a half-life of 17 h, far too slowly to account for the rapid drop of enzyme activity when food is withdrawn (7Bergö M. Wu G. Ruge T. Olivecrona T. J. Biol. Chem. 2002; 277: 11927-11932Google Scholar). Several groups have found that LPL mRNA and/or relative rate of LPL synthesis (8Semb H. Olivecrona T. Biochim. Biophys. Acta. 1986; 878: 330-337Google Scholar, 9Doolittle M.H. Ben-Zeev O. Elovson J. Martin D. Kirchgessner T.G. J. Biol. Chem. 1990; 265: 4570-4577Google Scholar, 10Tavangar K. Murata Y. Pedersen M.E. Goers J.F. Hoffman A.R. Kraemer F.B. J. Clin. Invest. 1992; 90: 1672-1678Google Scholar, 11Ong J.M. Simsolo R.B. Saghizadeh M. Goers J.W.F. Kern P.A. Metabolism. 1995; 44: 1596-1605Google Scholar, 12Lee J.J. Smith P.J. Fried S.K. J. Nutr. 1998; 128: 940-946Google Scholar) as well as LPL mass ((9Doolittle M.H. Ben-Zeev O. Elovson J. Martin D. Kirchgessner T.G. J. Biol. Chem. 1990; 265: 4570-4577Google Scholar, 13Terrettaz J. Cusin I. Etienne J. Jeanrenaud B. Int. J. Obes. 1994; 18: 9-15Google Scholar, 14Erskine J.M. Jensen D.R. Eckel R.H. J. Nutr. 1994; 124: 500-507Google Scholar, 15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar) remain essentially unchanged during short-term fasting. There are two forms of the lipase in tissues (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar). One form is catalytically active and has high affinity for heparin. This is the predominant form in adipose tissue of fed rats. The other form is catalytically inactive, has low affinity to heparin and predominates in adipose tissue during fasting. When fasted rats are given food, the LPL activity returns to fed levels in 4–6 h. This increase of activity requires synthesis of new LPL molecules; there is no evidence that the inactive lipase protein can be recruited into the active form (7Bergö M. Wu G. Ruge T. Olivecrona T. J. Biol. Chem. 2002; 277: 11927-11932Google Scholar,16Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Google Scholar). Many studies have shown that the LPL activity that is released when pieces of adipose tissue are incubated in heparin-containing medium (4Eckel R.H. Borensztajn J. Lipoprotein Lipase. Evener Publishers, Chicago1987: 79-132Google Scholar,12Lee J.J. Smith P.J. Fried S.K. J. Nutr. 1998; 128: 940-946Google Scholar) or when adipose tissue is perfused with heparin (17Kuwajima M. Foster D.W. McGarry J.D. Metabolism. 1988; 37: 597-601Google Scholar) (舠heparin-releasable LPL舡) changes more than total tissue LPL activity. This indicates that the regulation is exerted mainly on the extracellular LPL (18Braun J.E.A. Severson D.L. Biochem. J. 1992; 287: 337-347Google Scholar). Short-term regulation of LPL in adipose tissue thus appears to involve changes both in the activity state of the LPL protein and in the distribution of LPL between intra- and extracellular locations in the tissue, but it is not known how these two parameters are related to each other. To resolve this, we have measured LPL activity and mass in adipose tissue and in adipocytes isolated from the tissue. In some experiments we have blocked synthesis of new enzyme to study the turnover of the different pools of LPL, and we have studied which pools of LPL (active/inactive, intra/extracellular) that are accessible to heparin release. All results point to extracellular LPL as the target for short-term nutritional regulation. Male Sprague-Dawley rats (23 days old) weighing around 60 g were from Möllegaard Breeding Center (Ejby, Denmark). They were allowed to acclimatize for 7–10 days by which time they had reached a weight of ∼120 g. The rats were kept in a well ventilated, temperature (21 °C)- and humidity (40–457)-controlled room with free access to a standard laboratory chow (Laktamin AB, Stockholm, Sweden) 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 rats were killed by decapitation. The adipose depot used in all experiments was the periepididymal one. In some experiments the tissue was cut into small pieces and digested with collagenase to isolate the adipocytes as described (19Semb H. Olivecrona T. Biochim. Biophys. Acta. 1986; 876: 249-255Google Scholar). The animal ethics committee in Umeå approved the animal experiments. Cycloheximide, bovine serum albumin, and collagenase were from Sigma, protease inhibitor mixture tablets 舠Complete Mini舡 was from Roche Diagnostics, Mannheim, Germany. Heparin was from Lövens (Malmö, Sweden).125I-LPL was prepared as described (20Olivecrona G. Lookene A. Methods Enzymol. 1997; 286: 102-116Google Scholar). All other reagents were of the highest commercial grade. LPL was extracted from tissues by homogenization in an ammonia buffer (pH 8.2) containing detergents and protease inhibitors as described (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar) but with the protease inhibitor mixture tablets Complete Mini (Roche Diagnostics) instead of the inhibitors previously used. 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. Most of the data on activity or mass were calculated relative to the amount of DNA in the sample, which was measured by fluorometry as described by Labarca and Paigen (21Labarca C. Paigen K. Anal. Biochem. 1980; 102: 344-352Google Scholar). In some experiments active and inactive forms of LPL in the extracts were separated on heparin-agarose as described (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar). LPL activity was measured as described (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar). The substrate was3H-labeled triolein in Intralipid (107) kindly prepared by Pharmacia-UpJohn (Stockholm, Sweden). Briefly, 2 ॖl of tissue homogenate (triplicate samples) was incubated for 60 min at 25 °C with substrate in the presence of 10 ॖl of heat-inactivated serum from fasted rats (as source of apolipoprotein CII) and 67 bovine serum albumin. The total volume was 200 ॖl. After termination of lipolysis by addition of organic solvents, the fatty acids were extracted and counted for radioactivity. One milliunit of lipase activity represents 1 nmol of fatty acids released per minute. LPL mass was determined with a sandwich enzyme-linked immunoadsorbent assay as described before (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar) but with bovine LPL as standard. 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, Seattle, WA) followed by a peroxidase-conjugated anti-mouse IgG antibody. Absorbance at 490 nm was measured in a Spectramax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). Data are presented as mean ± S.D. Student's t test was used. As reported earlier (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar) LPL mass was similar, but LPL activity was higher in adipose tissue from fed compared with fasted rats (Table I). To study the distribution of LPL within the tissue we used collagenase to digest the extracellular matrix. The adipocytes were then isolated by floatation. LPL activity was similar in adipocytes from fed and fasted rats (TableI). Hence, the higher LPL activity in fed rats was due entirely to higher extracellular LPL activity.Table IDistribution of LPL in rat adipose tissueNutritional state and sampleLPL activityLPL massmilliunit/gmilliunit/ॖg DNAng/gng/ॖg DNAFed Tissue1663 ± 2231-ap < 0.001 comparing fed and fasted.6.26 ± 1.81-ap < 0.001 comparing fed and fasted.1789 ± 31417.4 ± 1.1 Adipocytes497 ± 1302.08 ± 1.0512 ± 1123.8 ± 1.3 Extracellular1166 ± 2111-ap < 0.001 comparing fed and fasted.1277 ± 214Fasted Tissue825 ± 1303.47 ± 0.71632 ± 24117.1 ± 2.5 Adipocytes495 ± 1222.07 ± 1.2567 ± 1363.6 ± 1.8 Extracellular330 ± 881065 ± 200Epididymal adipose tissue was collected from fed or 24-hour fasted rats. One portion of the tissue was cut into pieces and incubated with collagenase. Adipocytes were then isolated by centrifugation. LPL activity and mass were determined in extracts from whole tissue and adipocytes, respectively. Extracellular LPL was calculated as the difference between whole tissue and adipocytes. In the left columns the values are the amounts present in 1 g of tissue or in adipocytes isolated from 1 g of tissue, respectively. In the right columns the values are per ॖg of DNA in tissue or adipocytes, respectively. This gives a better comparison since the loss of triglyceride weight on fasting does not influence the values and since direct determination of DNA in the isolated adipocytes adjusts for variations in recovery. No value for extracellular LPL can be calculated from these values. All values are mean ± S.D. for six rats in each group.1-a p < 0.001 comparing fed and fasted. Open table in a new tab Epididymal adipose tissue was collected from fed or 24-hour fasted rats. One portion of the tissue was cut into pieces and incubated with collagenase. Adipocytes were then isolated by centrifugation. LPL activity and mass were determined in extracts from whole tissue and adipocytes, respectively. Extracellular LPL was calculated as the difference between whole tissue and adipocytes. In the left columns the values are the amounts present in 1 g of tissue or in adipocytes isolated from 1 g of tissue, respectively. In the right columns the values are per ॖg of DNA in tissue or adipocytes, respectively. This gives a better comparison since the loss of triglyceride weight on fasting does not influence the values and since direct determination of DNA in the isolated adipocytes adjusts for variations in recovery. No value for extracellular LPL can be calculated from these values. All values are mean ± S.D. for six rats in each group. When bovine or rat LPL was incubated with collagenase, LPL activity and mass decreased rapidly. To study why this happened,125I-labeled bovine LPL was added to the medium, which after incubation for a few minutes was analyzed by SDS-PAGE (data not shown). Radioautography showed that a number of low-molecular weight bands had been formed, demonstrating that LPL is rapidly cleaved by collagenase under the conditions used to isolate adipocytes. Hence, it was not possible to recover and directly measure extracellular LPL. The pellet of sedimented cells (often called the stromal-vascular cells) contained only 37 of tissue LPL activity and 47 of tissue LPL mass. Hence, virtually all LPL was either in adipocytes or extracellular. The amount of extracellular LPL could therefore be estimated as the difference between tissue total and adipocytes. This activity was 3.5-fold higher in fed compared with fasted rats (Table I). In contrast, extracellular LPL mass did not differ significantly between the nutritional states. This experiment was repeated six times with some variations (age of rats, time of fasting). The difference in tissue LPL activity between fed and fasted rats ranged from 2-fold (as in this experiment) to more than 4-fold. It was a consistent finding that LPL activity within adipocytes was the same irrespective of the nutritional state so that the difference in tissue LPL activity was always due to a difference in extracellular activity. LPL mass on the other hand did not differ significantly with nutritional state, in tissue total, in adipocytes, or extracellular (Table I). Earlier studies have shown that rat tissues contain at least two forms of LPL (15Bergö M. Olivecrona G. Olivecrona T. Biochem. J. 1996; 313: 893-898Google Scholar). The catalytically active form has high heparin affinity and elutes at about 1 m NaCl from heparin-agarose. Inactive LPL elutes earlier in the gradient. There is more active than inactive LPL in adipose tissue from fed rats, whereas the reverse is true for fasted rats. We obtained similar results in the present study (Fig.1, upper panels). The peak ratio (area of mass in the first peak divided by that in the second peak) was 0.49 ± 0.06 and 2.26 ± 0.14 in tissue extracts from fed and fasted rats, respectively. In contrast, there was no difference in the distribution of LPL between the active and inactive forms within the adipocytes (Fig. 1, lower panels). The peak ratios were 2.05 ± 0.15 and 2.25 ± 0.04 in fed and fasted rats, respectively. The specific activity for the enzyme that eluted in peak two was similar (about 1 milliunit/ng) for all four separations,i.e. for total tissue LPL as well as for adipocyte LPL in fed and in fasted rats. The size of the pools of LPL are determined in part by the turnover rates, which might change with the nutritional state. To explore this, rats were injected with cycloheximide. In the experiment in Fig. 2, LPL mass decreased by 65–757 in 2 h, indicating a half-life in the order of 1 h. There was no difference between fed and fasted rats (Fig. 2, lower panel). This rules out the possibility that the relatively large amount of inactive LPL in adipose tissue of fasted rats represents dead-end material that turns over slowly. After the first 2 h, the decrease of LPL became much slower, presumably because incomplete inhibition of protein synthesis allowed some continued production of the enzyme. LPL activity dropped rapidly after cycloheximide was given. This is in accord with earlier studies in vivo (7Bergö M. Wu G. Ruge T. Olivecrona T. J. Biol. Chem. 2002; 277: 11927-11932Google Scholar, 22Schotz M.C. Garfinkel A.S. Biochim. Biophys. Acta. 1965; 106: 202-205Google Scholar, 23Cryer A. Foster B. Wing D.R. Robinson D.S. Biochem. J. 1973; 132: 833-836Google Scholar) and with cells (16Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Google Scholar, 24Semb H. Olivecrona T. Biochim. Biophys. Acta. 1987; 921: 104-115Google Scholar). In the fed rats, LPL activity dropped at a similar rate as LPL mass (Fig. 2, upper panel). The much lower activity in the fasted rats also dropped. To be able to compare the rates between the nutritional states we recalculated the data as percent of initial activity (Fig. 2, inset). When expressed in this way, the LPL activities decreased at virtually the same rate in the two nutritional states. The loss of LPL mass and activity during the initial rapid phase (about 2 h) and the height of the subsequent slow phase differed between experiments, presumably due to variation in the extent to which protein synthesis was inhibited. It was not feasible to isolate adipocytes from all the animals in the experiment in Fig. 2. Therefore a separate experiment was carried out at a single time point (Fig. 3). In this experiment, LPL mass decreased by 45–557 in 90 min, compared with about 757 in 2 h in the experiment in Fig. 2. The percentage loss of LPL mass was similar for tissue total, for adipocytes, and for calculated extracellular LPL mass (Fig. 3, lower panel). LPL activity decreased by 53 and 477 in whole tissue and by 52 and 427 in adipocytes from fed and fasted rats, respectively (Fig. 3, upper panel). These differences were not statistically significant. In any case, this experiment shows that all four pools of LPL, active/inactive, intra/extracellular, turn over rapidly and at similar fractional rates. Many studies have shown that LPL is released from its endothelial binding sites by heparin (1Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Google Scholar, 2Olivecrona T. Olivecrona G. Betteridge D.J. Illingworth D.R. Shepherd J. Lipoproteins in Health and Disease. Arnold, London1999: 223-246Google Scholar). We questioned how much and what fraction of the total tissue LPL could be released by perfusion with heparin. For this we injected heparin and measured tissue LPL mass and activity at a series of times (Fig.4). There was a gradual decline of LPL activity with time. This was more pronounced in tissues from fed rats, from 1700–800 milliunits/g in 60 min compared with 700–600 milliunits/g in fasted rats. In contrast, there was no change in the activity within adipocytes. Hence, heparin mobilized a large fraction of the extracellular enzyme, but none of the intracellular. The inset in Fig. 4 shows the percent change in extracellular activity with time after heparin injection. When the data were expressed in this way, the decline of activity followed the same time course for fed and fasted rats, and the percent of the initial extracellular activity that was lost over the 60 min studied was also the same, about 607 in both fed and fasted rats. The changes in LPL mass (Fig. 4, lower panel) followed the same trends as for LPL activity. Heparin extracted more LPL mass in the fed compared with the fasted rats, and the changes occurred only in the extracellular portion. Within the adipocytes, LPL mass remained constant. The specific activity of tissue LPL was 0.93 milliunit/ng in the fed rats and 0.39 milliunit/ng in the fasted rats. A calculation of the specific activity for the enzyme that was lost from the tissue in 60 min after heparin injection returns values of 1.8 milliunits/ng in the fed and 1.7 milliunits/ng in the fasted rats. These figures carry a large uncertainty since they were calculated as the ratio of differences between rather large values. Furthermore, they do not take into account the LPL that was produced and released from the adipocytes during the 1-h experiment. Nonetheless, the figures indicate that heparin released only, or at least mainly, the active species of LPL. This study shows that most of the LPL protein in adipose tissue is located extracellularly, irrespective of the nutritional state. Some earlier studies had indicated that regulation of LPL activity is exerted by changes in the proportion of newly synthesized enzyme that is secreted from the cells (9Doolittle M.H. Ben-Zeev O. Elovson J. Martin D. Kirchgessner T.G. J. Biol. Chem. 1990; 265: 4570-4577Google Scholar, 12Lee J.J. Smith P.J. Fried S.K. J. Nutr. 1998; 128: 940-946Google Scholar, 25Garfinkel A.G. Nilsson-Ehle P. Schotz M.C. Biochim. Biophys. Acta. 1976; 424: 264-273Google Scholar). If so, the larger amount of inactive LPL in adipose tissue of fasted rats would be expected to be present within adipocytes, retained in the ER. The specific activity of intracellular LPL would be lower in fasted compared with fed rats, whereas the specific activity of the extracellular enzyme would be similar in both nutritional states. In contrast to these predictions, we found that the specific activity of intracellular LPL remained the same. It was the specific activity of the extracellular enzyme that changed. A confounding factor could have been that the turnover rates for the different pools of LPL changed with the nutritional state. In fasted rats an increased turnover rate for inactive LPL in adipocytes might compensate so that mass remained relatively constant even though more enzyme was retained in the ER. Conversely, the turnover rate of extracellular LPL would have to be slower in fasted rats so that mass remained high even though secretion was reduced. We did not, however, find any marked differences with nutritional state in the turnover rates for any of the pools of LPL. Our data thus indicate that the nutritional regulation of adipose LPL is exerted on the activity state of the extracellular enzyme. Ben-Zeev et al. recently studied the synthesis and maturation of human LPL transfected into Chinese hamster ovary cells (16Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Google Scholar). A large fraction of newly synthesized LPL was retained in the ER, formed disulfide-linked aggregates, and was degraded through an energy-dependent pathway. In concert with this, we found that about 707 of LPL protein within the adipocytes was inactive. In a pulse-chase study with guinea pig adipocytes, Semb and Olivecrona found that 657 of newly synthesized LPL was degraded within 4 h (24Semb H. Olivecrona T. Biochim. Biophys. Acta. 1987; 921: 104-115Google Scholar). Speake et al. (26Speake B.K. Parkin S.M. Robinson D.S. Biochim. Biophys. Acta. 1985; 840: 419-422Google Scholar) and Lee et al. (12Lee J.J. Smith P.J. Fried S.K. J. Nutr. 1998; 128: 940-946Google Scholar) have also shown that LPL is degraded in rat adipose tissue. All these data are in agreement with the hypothesis that a large fraction of newly synthesized LPL protein does not fold correctly, is therefore rejected by ER quality control mechanisms (27Ellgaard L. Helenius A. Curr. Opin. Cell Biol. 2001; 13: 431-437Google Scholar), aggregates, and is degraded within the ER. The active species made up about 307 of total LPL protein in the adipocytes. From the pattern of glycosylation Been-Zeev et al. concluded that most of the catalytically active LPL molecules were still in the ER (16Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Google Scholar). Roh et al. separated different intracellular structures from rat adipocytes by centrifugation (28Roh C. Roduit R. Thorens B. Fried S. Kandror K.V. J. Biol. Chem. 2001; 276: 35990-35994Google Scholar). They found that LPL was mainly in the ER with a small pool being present in low-density membrane vesicles that probably represent a secretory compartment in adipocytes. In pulse-chase experiments with guinea pig adipocytes, Semb and Olivecrona found that about one-third of pulse-labeled LPL had appeared in the medium within 30 min of chase (24Semb H. Olivecrona T. Biochim. Biophys. Acta. 1987; 921: 104-115Google Scholar). This figure rose to 497 by 1 h. After that there was no further appearance of labeled LPL in the medium. These data suggest that when the LPL molecules have been cleared by the quality control system, they move rapidly to the cell surface. Several laboratories have reported that short-term changes of LPL activity in adipose tissue occur without corresponding changes in LPL mRNA (9Doolittle M.H. Ben-Zeev O. Elovson J. Martin D. Kirchgessner T.G. J. Biol. Chem. 1990; 265: 4570-4577Google Scholar, 29Enerbäck S. Semb H. Tavernier J. Bjursell G. Olivecrona T. Gene. 1988; 64: 97-106Google Scholar), relative rates of LPL biosynthesis (8Semb H. Olivecrona T. Biochim. Biophys. Acta. 1986; 878: 330-337Google Scholar, 12Lee J.J. Smith P.J. Fried S.K. J. Nutr. 1998; 128: 940-946Google Scholar), or LPL mass ((9, 15). There are also reports that LPL activity in adipocytes isolated from fed or fasted rats is about the same (30Cunningham V.J. Robinson D.S. Biochem. J. 1969; 112: 203-209Google Scholar). In concert with this, we found no difference in adipocyte LPL activity with nutritional state. In addition, our data show that there was no difference with nutritional state in LPL mass or in the distribution of LPL protein between inactive and active forms within the adipocytes. To gain information on where in the tissue the extracellular LPL is localized we injected heparin. Tissue LPL activity decreased by only about 107 during the first 2 min. This presumably represented the LPL molecules that were at the endothelial surface (17Kuwajima M. Foster D.W. McGarry J.D. Metabolism. 1988; 37: 597-601Google Scholar, 31Nilsson-Ehle P. Garfinkel A.S. Schotz M.C. Annu. Rev. Biochem. 1980; 49: 667-693Google Scholar, 32Borensztajn J. Borensztajn J. Lipoprotein Lipase. Evener, Chicago1987: 133-148Google Scholar). Over the next 60 min an additional 407 of the LPL activity disappeared from the tissue. That tissue LPL activity continued to decrease long after of the endothelial-bound enzyme had been released supports the view that there is a relatively large pool of LPL molecules that recirculate between the luminal surface of the endothelium and extravascular sites in the tissue (1Goldberg I.J. J. Lipid Res. 1996; 37: 693-707Google Scholar, 2Olivecrona T. Olivecrona G. Betteridge D.J. Illingworth D.R. Shepherd J. Lipoproteins in Health and Disease. Arnold, London1999: 223-246Google Scholar). Calculation of the specific activity indicated that heparin extracted mainly, or perhaps only, the active form of the enzyme. This is expected since only this form has high affinity for heparin/heparan sulfate (33Lookene A. Chevreuil O. Ostergaard P. Olivecrona G. Biochemistry. 1996; 35: 12155-12163Google Scholar). That heparin releases primarily the active form of LPL is in concert with an earlier study on the specific activity of the LPL released into blood after injection of heparin (34Olivecrona G. Hultin M. Savonen R. Skottova N. Lookene A. Tugrul Y. Olivecrona T. Woodford F.P. Davignon J. Sniderman A.D. Atherosclerosis X. Elsevier Science Publishing Co., Inc., New York1995Google Scholar). One hour after injection of heparin, tissue LPL activity had decreased by about 507. This corresponds to more than two-thirds of the extracellular LPL activity. In contrast, LPL activity within the adipocytes did not change. This differs from what is usually seen in experiments with cultured cells where heparin increases the release of LPL activity to the medium at the expense of cell-associated LPL (16Ben-Zeev O. Mao H.Z. Doolittle M.H. J. Biol. Chem. 2002; 277: 10727-10738Google Scholar,24Semb H. Olivecrona T. Biochim. Biophys. Acta. 1987; 921: 104-115Google Scholar, 35Cryer A. Davies P. Williams E.R. Robinson D.S. Biochem. J. 1975; 146: 481-488Google Scholar). The mechanism for this enhanced release is presumably that heparin acts as a soluble ligand for the enzyme and prevents its recycling into the cells (36Cupp M. Bensadoun A. Melford K. J. Biol. Chem. 1987; 262: 6383-6388Google Scholar, 37Friedman G. Chajek-Shaul T. Olivecrona T. Stein O. Stein Y. Biochim. Biophys. Acta. 1982; 711: 114-122Google Scholar). One might have expected a similar process in the tissue, i.e. that heparin would drive the enzyme out from the cells toward the endothelium. That this did not happen indicates that even without exogenous heparin there is an excess of liganding sites in the tissue to receive the enzyme as it emerges from the adipocytes. Heparin released more than five times as much LPL activity from the adipose tissue of fed compared with fasted rats. When we calculated the release as fraction of the active, extracellular LPL in the tissue (Fig. 4, inset), the time course and extent of release was similar for the two nutritional states. This indicates that the active, extracellular LPL was equally accessible to release by heparin in both nutritional states. Hence, the larger release in the fed rats was a reflection of the larger amount of active extracellular LPL rather than due to some difference in how the enzyme was located in the tissue. In a recent study we found that down-regulation of LPL activity in adipose tissue during fasting requires that a gene, separate from the lipase gene, is switched on (7Bergö M. Wu G. Ruge T. Olivecrona T. J. Biol. Chem. 2002; 277: 11927-11932Google Scholar). The mechanism by which the product of this gene accomplishes the switch from predominantly active to predominantly inactive form(s) of LPL is not clear. Here we show that the switch primarily affects the extracellular LPL. In vivoexperiments, as used here, are too complex to unravel the mechanism. Studies with adipocytes have shown relatively small or no differences in LPL activity and secretion between cells from fasted compared with fed rats (12Lee J.J. Smith P.J. Fried S.K. J. Nutr. 1998; 128: 940-946Google Scholar, 24Semb H. Olivecrona T. Biochim. Biophys. Acta. 1987; 921: 104-115Google Scholar, 25Garfinkel A.G. Nilsson-Ehle P. Schotz M.C. Biochim. Biophys. Acta. 1976; 424: 264-273Google Scholar, 35Cryer A. Davies P. Williams E.R. Robinson D.S. Biochem. J. 1975; 146: 481-488Google Scholar, 38Spencer I.M. Hutchinson A. Robinson D.S. Biochim. Biophys. Acta. 1978; 530: 375-384Google Scholar, 39Lasuncion M.A. Herrera E. Biochem. J. 1983; 210: 639-643Google Scholar). The next step must therefore be to find a suitable cell system and/or conditions where the putative LPL-regulating gene can be switched on and off and the effects on LPL maturation, trafficking, and stability can be studied in detail.