The role of lipoprotein lipase in adipose tissue development ... - Nature

International Journal of Obesity (2000) 24, Suppl 4, S53±S56 ß 2000 Macmillan Publishers Ltd All rights reserved 0307±0565/00 $15.00 www.nature.com/ijo

The role of lipoprotein lipase in adipose tissue development and metabolism R Zechner1*, J Strauss1, S Frank2, E Wagner1, W Hofmann1, D Kratky1, M Hiden1 and S Levak-Frank2 1

Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Graz, Austria; and 2Institute of Medical Biochemistry and Medical Molecular Biology, University of Graz, Graz, Austria

Lipoprotein lipase (LPL) is essential for the hydrolysis and distribution of triglyceride-rich lipoprotein-associated fatty acids among extrahepatic tissues. Additionally, the enzyme facilitates several non-lipolysis associated functions including the cellular uptake of whole lipoprotein particles and lipophilic vitamins. The tissue-speci®c variations of LPL expression have been implicated in the pathogenesis of various lipid disorders, obesity and atherosclerosis. Transgenic technology provided the means to study the physiological response to the overexpression or absence of the enzyme in adipose tissue, muscle and macrophages. The effects of varying LPL expression in adipose tissue and muscle are summarized in this article. International Journal of Obesity (2000) 24, Suppl 4, S53±S56 Keywords: lipoprotein lipase; adipose tissue; muscle; transgenic mouse

Introduction Fat accumulation in adipose tissue (AT) largely depends on the ef®cient uptake of fatty acids from the circulation. Since the majority of fatty acids in plasma are present in esteri®ed form as lipoprotein associated triglycerides (TG), hydrolysis is a mandatory initial step for the tissue absorption of free fatty acids (FFA). In peripheral tissues, such as AT, the rate-limiting step for TG catabolism is catalyzed by lipoprotein lipase (LPL), an enzyme which is bound to glucosaminoglycans at the luminal side of the capillary endothelium. The activity of LPL in AT relative to its expression in other tissues, such as skeletal muscle (SM) and cardiac muscle (CM), determines the in¯ux of FFA and, possibly, the amount of fat deposited. LPL is therefore viewed as a `gate keeping' enzyme for the entry and reesteri®cation of FFA in AT.1 According to this hypothesis, decreased LPL activity in AT could reduce FFA uptake and lipid accumulation. Alternatively, overexpression of LPL in muscle might be associated with the redirection of nutrient fats from AT to muscle (`substrate steal'), again preventing FFA uptake in adipocytes. It is likely, therefore, that LPL and its tissue-speci®c regulation are centrally involved in the pathogenesis of obesity. In addition, LPL plays a key role in the metabolism of lipoproteins, which makes it an impor*Correspondence: R Zechner, Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Heinrichstrasse 31a, 8010 Graz, Austria. E-mail: [email protected]

tant factor in vascular disease as well. The advent of transgenic technology provided the means to directly modulate LPL expression levels in mice in a tissuedependent manner and study the tissue-speci®c role of the enzyme. This article will summarize some of the ®ndings that emerged from these studies.

Disruption of the mouse LPL gene Familial LPL de®ciency is a rare autosomal recessive disorder in humans, characterized by a massive accumulation of chylomicrons (type I hyperlipoproteinemia).2 Homozygous patients, when not kept under strict dietary control, exhibit plasma triglycerides above 3000 mg=dl, essentially lack HDL and suffer from recurring episodes of pancreatitis. To date more than 80 different mutations within the LPL structural gene are known to cause the disorder. Generally, LPL de®ciency is not associated with an increased incidence of atherosclerosis; however, in some families an increased prevalence of heart disease has been reported.3 This observation indicated that mutationspeci®c variations exist with regard to atherosclerosis susceptibility. Interestingly, patients with LPL de®ciency have normal amounts of AT, suggesting alternative, LPL-independent pathways to accumulate TG in adipocytes.4 Several years ago two laboratories reported independently the generation of LPL knock-out mice.5,6 Homozygous LPL-de®cient animals also developed severe hypertriglyceridemia, hypercholesterolemia and low HDL, but in contrast to humans all knock-out animals

Lipoprotein lipase in adipose tissue development R Zechner et al

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died within 30 h after birth. This phenotype was similar to the one observed in the well-studied cld=cld-mouse, namely severe hypertriglyceridemia and post-natal death as a result of the de®ciency of enzymatically active LPL and hepatic lipase.7 Both mouse models, cld=cld and LPL knock-out, suggested that LPL-de®cient mice, in contrast to humans, cannot tolerate the massive amount of TG that accumulates in the blood as soon as the newborn animals begin to suckle. Although the exact cause of death has not been elucidated, it has been speculated that the excessive blood-lipid content could clog the lung capillaries and interfere with a functional gas exchange, resulting in alveolar dysfunction and cyanosis.5 Hypoglycemia was proposed as an alternative cause of death by Merkel et al,8 when the authors observed that newborn LPL knock-out mice had unusually low blood glucose levels. Recent experiments in our laboratory utilizing the transient post-natal adenovirus mediated expression of LPL has enabled us to rescue LPL knock-out mice during the suckling period and generate adult, LPL-de®cient animals to an age well over 12 months. These mice exhibited increased TG levels, extremely low HDL concentrations and increased FFA and ketones in plasma when kept on a chow diet. Gross morphological examination revealed the presence of roughly normal amounts of AT.

Tissue-speci®c overexpression of LPL Skeletal muscle

LPL overexpression in skeletal muscle (SM) was achieved by fusing an LPL minigene to the promoter of the mouse muscle creatine kinase gene.9 ± 11 A 5fold overexpression of LPL in SM caused a marked decrease in TG rich lipoproteins, decreased HDL levels, and low plasma FFA concentrations.9,10 The work by Jensen et al10 provided convincing evidence that low-level overexpression of LPL in SM can decrease a diet-induced weight gain in agreement with the `substrate steal' concept. Higher levels of LPL overexpression, however, resulted in the proliferation of mitochondria and peroxisomes in SM, which eventually led to a severe myopathy and premature death.9 These experiments indicated that TG hydrolysis by SM-LPL and the subsequent uptake of the FFA by muscle tissue is very ef®cient. The apparently unrestricted FFA uptake that leads to myopathy suggested that LPL is the major determinant for FFA uptake in muscle and that no additional regulatory mechanism exists to prevent the ultimately lethal FFA accumulation in SM. Muscle-speci®c LPL overexpression in transgenic mice also enabled an investigation of the role of LPL in the uptake of apolar lipids (cholesteryl esters) and lipophilic vitamins (vitamin E).12,13 A clear linear relationship was observed between LPL activity in International Journal of Obesity

SM and the uptake of a-tocopherol, providing in vivo evidence for the involvement of LPL in cellular vitamin E uptake.

Cardiac muscle

LPL overexpression in cardiac muscle (CM) was achieved by the fusion of the LPL minigene with promoter sequences from the mouse LPL gene.14 0 Eight kilobases (kb) of 5 ¯anking region of the mouse LPL gene were suf®cient for CM-speci®c LPL expression, but were unable to drive transgene transcription in SM or AT. Even moderate levels of CM-speci®c LPL overexpression had a pronounced effect on VLDL catabolism and plasma TG levels, indicating a particularly powerful role of CM-LPL in the catabolism of TG-rich lipoproteins.

Liver

Liver-speci®c overexpression of LPL was achieved by the fusion of the minigene with the promoter for apolipoprotein AI.8 Despite a 4 ± 9-fold overexpression of the human enzyme in the liver, the effects on plasma lipids and liver lipid accumulation were quite moderate. Interestingly, however, these animals exhibited a marked increase in plasma ketones in response to increased FFA uptake in the liver.

Tissue-speci®c LPL transgene expression on an LPL knock-out background Crossbreeding of tissue-speci®c LPL overexpressing transgenic animals with heterozygous LPL knock-out animals resulted in mice that expressed only the transgene but not the endogenous LPL gene. Depending on the tissue of transgene expression, these animals produced LPL activity only in SM, CM or liver but lacked the enzyme in all other tissues, including AT and macrophages. Independent of the site of LPL expression, all mouse lines were rescued from the lethal hypertriglyceridemia present in LPL knock-out mice. This ®nding indicated that LPL expression in any one of these tissues is adequate for survival. Conversely, complete loss of LPL expression in any of these tissues does not cause lethality. In fact, all of the above mouse lines remain grossly healthy and have a normal life expectancy. Additionally, LPL activity measurements in various tissues of these `single tissue' expressors demonstrated that LPL predominantly remains in the tissue where it was originally synthesized. No evidence was obtained for the translocation of a considerable amount of enzymatically active enzyme from the site of synthesis to other tissues.


LPL-de®cient AT

The lack of LPL in AT of mice expressing LPL exclusively in SM or CM14 ± 16 did not cause a massive loss of AT mass or altered body mass when the animals were kept on a chow diet. However, leptinde®cient animals were found to be much more resistant to obesity when they lacked LPL in AT.16 The drastically reduced content of polyunsaturated fatty acids (PUFA) in the AT lipid moiety of AT-LPL de®cient mice suggested, however, that the lack of import of exogenous FFA was replaced by endogenous FFA and TG synthesis. Recent efforts in this laboratory to analyze the activity of glycolytic and lipogenic enzymes support this hypothesis. Additionally, other mechanisms are conceivable that could alleviate LPL de®ciency and facilitate lipid synthesis in AT under normal dietary conditions. These include the increased receptor-mediated uptake of whole lipoprotein particles (eg VLDL receptor) as well as the action of alternative lipases such as the recently discovered endothelial derived lipase.17,18 LPL-de®cient SM and AT

The loss of 80% of total body LPL due to the LPL de®ciency in AT and SM in mice expressing LPL exclusively in CM did not cause hypertriglyceridemia or decreased HDL cholesterol levels when kept on normal chow diet.14 This unexpected result again illustrates the powerful role of CM-LPL in the clearance of plasma TG and suggests that the site of LPL expression is more important than the overall amount of LPL for a functional metabolism of lipids and lipoproteins. Furthermore, the lack of LPL in SM in mice that expressed LPL exclusively in the heart did not cause marked morphological or histopathological changes in either muscle type, indicating that the alternative use of glucose and nonesteri®ed fatty acids (derived from adipose tissue lipolysis) as energy substrate was suf®cient to sustain normal energy metabolism. The effects of decreased PUFA content on membrane properties and function, or prostaglandin metabolism, remain to be investigated. LPL-de®cient macrophages

The role of macrophage LPL in the formation of foam cells and atherosclerotic lesions is not well understood and has received signi®cant attention recently. In the absence of LPL, the ability of macrophages to bind and take up TG-rich lipoproteins (VLDL) or modi®ed lipoproteins (oxidized LDL) is strongly reduced.19 Importantly, bone marrow transplantation experiments revealed that the absence of LPL in monocytes=macrophages markedly reduces the susceptibility of C57B16 mice to develop atherosclerotic lesions when fed a high cholesterol diet.20,21 In contrast to the bene®cial role of high LPL activities in peripheral tissues to ensure low TG and high HDL cholsterol levels, thereby decreasing the atherosclerotic risk, high LPL activities in macrophages of mice

appear to induce lipid accumulation, foam cell formation and atherosclerosis susceptibility.

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Future directions The initial experiments using induced mutant mouse lines have clearly demonstrated that variations in the tissue speci®c expression pattern of LPL can affect the lipid transport system, the distribution of energy substrate and the pathophysiology of obesity and atherosclerosis. In fact, the site of enzyme expression might be more important than the undifferentiated, additive activity that is generally measured in postheparin plasma. To strengthen the evidence provided from these initial observations, future work will have to address the following topics. 1. It will be necessary to generate and study conditional knock-out mice by utilizing the cre-loxP technology. This will permit more physiologically relevant modulations of LPL activities in single tissues that resemble more closely the in vivo situation of varying tissue-speci®c LPL activities in response to nutritional and hormonal changes compared to current animal models. Additionally, to date no transgenic mouse lines have been reported which overexpress LPL in AT. It will be interesting to observe whether a marked overexpression of the enzyme in adipocytes can cause obesity in mice or whether alternative mechanisms can prevent fat accumulation. 2. It will be important to elucidate the metabolic changes in adipocytes that lack LPL. Preliminary experiments indicate that the decreased FFA import under this condition can initiate a whole spectrum of metabolic adaptations which allow the fat cell to continue to produce considerable amounts of fat. These alternative pathways of lipogenesis need to be characterized with regard to their initiation and regulation. 3. The gene regions that regulate tissue-speci®c transcription of LPL and the corresponding transcription factors will have to be characterized in more detail. Presently, insuf®cient information is available describing the major enhancer regions that control LPL expression in AT, SM, CM and macrophages. Yet, only a detailed understanding of the transcriptional mechanisms that govern LPL expression in distinct tissues will enable the development of drugs for potential pharmacological interventions.

Acknowledgement

This work was supported by the Austrian FWF grants F701, F713 and P11696. International Journal of Obesity


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References

1 Greenwood MRC, Cleary MP, Steingrimsdottir L, Vassell JR. Adipose tissue metabolism and genetic obesity: the LPL hypothesis. Recent Adv Obes Res 1981; 3: 75 ± 79. 2 Brunzell JD. Familial lipoprotein lipase de®ciency and other causes of the chylomicronemia syndrome. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill: New York, 1995, pp 1913 ± 1932. 3 Benlian P, De Gennes JL, Foubert L, Zhang H, Gagne SE, Hayden M. Premature atherosclerosis in patients with familial chylomicronemia caused by mutations in the lipoprotein lipase gene. New Engl J Med 1996; 335: 848 ± 854. 4 Brun LD, Gagne C, Julien P, Tremblay A, Moorjani S, Bouchard C, Lupien PJ. Familial lipoprotein lipase-activity de®ciency: study of total body fatness and subcutaneous fat tissue distribution. Metabolism 1989; 38: 1005 ± 1009. 5 Weinstock PH, Bisgaier CL, Aalto-SetaÈla K, Radner H, Ramakrishnan R, Levak-Frank S, Essenburg AD, Zechner R, Breslow JL. Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. J Clin Invest 1995; 96: 2555 ± 2568. 6 Coleman T, Seip RL, Gimble JM, Lee D, Maeda N, Semenkovich CF. COOH-disruption of lipoprotein lipase in mice is lethal in homozygotes, but heterozygotes have elevated triglycerides and impaired enzyme activity. J Biol Chem 1995; 270: 12518 ± 12525. 7 Paterniti JR Jr, Brown WV, Ginsberg HN, Artzt K. Combined lipase de®ciency (cld): a lethal mutation on chromosome 17 of the mouse. Science 1983; 221: 167 ± 169. 8 Merkel M, Weinstock PH, ChajekShaul T, Radner H, Yin BY, Breslow JL, Goldberg IJ. Lipoprotein lipase expression exclusively in liver Ð a mouse model for metabolism in the neonatal period and during cachexia. J Clin Invest 1998; 102: 893 ± 901. 9 Levak-Frank S, Radner H, Walsh AM, Stollberger R, Knipping G, Hoe¯er G, Sattler W, Breslow JL, Zechner R. Muscle speci®c overexpression of lipoprotein lipase causes a severe myopathy characterized by proliferation of mitochondria and peroxisomes in transgenic mice. J Clin Invest 1995; 96: 976 ± 986. 10 Jensen DR, Schlaepfer IR, Morin CL, Pennington DS, Marcell T, Ammon SM, Gutierrez-Hartmann A, Eckel RH. Prevention of diet-induced obesity in transgenic mice overexpressing skeletal muscle lipoprotein lipase. Am J Physiol 1997; 42: R683 ± R689. 11 Schlaepfer IR, Eckel RH. Plasma triglyceride reduction in mice after direct injections of muscle-speci®c lipoprotein lipase DNA. Diabetes 1999; 48: 223 ± 227.

International Journal of Obesity

12 PanzenboÈck U, Wintersberger A, Levak-Frank S, Zimmermann R, Zechner R, Kostner GM, Malle E, Sattler W. Implications of endogenous and exogenous lipoprotein lipase for the selective uptake of HDL-3 associated cholesterylesters by mouse peritoneal macrophages. J Lipid Res 1997; 38: 239 ± 253. 13 Sattler W, Levak-Frank S, Radner H, Kostner GM, Zechner R. Muscle speci®c overexpression of lipoprotein lipase in transgenic mice results in increased a-tocopherol levels in skeletal muscle. Biochem J 1996; 318: 15 ± 19. 14 Levak-Frank S, Hofmann W, Weinstock PH, Radner H, Sattler W, Breslow JL, Zechner R. Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle but not in skeletal muscle and adipose tissue have normal plasma triglyceride and HDL-cholesterol levels. Proc Natl Acad Sci USA 1999; 96: 3165 ± 3170. 15 Levak-Frank S, Weinstock PH, Hayek T, Hofmann W, Verdery R, Breslow JL, Zechner R. Induced mutant mice expressing lipoprotein lipase exclusively in muscle have subnormal triglycerides yet reduced HDL-cholesterol levels in plasma. J Biol Chem 1997; 272: 17182 ± 17190. 16 Weinstock PH, Levak-Frank S, Hudgins LC, Radner H, Friedman JM, Zechner R, Breslow JL. Lipoprotein lipase controls fatty acid entry adipose tissue, but fat mass is preserved by endogenous synthesis in mice de®cient in adipose tissue lipoprotein lipase. Proc Natl Acad Sci USA 1997; 94: 10261 ± 10266. 17 Jaye M, Lynch KJ, Krawiec T, Marchadier D, Maugeais C, Doan K, South V, Amin D, Perrone M, Rader DJ. A novel endothelial-derived lipase that modulates HDL metabolism. Nature Genet 1999; 21: 424 ± 428. 18 Hirata K, Dichek HL, Ciof® JA, Choi SY, Leeper NJ, Quintana L, Kronmal GS, Cooper AD, Quertermous T. Cloning of a unique lipase from endothelial cells extends the lipase gene family. J Biol Chem 1999; 274: 14170 ± 14175. 19 Wang X, Greilberger J, Levak-Frank S, Zimmermann R, Zechner R, JuÈrgens G. Endogenously produced lipoprotein lipase enhances the binding and uptake of mildly and moderately oxidized low density lipoprotein in mouse peritoneal macrophages. Biochem J 1999; 343: 347 ± 353. 20 Babaev VR, Fazio S, Gleaves LA, Carter KJ, Semenkovich CF, Linton MF. Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo. J Clin Invest 1999; 103: 1697 ± 1705. 21 van Eck M, Zimmermann R, Groot P, Zechner R, van Berkel T. Role of macrophage derived lipoprotein lipase in lipoprotein metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol 2000; 20: E53 ± E62.