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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 in development and metabolism

R Zechner1*, J Strauss1, S Frank2, E Wagner1, W Hofmann1, D Kratky1, M Hiden1 and S Levak-Frank2

1Institute 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 and distribution of -rich lipoprotein-associated fatty acids among extrahepatic tissues. Additionally, the 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 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 tant factor in vascular disease as well. The advent of transgenic technology provided the means to directly Fat accumulation in adipose tissue (AT) largely modulate LPL expression levels in mice in a tissue- depends on the ef®cient uptake of fatty acids from dependent manner and study the tissue-speci®c role of the circulation. Since the majority of fatty acids in the enzyme. This article will summarize some of the plasma are present in esteri®ed form as lipoprotein ®ndings that emerged from these studies. associated (TG), hydrolysis is a manda- tory 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 Disruption of the mouse LPL gene lipoprotein lipase (LPL), an enzyme which is bound to Familial LPL de®ciency is a rare autosomal recessive glucosaminoglycans at the luminal side of the capil- disorder in humans, characterized by a massive accu- lary . The activity of LPL in AT relative mulation of (type I hyperlipoprotein- to its expression in other tissues, such as skeletal emia).2 Homozygous patients, when not kept under muscle (SM) and (CM), determines strict dietary control, exhibit plasma triglycerides the in¯ux of FFA and, possibly, the amount of fat above 3000 mg=dl, essentially lack HDL and suffer deposited. LPL is therefore viewed as a `gate keeping' from recurring episodes of pancreatitis. To date more enzyme for the entry and reesteri®cation of FFA in than 80 different mutations within the LPL structural 1 AT. According to this hypothesis, decreased LPL gene are known to cause the disorder. Generally, LPL activity in AT could reduce FFA uptake and lipid de®ciency is not associated with an increased inci- accumulation. Alternatively, overexpression of LPL in dence of atherosclerosis; however, in some families muscle might be associated with the redirection of an increased prevalence of heart disease has been nutrient fats from AT to muscle (`substrate steal'), reported.3 This observation indicated that mutation- again preventing FFA uptake in . It is speci®c variations exist with regard to atherosclerosis likely, therefore, that LPL and its tissue-speci®c susceptibility. Interestingly, patients with LPL de®- regulation are centrally involved in the pathogenesis ciency have normal amounts of AT, suggesting alter- of obesity. In addition, LPL plays a key role in the native, LPL-independent pathways to accumulate TG metabolism of , which makes it an impor- in adipocytes.4 Several years ago two laboratories reported indepen- dently the generation of LPL knock-out mice.5,6 Homo- *Correspondence: R Zechner, Institute of Molecular Biology, zygous LPL-de®cient animals also developed severe Biochemistry and Microbiology, University of Graz, Heinrichstrasse 31a, 8010 Graz, Austria. , hypercholesterolemia and low E-mail: [email protected] HDL, but in contrast to humans all knock-out animals Lipoprotein lipase in adipose tissue development R Zechner et al S54 died within 30 h after birth. This phenotype was SM and the uptake of a-tocopherol, providing in vivo similar to the one observed in the well-studied evidence for the involvement of LPL in cellular cld=cld-mouse, namely severe hypertriglyceridemia vitamin E uptake. and post-natal death as a result of the de®ciency of enzymatically active LPL and .7 Both mouse models, cld=cld and LPL knock-out, suggested that LPL-de®cient mice, in contrast to humans, cannot Cardiac muscle tolerate the massive amount of TG that accumulates in LPL overexpression in cardiac muscle (CM) was the blood as soon as the newborn animals begin to achieved by the fusion of the LPL minigene with suckle. Although the exact cause of death has not been promoter sequences from the mouse LPL gene.14 elucidated, it has been speculated that the excessive Eight kilobases (kb) of 50 ¯anking region of the blood-lipid content could clog the lung and mouse LPL gene were suf®cient for CM-speci®c interfere with a functional gas exchange, resulting in LPL expression, but were unable to drive transgene alveolar dysfunction and cyanosis.5 Hypoglycemia transcription in SM or AT. Even moderate levels of was proposed as an alternative cause of death by CM-speci®c LPL overexpression had a pronounced Merkel et al,8 when the authors observed that new- effect on VLDL catabolism and plasma TG levels, born LPL knock-out mice had unusually low blood indicating a particularly powerful role of CM-LPL in glucose levels. Recent experiments in our laboratory the catabolism of TG-rich lipoproteins. utilizing the transient post-natal adenovirus mediated expression of LPL has enabled us to rescue LPL knock-out mice during the suckling period and gen- erate adult, LPL-de®cient animals to an age well over Liver 12 months. These mice exhibited increased TG levels, Liver-speci®c overexpression of LPL was achieved by extremely low HDL concentrations and increased the fusion of the minigene with the promoter for FFA and ketones in plasma when kept on a chow AI.8 Despite a 4 ± 9-fold overexpres- diet. Gross morphological examination revealed the sion of the human enzyme in the liver, the effects on presence of roughly normal amounts of AT. plasma 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 overexpression of LPL Tissue-speci®c LPL transgene LPL overexpression in skeletal muscle (SM) was expression on an LPL knock-out achieved by fusing an LPL minigene to the promoter background of the mouse muscle creatine kinase gene.9±11 A5- fold overexpression of LPL in SM caused a marked Crossbreeding of tissue-speci®c LPL overexpressing decrease in TG rich lipoproteins, decreased HDL transgenic animals with heterozygous LPL knock-out levels, and low plasma FFA concentrations.9,10 The animals resulted in mice that expressed only the work by Jensen et al10 provided convincing evidence transgene but not the endogenous LPL gene. Depend- that low-level overexpression of LPL in SM can ing on the tissue of transgene expression, these ani- decrease a diet-induced weight gain in agreement mals produced LPL activity only in SM, CM or liver with the `substrate steal' concept. Higher levels of but lacked the enzyme in all other tissues, including LPL overexpression, however, resulted in the pro- AT and macrophages. Independent of the site of LPL liferation of mitochondria and peroxisomes in SM, expression, all mouse lines were rescued from the which eventually led to a severe myopathy and pre- lethal hypertriglyceridemia present in LPL knock-out mature death.9 These experiments indicated that TG mice. This ®nding indicated that LPL expression in hydrolysis by SM-LPL and the subsequent uptake of any one of these tissues is adequate for survival. the FFA by muscle tissue is very ef®cient. The Conversely, complete loss of LPL expression in any apparently unrestricted FFA uptake that leads to of these tissues does not cause lethality. In fact, all of myopathy suggested that LPL is the major determi- the above mouse lines remain grossly healthy and nant for FFA uptake in muscle and that no additional have a normal life expectancy. Additionally, LPL regulatory mechanism exists to prevent the ultimately activity measurements in various tissues of these lethal FFA accumulation in SM. `single tissue' expressors demonstrated that LPL pre- Muscle-speci®c LPL overexpression in transgenic dominantly remains in the tissue where it was origin- mice also enabled an investigation of the role of LPL ally synthesized. No evidence was obtained for the in the uptake of apolar lipids (cholesteryl esters) and translocation of a considerable amount of enzymati- lipophilic vitamins (vitamin E).12,13 A clear linear cally active enzyme from the site of synthesis to other relationship was observed between LPL activity in tissues.

International Journal of Obesity Lipoprotein lipase in adipose tissue development R Zechner et al S55 LPL-de®cient AT appear to induce lipid accumulation, foam cell The lack of LPL in AT of mice expressing LPL formation and atherosclerosis susceptibility. 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, leptin- de®cient animals were found to be much more resis- tant to obesity when they lacked LPL in AT.16 The Future directions drastically reduced content of polyunsaturated fatty acids (PUFA) in the AT lipid moiety of AT-LPL The initial experiments using induced mutant mouse de®cient mice suggested, however, that the lack of lines have clearly demonstrated that variations in the import of exogenous FFA was replaced by endogen- tissue speci®c expression pattern of LPL can affect the ous FFA and TG synthesis. Recent efforts in this lipid transport system, the distribution of energy laboratory to analyze the activity of glycolytic and substrate and the pathophysiology of obesity and lipogenic support this hypothesis. Addition- atherosclerosis. In fact, the site of enzyme expression ally, other mechanisms are conceivable that could might be more important than the undifferentiated, alleviate LPL de®ciency and facilitate lipid synthesis additive activity that is generally measured in post- in AT under normal dietary conditions. These include plasma. To strengthen the evidence provided the increased receptor-mediated uptake of whole lipo- from these initial observations, future work will have protein particles (eg VLDL receptor) as well as the to address the following topics. action of alternative such as the recently 1. It will be necessary to generate and study condi- 17,18 discovered endothelial derived lipase. tional knock-out mice by utilizing the cre-loxP technology. This will permit more physiologically LPL-de®cient SM and AT relevant modulations of LPL activities in single The loss of 80% of total body LPL due to the LPL tissues that resemble more closely the in vivo de®ciency in AT and SM in mice expressing LPL situation of varying tissue-speci®c LPL activities exclusively in CM did not cause hypertriglyceridemia in response to nutritional and hormonal changes or decreased HDL levels when kept on compared to current animal models. Additionally, normal chow diet.14 This unexpected result again to date no transgenic mouse lines have been illustrates the powerful role of CM-LPL in the clear- reported which overexpress LPL in AT. It will be ance of plasma TG and suggests that the site of LPL interesting to observe whether a marked over- expression is more important than the overall amount expression of the enzyme in adipocytes can cause of LPL for a functional metabolism of lipids and obesity in mice or whether alternative mechanisms lipoproteins. Furthermore, the lack of LPL in SM in can prevent fat accumulation. mice that expressed LPL exclusively in the heart did 2. It will be important to elucidate the metabolic not cause marked morphological or histopathological changes in adipocytes that lack LPL. Preliminary changes in either muscle type, indicating that the experiments indicate that the decreased FFA alternative use of glucose and nonesteri®ed fatty import under this condition can initiate a whole acids (derived from adipose tissue lipolysis) as spectrum of metabolic adaptations which allow the energy substrate was suf®cient to sustain normal fat cell to continue to produce considerable energy metabolism. The effects of decreased PUFA amounts of fat. These alternative pathways of content on membrane properties and function, or lipogenesis need to be characterized with regard prostaglandin metabolism, remain to be investigated. to their initiation and regulation. 3. The gene regions that regulate tissue-speci®c tran- scription of LPL and the corresponding transcrip- LPL-de®cient macrophages tion factors will have to be characterized in more The role of macrophage LPL in the formation of foam detail. Presently, insuf®cient information is avail- cells and atherosclerotic lesions is not well understood able describing the major enhancer regions that and has received signi®cant attention recently. In the control LPL expression in AT, SM, CM and absence of LPL, the ability of macrophages to bind macrophages. Yet, only a detailed understanding and take up TG-rich lipoproteins (VLDL) or modi®ed of the transcriptional mechanisms that govern LPL lipoproteins (oxidized LDL) is strongly reduced.19 expression in distinct tissues will enable the devel- Importantly, bone marrow transplantation experiments opment of drugs for potential pharmacological revealed that the absence of LPL in mono- interventions. cytes=macrophages markedly reduces the suscepti- bility 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 peri- pheral tissues to ensure low TG and high HDL Acknowledgement cholsterol levels, thereby decreasing the atherosclerotic This work was supported by the Austrian FWF grants risk, high LPL activities in macrophages of mice F701, F713 and P11696.

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International Journal of Obesity