Advertisement
J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.D700021-JLR200 on September 13, 2007

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
D700021-JLR200v1
48/12/2788    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dallinga-Thie, G. M.
Right arrow Articles by van Tol, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dallinga-Thie, G. M.
Right arrow Articles by van Tol, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 48, 2788-2791, December 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Appraisal of hepatic lipase and lipoprotein lipase activities in mice

G. M. Dallinga-Thie1,*, A. J. Zonneveld-de Boer{dagger}, L. C. van Vark-van der Zee§, R. van Haperen**, T. van Gent**, H. Jansen{dagger}, R. De Crom** and A. van Tol**

* Department of Vascular Medicine, Academic Medical Center, Amsterdam, The Netherlands
{dagger} Department of Clinical Chemistry, Erasmus University Medical Center, Rotterdam, The Netherlands
§ Department of Vascular Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands
** Department of Cell Biology and Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands

Published, JLR Papers in Press, September 13, 2007.

1 To whom correspondence should be addressed. e-mail: g.m.dallinga{at}amc.uva.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.

Supplementary key words high salt • preheparin plasma • postheparin plasma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
LPL and HL play an essential role in lipoprotein metabolism. Hepatic lipase is a lipolytic enzyme synthesized and secreted by hepatocytes that binds extracellularly to parenchymal and endothelial cell surfaces in the space of Disse (1, 2). HL affects the lipid composition of all lipoprotein classes by hydrolyzing phospholipids and triglycerides. It has been demonstrated that mice have HL circulating in preheparin plasma (3), suggesting that the affinity between mouse HL and its cellular binding sites is low. In contrast, rat and human preheparin plasma do not contain any measurable HL activity (3).

LPL is synthesized by parenchymal cells in a variety of tissues, including adipose tissue, skeletal muscle, and heart, and is subsequently translocated to its site of action, the endothelium (4). The major target lipoprotein is the triglyceride-rich apolipoprotein B containing VLDL and chylomicrons. LPL hydrolyzes triglycerides into free fatty acids for direct use in muscle and adipose tissue (5, 6). As a result of the action of LPL, VLDL, and chylomicrons will become triglyceride-depleted, cholesterol-enriched lipoprotein particles, which are potentially atherogenic. LPL may act as a bridging molecule between these atherogenic lipoproteins and hepatic receptors (7, 8).

The mouse is a frequently used animal model in atherosclerosis research, even though large differences exist in lipoprotein metabolism between mice and humans. However, the possibilities to create specific genetic models by overexpression, deletion, specific inducible gene expression, and specific tissue expression of a target gene have led to a spectacular increase in the use of transgenic mice. Recently, newly identified proteins turned out to have LPL-regulating properties [e.g., apolipoprotein A-V (9), the angiopoetin-like proteins (10, 11), and the recently discovered glycosylphosphatidylinositol-anchored HDL binding protein 1 (12)]. This further emphasizes the need for accurate and relatively simple assays of LPL and HL activities.

In humans, LPL and HL are bound to the vascular endothelium, and their activities have been analyzed in plasma obtained from blood samples collected after intravenous injection of heparin, a procedure to release LPL and HL from cellular proteoglycans. Total lipase activity is then analyzed in the presence of the LPL activator apolipoprotein C-II and at low salt concentrations. LPL activity may be inhibited by high salt (1 M NaCl) in the assay mixture, thereby enabling the measurement of HL activity (13). In addition, specific antibodies have been used to inhibit either LPL activity or HL activity in human postheparin plasma (14, 15).

In the present study, we compare two approaches to analyze HL and LPL activity in mouse plasma. In the first approach, we use specific antibodies to inhibit either LPL or HL activity. In the second approach, we test the use of 1 M NaCl to inhibit LPL activity, thereby enabling the assay of HL activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Animals
Male C57BL/6 mice were housed in a temperature-controlled room operating under a 12 h/12 h light/dark cycle. Animals were fed a regular chow diet and obtained water ad libitum. HL-deficient mice with a C57BL/6 background were obtained from the Jackson Laboratories. Preheparin blood was obtained by orbital puncture under isoflurane anesthesia. Postheparin blood was obtained at 5 min after heparin injection (200 U/kg body weight injected into a tail vein). Plasma was isolated by centrifugation at 3,000 rpm for 15 min and frozen at –80°C before analysis of lipase activities. Plasma LPL activity was determined in both preheparin and postheparin plasma obtained from three individual mice with hepatic lipase deficiency. Ten control C57BL/6 mice were used to create a pool of both preheparin and postheparin plasma. All animal experiments were performed in accordance with institutional and national guidelines.

Antibodies
Rat HL and LPL were isolated as described (16–18). Polyclonal antibodies against rat HL and LPL were generated in rabbit (anti-HL) and goat (anti-LPL). The IgG fractions of both antibodies were isolated by protein G affinity chromatography according to the manufacturer's procedure. The IgG fraction was dialyzed against 5 mM (NH4)HCO3 and lyophilized. The obtained pellets were resuspended in 0.15 M NaCl and stored at –80°C (17). Total protein concentration was 18.7 g/l in the anti-HL preparation and 95.3 g/l in the anti-LPL preparation.

Treatment of plasma with antibodies
Immunoinhibition of HL and LPL was accomplished by mixing a mouse plasma sample with anti-LPL and anti-HL and subsequent incubation at 4°C for 2.5 h. As a control, plasma was incubated with a saline solution. For LPL analyses, 25 µl of plasma was incubated with 6.25 or 25 µl of anti-HL in a total volume of 125 µl. For HL analyses, 25 µl of plasma was incubated with 25 or 50 µl of anti-LPL in a total volume of 125 µl. Subsequently, after the preincubation, 50 µl of the mixture was used in the lipase assay.

Lipase assay
Preheparin and postheparin total lipase activities were assayed using an artificial glycerol-[1-14C]trioleate (Sigma T-7140)-containing lipid emulsion (13, 19). A mixture of 20 µCi of [1-14C]trioleylglycerol and 35 mg of unlabeled trioleylglycerol was dried under nitrogen and emulsified by sonification in 2.5 ml of 5% (w/v) gum acacia (BDH Chemicals) containing 1% NaHCO3 (pH 7). The emulsion was mixed with 3 ml of 10% (w/v) fat-free BSA (Fluka BioChemika 05490), 0.5 ml of 1 M Tris-HCl (pH 8.5), and 0.5 ml of 2.5 M NaCl + 4.5 ml of water (low-salt assay) or 5 ml of 2.5 M NaCl (high-salt assay). Lipase activity was analyzed in the presence of heat-inactivated normolipidemic human plasma as a source of apolipoprotein C-II (10%, v/v) (20). A substrate mixture (200 µl), a 10 µl serum sample, and 40 µl of 0.9% NaCl were incubated for 30 min at 30°C. The reaction was stopped by adding 3.25 ml of a mixture of chloroform-methanol-heptane (33:40:27) and 1.05 ml of 0.1 M K2CO3 and 0.1 M H3BO3, pH 10.5, and putting the tubes on ice. Free fatty acids were measured in the water phase. Lipase activities are given in mU/ml, where 1 mU represents 1 nmol of free fatty acid released from the triglyceride substrate per minute at 30°C.

Statistical analyses
Lipase values are expressed as means ± SD and were compared using t-test statistics.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
It is generally accepted that lipase activity in murine preheparin plasma represents HL activity (3), reflecting its low affinity for heparin sulfate proteoglycans. Virtually zero lipase activity was measured in preheparin plasma from HL-deficient mice (data not shown). Subsequently, we analyzed preheparin lipase activity in the presence of different antibodies. First, it was observed that incubation with the anti-LPL antibody does not affect lipase activity in preheparin plasma from wild-type mice (Fig. 1 ). Hence, this antibody does not interfere with mouse HL activity, illustrating its specificity. The efficacy of the antibody directed against HL (anti-HL) to inhibit its activity was tested using the same batch of preheparin plasma. Figure 1 shows that the total lipase activity in preheparin plasma of wild-type mice was 92 ± 2 mU/ml. Treatment with anti-LPL did not result in a significant change in total lipase activity (86 ± 3 mU/ml), whereas treatment with anti-HL inhibited lipase activity by 97% (remaining activity, 3 ± 2 mU/ml). Different antibody dilutions were tested. These experiments illustrate the specificity of the antibody and demonstrate that virtually all lipase circulating in preheparin plasma in mice is indeed HL.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 1. Effects of anti-LPL and anti-HL antibodies on lipase activities in preheparin plasma from wild-type mice. Experiments were performed in triplicate. The indicated amounts of IgG (mg) were present in the lipase assay. Values are expressed as means ± SD.

 
In the experiments described above, all assays were performed at low NaCl concentration. In humans, both HL and LPL activities are present in postheparin plasma, and the contribution of HL can be measured by blocking LPL activity with 1 M NaCl (21). However, in our experiments with mouse plasma, increasing the final salt concentration in the assay mixture to 1 M NaCl led to a significant 61% increase in HL activity in preheparin mouse plasma (from 92 ± 2 to 148 ± 3 mU/ml). Total lipase activity in postheparin plasma of wild-type mice was 717 ± 10 mU/ml (Fig. 2 ). Surprisingly, in the presence of 1 M NaCl, the residual lipase activity remained 579 ± 30 mU/ml. This suggests that either most of the total lipase activity is derived from HL in postheparin mouse plasma or that 1 M NaCl does not completely inhibit mouse LPL. We also have to keep in mind that HL activity is increased in the presence of high salt (see above).


Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. Effects of incubation with anti-LPL antibody on lipase activities in postheparin plasma from wild-type mice and HL-deficient mice. Experiments were performed in triplicate. The indicated amounts of IgG (mg) were present in the lipase assay. Values are expressed as means ± SD.

 
To obtain conclusive evidence regarding whether 1 M NaCl inhibits LPL activity in mouse plasma, we analyzed total lipase activity in postheparin plasma of the HL-deficient mouse with and without 1 M NaCl. In these knockout mice, HL was not present in postheparin plasma. Total lipase activity was 755 ± 17 mU/ml when measured in low NaCl, whereas plasma lipase activity in 1 M NaCl was 456 ± 14 mU/ml (Fig. 3 ). Thus, 1 M NaCl only partly inhibited postheparin plasma lipase activity in HL-deficient mice, suggesting that 1 M NaCl does not completely inhibit mouse LPL. Alternatively, another lipase could contribute to total lipase activity in these animals, although no evidence for an additional lipase in mice could be found (3). Recently, endothelial lipase (EL) was described as a new member of the lipase family (22). Despite the fact that a significant degree of sequence homology exists between EL and LPL, its substrate specificity is quite different (23). In contrast to LPL, which has the capacity to hydrolyze triglycerides, EL is active as a phospholipase. Because our assay measures the capacity of the enzymes to hydrolyze triglycerides, it is unlikely that we measured EL activity.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Effects of incubation with anti-HL antibody on lipase activities in postheparin plasma from wild-type mice and HL-deficient mice. Experiments were performed in triplicate. The indicated amounts of IgG (mg) were present in the lipase assay. Values are expressed as means ± SD.

 
In another series of experiments, we used anti-LPL and anti-HL in postheparin mouse plasma of both wild-type animals and HL-deficient animals. All experiments were again performed under low-salt conditions and in the presence of heat-inactivated plasma as a source of apolipoprotein C-II. Incubation of postheparin plasma from HL-deficient mice with anti-LPL resulted in a 95% inhibition of lipase activity, as shown in Fig. 2. Total lipase activity decreased from 755 ± 17 to 37 ± 2 mU/ml. Therefore, it can be concluded that there is no alternative lipase that significantly contributes to postheparin lipase activity other than LPL. When using plasma from wild-type animals, plasma total lipase activity in postheparin plasma after treatment with anti-LPL was inhibited from 717 ± 10 to 110 ± 2 mU/ml. As shown in Fig. 2, no further inhibition can be achieved by increasing the antibody concentration. Figure 3 shows the results of similar experiments with postheparin plasma from wild-type mice, but after treatment with anti-HL. We already showed (Fig. 1) that the decrease in HL activity in preheparin plasma was almost complete under these conditions. In postheparin plasma from wild-type mice, treatment with anti-HL resulted in a decrease of the lipase activity of only ~10% (from 657 ± 10 to 590 ± 8 mU/ml). Therefore, HL activity in postheparin plasma was 67 mU/ml. This value is similar to the preheparin plasma HL activity shown in Fig. 1. Figure 3 shows that little if any effect of anti-HL treatment was evident in postheparin plasma from HL-deficient mice.

It can be concluded that virtually all active HL is circulating in wild-type mouse plasma, even before heparin injection, and that liver-bound HL is hardly present. Based on these results, we propose to measure LPL activity in postheparin plasma by analyzing total lipase activity in the presence of apolipoprotein C-II as activator and under low-salt concentration. Of course, this activity measured in postheparin plasma has to be corrected for the small amount of HL activity already present in preheparin plasma to obtain the actual postheparin LPL activity. Thus, total postheparin plasma activity minus preheparin plasma activity equals postheparin plasma LPL activity.

Manuscript received July 27, 2007 and in revised form August 17, 2007 and in re-revised form September 4, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
  1. Jansen, H., A. J. Verhoeven, and E. J. Sijbrands. 2002. Hepatic lipase: a pro- or anti-atherogenic protein? J. Lipid Res. 43: 1352–1362.[Abstract/Free Full Text]

  2. Olivecrona, T., and G. Bengtsson-Olivecrona. 1993. Lipoprotein lipase and hepatic lipase. Curr. Opin. Lipidol. 4: 187–196.

  3. Peterson, J., G. Bengtsson-Olivecrona, and T. Olivecrona. 1986. Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin. Biochim. Biophys. Acta. 878: 65–70.[Medline]

  4. Mead, J. R., S. A. Irvine, and D. P. Ramji. 2002. Lipoprotein lipase: structure, function, regulation, and role in disease. J. Mol. Med. 80: 753–769.[CrossRef][Medline]

  5. Eckel, R. H. 1989. Lipoprotein lipase: a multifactorial enzyme relevant to common metabolic disease. N. Engl. J. Med. 320: 1060–1068.[Abstract]

  6. Goldberg, I. J. 1996. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. J. Lipid Res. 37: 693–707.[Abstract]

  7. Beisiegel, U., W. Weber, and G. Bengtsson Olivecrona. 1991. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc. Natl. Acad. Sci. USA. 88: 8342–8346.[Abstract/Free Full Text]

  8. Heeren, J., A. Niemeier, M. Merkel, and U. Beisiegel. 2002. Endothelial-derived lipoprotein lipase is bound to postprandial triglyceride-rich lipoproteins and mediates their hepatic clearance in vivo. J. Mol. Med. 80: 576–584.[CrossRef][Medline]

  9. Merkel, M., B. Loeffler, M. Kluger, N. Fabig, G. Geppert, L. A. Pennacchio, A. Laatsch, and J. Heeren. 2005. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase. J. Biol. Chem. 280: 21553–21560.[Abstract/Free Full Text]

  10. Koster, A., Y. B. Chao, M. Mosior, A. Ford, P. A. Gonzalez-DeWhitt, J. E. Hale, D. Li, Y. Qiu, C. C. Fraser, D. D. Yang, et al. 2005. Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism. Endocrinology. 146: 4943–4950.[Abstract/Free Full Text]

  11. Shimizugawa, T., M. Ono, M. Shimamura, K. Yoshida, Y. Ando, R. Koishi, K. Ueda, T. Inaba, H. Minekura, T. Kohama, et al. 2002. ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase. J. Biol. Chem. 277: 33742–33748.[Abstract/Free Full Text]

  12. Beigneux, A. P., B. S. Davies, P. Gin, M. M. Weinstein, E. Farber, X. Qiao, F. Peale, S. Bunting, R. L. Walzem, J. S. Wong, et al. 2007. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons. Cell Metab. 5: 279–291.[CrossRef][Medline]

  13. Huttunen, J. K., C. Ehnholm, P. K. Kinnunen, and E. A. Nikkila. 1975. An immunochemical method for the selective measurements of two triglyceride lipases in human postheparin plasma. Clin. Chim. Acta. 63: 335–347.[CrossRef][Medline]

  14. Greten, H., V. Laible, G. Zipperle, and J. Augustin. 1977. Comparison of assay methods for selective measurement of plasma lipase. The effect of clofibrate on hepatic and lipoprotein lipase in normals and patients with hypertriglyceridemia. Atherosclerosis. 26: 563–572.[CrossRef][Medline]

  15. Olivecrona, T., and G. Olivecrona. 1997. Determination and clinical significance of lipoprotein lipase and hepatic lipase. In Handbook of Lipoprotein Testing. N. Rifai, G. R. Warnick, and M. H. Dominiczak, editors. AACC Press, Washington, DC. 373–392.

  16. Persoon, N. L., W. C. Hulsmann, and H. Jansen. 1986. Localization of the salt-resistant heparin-releasable lipase in the rat liver, adrenal and ovary. Eur. J. Cell Biol. 41: 134–137.[Medline]

  17. Vieira-van Bruggen, D., I. Kalkman, T. van Gent, A. van Tol, and H. Jansen. 1998. Induction of adrenal scavenger receptor BI and increased high density lipoprotein-cholesteryl ether uptake by in vivo inhibition of hepatic lipase. J. Biol. Chem. 273: 32038–32041.[Abstract/Free Full Text]

  18. Persoon, N. L., W. C. Hulsmann, and H. Jansen. 1987. Structural modulation of salt-resistant rat-liver lipase alters the relative phospholipase and triacylglycerol hydrolase activities. Biochim. Biophys. Acta. 917: 186–193.[Medline]

  19. Jansen, H., W. Hop, A. van Tol, A. V. G. Bruschke, and J. C. Birkenhäger. 1994. Hepatic lipase and lipoprotein lipase are not major determinants of the low density lipoprotein subclass pattern in human subjects with coronary heart disease. Atherosclerosis. 107: 45–54.[CrossRef][Medline]

  20. Bengtsson, G., and T. Olivecrona. 1982. Activation of lipoprotein lipase by apolipoprotein CII. Demonstration of an effect of the activator on the binding of the enzyme to milk-fat globules. FEBS Lett. 147: 183–187.[CrossRef][Medline]

  21. Bensadoun, A. 1991. Lipoprotein lipase. Annu. Rev. Nutr. 11: 217–237.[CrossRef][Medline]

  22. Jaye, M., K. J. Lynch, T. Krawiec, D. Marchadier, C. Maugeais, K. Doan, V. South, D. Amin, M. Perrone, and D. J. Rader. 1999. A novel endothelial-derived lipase that modulates HDL metabolism. Nat. Genet. 21: 424–428.[CrossRef][Medline]

  23. Griffon, N., E. C. Budreck, C. J. Long, U. C. Broedl, D. H. Marchadier, J. M. Glick, and D. J. Rader. 2006. Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras. J. Lipid Res. 47: 1803–1811.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
D700021-JLR200v1
48/12/2788    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dallinga-Thie, G. M.
Right arrow Articles by van Tol, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dallinga-Thie, G. M.
Right arrow Articles by van Tol, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today 
Advertisement
spacer
Advertisement
Advertisement