Localization of Lipoprotein Lipase in Mouse Pancreas, Kidney and Placenta Impact of Metabolic Disturbances on Cellular Distribution and Activity Regulation

Home , Blood lipids

Rakel Nyrén

Department of Medical Biosciences Umeå 2020 This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD ISBN: 978-91-7855-295-5 (print) ISBN: 978-91-7855-296-2 (PDF) ISSN: 0346-6612 New Series Number: 2087 Cover design by Rakel Nyrén, Lipoprotein lipase in mouse kidney. Electronic version available at: http://umu.diva-portal.org/ Printed by: CityPrint i Norr AB Umeå, Sweden 2020

Tell me and I forget. Teach me and I remember. Involve me and I learn.

- Benjamin Franklin

To my family

Table of Contents

Abstract ...... iii Original papers ...... v Abbreviations ...... vii Populärvetenskaplig sammanfattning ...... viii Background ...... 1 Metabolism ...... 1 Lipid metabolism ...... 2 Transport of dietary lipids ...... 2 Transport of endogenous lipids ...... 3 Apolipoproteins ...... 5 Mobilization of stored lipids ...... 6 Lipoprotein lipase (LPL) ...... 6 Non-catalytic functions of LPL ...... 8 Proteins involved in regulation of LPL ...... 9 GPIHBP1 ...... 9 Angiopoietin-like proteins ...... 10 Obesity and insulin resistance ...... 12 Hyperlipidemia ...... 13 Kidney disease and dyslipidemia ...... 14 The role of LPL in different tissues ...... 15 Adipose tissue ...... 15 Skeletal muscle ...... 16 Heart ...... 18 The central nervous system ...... 18 Aims of the thesis ...... 19 Materials and Methods ...... 20 Mouse models with metabolic dysfunction ...... 20 Diet-induced obesity (DIO) ...... 20 Ob/ob ...... 21 Angptl4-tg+/- and Angptl4-/- ...... 21 Implications of circadian rhythm, nutritional state and sex ...... 21 Tissue handling and analyses ...... 22 Blood biochemistry ...... 24 Small-animal PET/CT ...... 24 Statistics ...... 24 Results and discussion ...... 25 Paper I ...... 25 Paper II and III ...... 27 LPL is localized to proximal tubules ...... 28 LPL is not involved in triglyceride uptake and accumulation ...... 29 LPL activity in kidney is regulated by ANGPTL4 ...... 29

Effects of obesity on LPL ...... 30 Obesity increases FDG and FTHA uptake ...... 30 Paper IV ...... 32 Major findings ...... 35 Perspectives ...... 36 Acknowledgements ...... 37 References ...... 39

Abstract

Lipoprotein lipase (LPL) is the key enzyme for metabolism of triglycerides in plasma lipoproteins. In recent years many new facts about the enzyme and its regulation have been uncovered. The endothelial membrane protein GPIHBP1 translocates LPL through endothelial cells and holds the enzyme in place at the luminal side of the capillary endothelium. Some of the angiopoietin-like proteins (ANGPTLs) bind to LPL and are responsible for tissue-specific regulation of the enzyme’s catalytic activity. Most studies in the past have focused on LPL in adipose and muscle tissues. LPL is also present in several other tissues, but the localization and function of LPL at these sites have not been fully elucidated.

One aim of the present thesis was to develop a protocol for immunolocalization of LPL in mouse tissues. In pancreas, the enzyme was localized to capillaries of the exocrine tissue, together with GPIHBP1, but also inside α- and β-cells. LPL in β-cells was absent in leptin-deficient ob/ob mice, but appeared after treatment with leptin. In kidney, LPL was mostly present within the proximal tubular cells of the nephron. In fed animals, LPL was also seen in intertubular vessels together with GPIHBP1. A LPL knock-out mouse model, MCKL0, was used to validate the specificity of our immuno-protocol. Kidneys from these mice showed no or very little staining for LPL. In mouse placenta, LPL was mostly found in capillaries of the labyrinth zone, where the exchange between fetal and maternal blood occurs.

A second aim was to gain better understanding for when, how and why LPL activity is regulated in mouse kidneys, and how obesity induced by high-fat diet (HFD) affects the LPL system. LPL activity in kidneys was regulated by ANGPTL4 in a similar manner as LPL in white adipose tissue, but in contrast to adipose tissue, the kidney LPL did not contribute to the uptake of fatty acids from chylomicron triglycerides. We found that obesity and insulin resistance, induced by long-term feeding of HFD, abolished the nutritional regulation of LPL activity in kidneys of male, but not of female, mice. To directly study the uptake of energy substrates in mouse kidneys, we developed a protocol for measurement of radiolabeled substrates in kidneys using PET/CT with the tracers [18F]FDG (a glucose analogue) and [18F]FTHA (a fatty acid analogue) injected to blood. There was an increase in uptake of both tracers in fasted male mice that had been on long-term HFD, compared to controls, as revealed by scanning of perfused organs, ex vivo, 3 hours after the injections.

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A third aim was to study LPL and the function of ANGPTL4 in pregnant mice and placentas. ANGPTL4 is known to increase in human plasma throughout pregnancy. As ANGPTL4 levels rise, triglyceride levels increase as well. We used mice that either lacked (Angptl4-/-) or overexpressed Angptl4 (Angptl4-tg+/-), and compared them to wild-type mice. Plasma triglycerides and VLDL levels increased during pregnancy both in wild-type and in Angptl4-/- mice. The lipid profile in Angptl4-tg+/- was high already before conception, and did not change. LPL activity in placenta was, however, similar in all genotypes. The increase in ANGPTL4 in maternal blood during pregnancy might originate from placenta, but Angptl4 expression was also increased in maternal liver and subcutaneous white adipose tissue. The pups from Angptl4-tg+/- had reduced birthweight compared to pups from wild-type and Angptl4-/- mice.

In conclusion, the present thesis provides information on the localization and possible functions of LPL and some of its regulator proteins in mouse pancreas, kidney and placenta. New data on the regulation of LPL activity in mouse kidney, and the effects of HFD and obesity, is presented, as well as insights into the potential role of ANGPTL4 for control of plasma triglyceride levels and fetal growth during mouse pregnancy.

Original papers

This thesis is based on the following papers, which are referred to in the text by their Roman numbers:

Paper I Nyrén R, Chang, CL, Lindström P, Barmina A, Vorrsjö E, Ali Y, Juntti-Berggren L, Bensadoun A, Young SG, Olivecrona T, Olivecrona G. Localization of lipoprotein lipase and GPIHBP1 in mouse pancreas: effects of diet and leptin deficiency. BMC Physiology 2012;12:14. DOI: 10.1186/1472-6793-12-14

Paper II Nyrén R, Makoveichuk E, Malla S, Kersten S, Nilsson SK, Ericsson M, Olivecrona G. Lipoprotein lipase in mouse kidney: effects of nutritional status and high-fat diet. American Journal of Physiology, Renal Physiology 2019;316(3):F558-571. DOI: 10.1152/ajprenal.00474.2018

Paper III Nyrén R, Scherman H, Axelsson J, Chang LC, Olivecrona G, Ericsson M. Visualizing uptake of [18F]FDG and [18F]FTHA in kidneys of metabolically challenged C57BL/6J male mice using PET/CT. Manuscript.

Paper IV Nyrén R, Olivecrona G, Ericsson M. Angiopoietin-like protein 4 in mouse pregnancy: effects on lipoprotein profile, fetal weight and lipoprotein lipase activity. Manuscript.

Papers I and II did not require permission to be reprinted.

Co-authored published original articles not included in thesis

A. Davies BSJ, Beigneux AP, Barnes RH, Tu Y, Gin P, Weinstein MM, Nobumori C, Nyrén R, Goldberg I, Olivecrona G, Bensadoun A, Young SG, Fong LG. GPIHBP1 is responsible for entry of lipoprotein lipase into capillaries. Cell Metabolism 2010;12(1):42-52 DOI: 10.1016/j.cmet.2010.04.016

B. Cang CL, Garcia-Arcos I, Nyrén R, Olivecrona G, Kim JY, Hu Y, Agrawal RR, Murphy AJ, Goldberg IJ, Deckelbaum RJ. Lipoprotein lipase deficiency impairs bone marrow myelopoiesis and reduces circulating monocyte levels. Atherosclerosis, Thrombosis and Vascular Biology 2018;38(3):509-519 DOI: 10.1161/ATVBAHA.117.310607

Abbreviations

Ad libitum Unlimited and free excess to food ANGPTLs Angiopoietin-like proteins ATGL Adipose triglyceride lipase ATP Adenosine triphosphate FA Fatty acid FDG 2-deoxy-2-[18F]fluoro-D-glucose FTHA 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid GLUT Glucose transporter GPIHBP1 Glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 HDL High-density lipoprotein HFD High-fat diet HOMA-IR Homeostasis model assessment of insulin resistance HSL Hormone sensitive lipase HSPG Heparan sulfate proteoglycan IF Immunofluorescence ISH In situ hybridization LDLR Low density lipoprotein receptor LPL Lipoprotein lipase LRP LDL receptor-related protein NAFLD Non-alcoholic fatty-liver disease NEFA Non-esterified fatty acids, also called free fatty acids OMIS Outer medulla inner stripe OMOS Outer medulla outer stripe PET/CT Positron emission tomography/computed tomography PPAR Peroxisome proliferator activated receptor RT-qPCR Reverse transcription quantitative polymerase chain reaction tg Transgene TG Triglyceride (or triacylglycerol) TRL Triglyceride rich lipoprotein (VLDLs and chylomicrons) VLDL Very low density lipoprotein WAT White adipose tissue pgWAT Perigonadal WAT scWAT Subcutaneous WAT WT Wild-type

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Populärvetenskaplig sammanfattning

Fett som vi får i oss via kosten eller som bildats i levern paketeras som triglycerider i fettdroppar, lipoproteiner, vilka släpps ut i blodbanan för att levereras till kroppens vävnader. Vävnaderna kan inte ta upp triglyceriderna direkt, utan är beroende av enzymet lipoproteinlipas (LPL) som klipper loss fettsyror från triglyceriderna i lipoproteinerna. Fettsyrorna tas upp i vävnaderna och används som källa till energi eller lagras i fettceller som triglycerider för att täcka senare behov. Individer som saknar LPL får kraftigt förhöjda nivåer av triglycerider i blodet då de inte kan nyttiggöras i vävnaderna. LPL produceras huvudsakligen i fettceller i fettvävnad och i muskelceller i hjärta och skelettmuskler. För att kunna passera till insidan av kapillärerna, där LPL verkar på blodets lipoproteiner, behöver LPL hjälp av transportproteinet GPIHBP1. Bundet till GPIHBP1 flyttas LPL till kärlets insida och det skyddas från inaktivering under transporten. Ett annat protein, som kallas ANGPTL4, är nämligen en kraftfull hämmare av LPL. ANGPTL4 sköter en stor del av den viktiga kontrollen av LPL, så att aktiviteten anpassas till hela kroppens behov.

LPL finns även i många andra vävnader, förutom i fett- och muskelvävnader. Vi har studerat var i några av dessa vävnader som LPL finns, hur enzymaktiviteten kontrolleras jämfört med i fettvävnad och muskler, samt vilken roll LPL spelar på respektive plats. I denna avhandling har vi studerat bukspottkörtel (pankreas), njure och moderkaka (placenta) från mus. För att lokalisera ett protein i vävnadssnitt med mikroskopi kan man använda sig av antikroppar som är märkta med en fluorescerande färg. Metoden är mycket användbar, men den måste optimeras och kontrolleras noga så att man inte drar felaktiga slutsatser. Vi kunde visa att i pankreas finns LPL framförallt inuti de insulinproducerande β- cellerna, men även i kapillärer i den del av pancreas som utsöndrar matspjälkningsenzymer. Genom att studera feta möss som saknar leptin, ett aptitreglerande hormon, fann vi att leptin krävs för bildning av LPL i β-cellerna.

Den funktionella enhet i njuren som filtrerar blodet och tillverkar urin kallas nefron. Proximala tubuli är en del av nefronet. Där återförs viktiga näringsämnen och salter från den primära urinen tillbaka till blodet. Proximala tubuli kan även vid behov tillverka glukos. Vi kunde visa att LPL framförallt finns inuti de celler som bildar proximala tubuli. LPL finns även i kapillärerna intill tubuli. Där sitter enzymet tillsammans med GPIHBP1. I den positionen har LPL kontakt med blodet och kan därmed frisätta fettsyror från lipoproteiner till njurens energikrävande processer. Fettsyror är nämligen njurens främsta energisubstrat. Efter att mössen ätit ökar LPL-aktiviteten i njuren, men efter att man tagit bort maten under några timmar sjunker den drastiskt. Vi kunde visa att effekten beror på ANGPTL4 som bildas i ökad mängd vid fasta, och som då verkar på LPL så att

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aktiviteten blir lägre. Det är välkänt sedan tidigare att LPL regleras på liknande sätt i fettvävnad. Där är den logiska funktionen att vid fasta ska blodfetter inte tas upp för lagring, utan användas för energiproduktion. Trots att LPL-aktiviteten är hög i musnjure kunde vi inte finna belägg för att den bidrar till upptaget av fettsyror. Detta beror sannolikt på att LPL huvudsakligen finns inuti tubulicellerna. Vilken funktion LPL kan ha där har vi tyvärr inte lyckats lösa.

För att studera hur njurarnas energiupptag påverkas av fetma lät vi möss äta en kost med mycket fett under 20 veckor. Därefter gav vi dem märkta testsubstanser som motsvarar glukos och fettsyror. Med PET/CT, en metod som också används på människa, kunde vi visa att högfettskost och fetma ledde till ett ökat upptag av båda energisubstraten i njuren, troligen p.g.a att den har ett ökat energibehov. En musnjure hos en ung vuxen mus väger endast kring 160 mg och ett hjärta kring 125 mg. Det innebär därför ganska stora tekniska utmaningar att studera upptag av substanser i musorgan med PET/CT, men vi hittade lösningar för detta.

Placentan var det tredje organet som undersöktes i avhandlingen. Under graviditet ökar mängden triglycerider i blodet, så att den är som högst mot slutet av graviditeten. Det har visats på människa att även nivåerna av ANGPTL4 i blodet stiger kraftigt. För att studera ANGPTL4 och dess effekter på LPL vid graviditet använde vi möss som antingen bildade mer ANGPTL4 än normalt, eller som helt saknade proteinet. Hos vanliga möss steg triglyceriderna i blodet som förväntat. De möss som hade mycket ANGPTL4 hade redan före graviditeten höga nivåer av triglycerider i blodet och de förändrades sedan inte mycket under graviditeten. Möss som saknade ANGPTL4 hade mycket låga nivåer av triglycerider i blodet, men de steg något under graviditeten. Båda typerna av möss bar på ungefär samma antal ungar, men hos möss med mer ANGPTL4 vägde fostren i genomsnitt lite mindre än normalt. Hos normala möss såg vi ett negativt förhållande mellan mängd av ANGPTL4 som kan bildas i placentorna och fostervikt. Eftersom lipoproteiner inte kan ta sig över placentan till fostrets blodcirkulation tror man, på goda grunder, att LPL behövs för att frisätta fettsyror som sedan kan tas upp. Med antikroppar kunde vi lokalisera LPL till den del av placentan där näringsutbytet sker, och där fanns även transportproteinet GPIHBP1. Den LPL-aktivitet vi kunde uppmäta i placentorna var densamma oavsett om mössen hade mycket ANGPTL4 eller inget alls. Därför kunde ingen klar koppling göras mellan LPL-aktivitet och fostervikt hos dessa möss.

Sammanfattningsvis bidrar avhandlingen med ny information om var LPL är lokaliserat i pankreas, njure och placenta hos mus, samt tankar kring lipasets funktion i dessa olika organ. Effekten av högfettsdiet och fetma på LPL-systemet har studerats liksom betydelsen av ANGPTL4 för regleringen av LPL i njure, samt för LPL i placenta under graviditet.

Background

Metabolism According to Merriam-Websters dictionary [1] the word metabolism means, “the chemical changes in living cells by which energy is provided for vital processes and activities and new material is assimilated”. In other words, living organisms convert food into energy and building blocks and excrete the waste. This is crucial for maintaining life, growth and development. Our three main energy sources, or macronutrients, are carbohydrates (sugar, glucose), proteins and fat. There is an intricate interplay in metabolism of these nutrients depending on the state of the organism. Most reactions are catalyzed by enzymes, which are proteins with very specific functions. The rate of each chemical reaction is to a large extent controlled by the amount and activity of the respective enzyme. Some reactions occur at a constant rate most of the time, while others are carefully regulated by several means. These can affect the amount and catalytic function of the enzyme, so that the reaction rate is well adapted to the requirements of the entire body. Metabolic reactions can be divided into two major processes, anabolism and catabolism. Anabolism, is the energy requiring part, where larger units are built from smaller ones. Catabolism is the energy releasing part through break-down of larger units. All three macronutrients mentioned can undergo both anabolic and catabolic reactions. The basic processes are summarized in Table 1. In the present thesis, the main focus has been on lipid metabolism, and specifically on the enzyme lipoprotein lipase (LPL), which is crucial for providing fatty acids (FA) from lipids in blood for uptake in tissues. Some of the other metabolic pathways will be mentioned in this thesis in relation to discussions of effects of obesity and insulin resistance.

Anabolic process Catabolic process

Carbohydrates Glycogenesis - building of Glycogenolysis – the breakdown glycogen of glycogen Gluconeogenesis - generation Glycolysis – breakdown of glucose of glucose from amino acids, to generate energy (ATP) glycerol, lactate Lipids Lipogenesis – generation of Lipolysis – breakdown of lipids lipids

Proteins Protein synthesis Proteolysis – breakdown of proteins to amino acids

Table 1: A summary of anabolic and catabolic processes of macronutrients.

Lipid metabolism Fat, which is the most energy dense of the macronutrients, consists of different types of lipids. The most abundant in our diet is the triglyceride (neutral fat). The triglyceride is built on a glycerol backbone with three fatty acid chains attached (Figure 1). The fatty acids may differ in length and be saturated or unsaturated. Other common lipids are phospholipids, cholesterol and cholesteryl esters. Lipids can be added to the blood stream by three different pathways. One is from the dietary fat absorbed in the gut, the second is release of lipids that are endogenously synthesized in the liver, and the third is mobilization of stored lipids from adipose tissue [2].

Figure 1: The chemical structure of a triglyceride (TG). The TG contains a glycerol backbone with three fatty acids attached via ester bonds.

Transport of dietary lipids When a balanced meal containing a combination of carbohydrate, fat and protein enters our gastrointestinal system, the nutrients are digested into smaller parts, absorbed and released into the blood. The lipids need to go through several steps in the digestive tract to allow absorption and later transport in the blood. First the fat is dispersed into droplets which are mixed with bile acids in a process called emulsification, generating even smaller droplets [3]. This allows enzymes (mainly pancreatic lipase) to work on the surface of the lipid droplets to hydrolyze their triglycerides into smaller, more polar, parts in a process called lipolysis. The lipolysis products, mainly monoglycerides and fatty acids, are then absorbed by enterocytes in the intestinal lumen [3]. In the enterocytes the lipids are re- esterified to form triglycerides that are packed with other lipids and proteins into new, lipid droplets called chylomicrons for secretion to blood [2]. A lipoprotein particle contains a hydrophobic core with nonpolar lipids, i.e. triglycerides and cholesteryl esters, that are surrounded by an amphiphilic monolayer of phospholipids, cholesterol and apolipoproteins (Figure 2). A particular apolipoprotein, ApoB48, is needed for chylomicron assembly [4]. With the help of microsomal transfer protein, lipids are loaded onto ApoB48 to form a lipoprotein particle that consists mainly of triglycerides and is the largest lipoprotein in the body [4]. The chylomicrons are transported in the lymphatic

system (via the thoracic duct), and enters the blood in the junction between the left jugular and subclavian veins [2]. In the bloodstream, the newly secreted, nascent, chylomicron adopts additional apolipoproteins to its surface (mainly from circulating high-density lipoproteins, HDL), whereby the particle is said to mature [5]. The cells cannot utilize the fatty acids as long as they are bound in triglycerides, because non-polar lipids cannot readily pass over cell membranes. Therefore, for unloading, chylomicrons must be acted on by the enzyme LPL which will rapidly hydrolyze a major part of the triglycerides from the chylomicron core [6,7]. This results in release of fatty acids and monoglycerides (glycerol with one remaining esterified fatty acid) for the cells to absorb. After the chylomicron core is more or less emptied from triglycerides, the remaining lipoprotein particle is now cholesterol-rich and is called a chylomicron remnant. Redundant components from the chylomicron surface layer, like some of the apolipoproteins, are recycled to HDL particles [8]. Chylomicron remnants are internalized by receptor-mediated endocytosis in the liver [9].

Figure 2: A schematic illustration of a plasma lipoprotein particle. The amphiphilic surface contains cholesterol, phospholipids and apolipoproteins, while the hydrophobic core carries triglycerides and cholesteryl esters.

Transport of endogenous lipids Chylomicrons are transporters of triglycerides from the gut, the exogenous lipid pathway originating from dietary lipids. In the endogenous lipid pathway, lipids are synthesized, or recycled, in the liver. The hepatocytes absorb chylomicron remnants, which still carry some remaining triglycerides and a heavy load of cholesteryl ester. The uptake occur via ApoE that remains on the surface of the remnant and interacts with members of the low density lipoprotein receptor (LDLR) family on the liver cell membranes [9–11]. In addition, the liver takes up a significant amount of albumin-bound free fatty acids from blood (called non- esterified fatty acids, NEFAs) and is the main organ for utilization of free glycerol [12]. By lipogenesis, the liver can convert a surplus of both carbohydrates and amino acids to lipids, which together with lipids from other sources can be

temporarily stored as lipid droplets within the hepatocytes. For secretion to blood, triglyceride from the liver is packed into very low density lipoprotein particles (VLDL) with the help of microsomal transfer protein [13]. VLDL is built on the full-length ApoB, ApoB100 [14]. Similar to chylomicrons, VLDL mature by acquiring apolipoproteins (ApoC and ApoE) from HDL. Triglycerides in VLDL are hydrolyzed by LPL and the products, fatty acids and monoglycerides, are taken up, stored or utilized by tissues [6,7]. When the triglyceride-core of VLDL is sufficiently emptied and the particle size has become smaller, the affinity for LPL is also reduced. This remnant particle is called an intermediate-density lipoprotein (IDL). IDL loses some apolipoproteins to HDL, but ApoE stays on the particle for docking in the liver and further hydrolysis of remaining triglycerides by hepatic lipase [15]. When almost no triglycerides are left, the lipoprotein contains a high proportion of protein and cholesterol/cholesteryl ester, and is now called low-density lipoprotein (LDL). LDL retains the ApoB100 and is the particle that transports cholesterol to extrahepatic tissues for use as building blocks for cell membranes or for synthesis of steroid hormones [16]. A summary of the lipoprotein subclasses and their apolipoproteins is found in Table 2.

Lipoprotein Composition Diameter Density Apolipoproteins particle (% dry weight) (µm) (g/ml)

Chylomicron 75-1200 <0.95 B48 (A,C,E)

VLDL 30-80 0.95- B100(A,C,E) 1.006

IDL 25-35 1.006- B100, E 1.019

LDL 18-25 1.019- B100 1.063

HDL 5-12 1.063- AI, AII (C,E) 1.210

Table 2: Characteristics and composition of lipoprotein subclasses. Data summarized from [2].

Apolipoproteins Apolipoproteins are proteins with specific properties that make them suitable to be incorporated into the surface layer of lipoproteins. They function as ligands for receptors for mediated particle uptake by endocytosis, as well as for control of lipoprotein metabolism as regulators of enzymes and transfer proteins [17]. The ApoBs are crucial for assembly of VLDL and chylomicrons and for their stability. There is one copy of ApoB on every lipoprotein particle, except for on HDL that do not contain ApoB. As mentioned above, VLDL carry ApoB100, while chylomicrons carry the truncated ApoB48. ApoB100 remains as the lipoprotein transforms into an IDL and an LDL. ApoA, ApoC and ApoE all differ from ApoB, because they can transfer from one lipoprotein to another, and because several copies of each can exist on the same lipoprotein [17,18]. ApoB100 is a ligand for the LDL-receptor and mediates receptor-mediated uptake of LDL in cells [19]. ApoB48 is a version lacking the carboxy-terminal half of the molecule where the receptor-binding structures reside [20]. Therefore, chylomicron remnants are dependent on ApoE for receptor interaction [21].

Below is a short description of the apolipoproteins that are known to directly control the rate of triglyceride hydrolysis by LPL:

ApoCII is crucial for LPL-mediated intravascular triglyceride hydrolysis and has recently become a novel target for drug development [22]. ApoCII acts as a cofactor for LPL, and is mainly produced in the liver. ApoCII is present both on the triglyceride-rich lipoproteins chylomicrons and VLDL (TRLs), and on HDL. Although ApoCII has been known and studied for many decades, the detailed mechanism by which ApoCII acts is not known. Individuals lacking ApoCII, or with non-functional ApoCII, have severe hypertriglyceridemia and problems similar to patients with total LPL deficiency [23]. The hypertriglyceridemia can be temporarily relieved by plasmapheresis with plasma from healthy donors to avoid incidences of life-threatening triglyceride-induced pancreatitis [24].

ApoCIII is like ApoCII mostly produced in the liver and becomes a surface component of chylomicrons, VLDL and HDL. Increased levels of ApoCIII are associated with increased levels plasma triglycerides [25]. The action of ApoCIII is important, but not fully understood. ApoCIII has been shown to inhibit LPL- mediated triglyceride hydrolysis, at least at high concentrations in vitro, to displace ApoCII from lipoproteins, as well as to inhibit uptake of remnant particles via LDLR or LDL receptor-related protein (LRP) [25–27]. Recent studies in LPL-deficient mice have demonstrated that antisense oligonucleotides against ApoCIII lowers triglycerides even in the absence of LPL, demonstrating that a main effect in vivo is likely on receptor-mediated uptake of lipoprotein remnants [28].

ApoAV is also synthesized in the liver, but is present on lipoproteins in much lower amounts than the ApoCs described above. Mutations in the ApoAV gene are associated with elevated triglyceride levels [29]. The effects of ApoAV somehow involves LPL, and increases the activity of the enzyme, but how this is accomplished is not known. A direct effect of ApoAV on hydrolysis by LPL has been difficult to demonstrate in vitro. Very low levels of ApoAV are found in plasma, meaning that only few lipoproteins carry this apolipoprotein, indicating that the protein is reused [29].

Mobilization of stored lipids Lipids stored as triglycerides in intracellular lipid droplets in adipose tissue can be mobilized from the tissue in the form of fatty acids and glycerol and released to the blood. The fatty acids are immediately bound to plasma albumin and included in the pool of NEFA that is rapidly turning over by uptake in cells for metabolic processes [30]. A large fraction of the NEFAs is taken up in the liver. The mobilization of stored triglycerides in adipose tissue by intracellular lipolysis is stimulated when the energy balance is negative, for example during fasting or exercise, when NEFAs are needed for ATP production [31]. Three major lipases are involved in this process; adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and monoglyceride lipase (MGL). They release one FA each from the glycerol backbone [31,32]. Intracellular lipolysis is strongly attenuated in the presence of insulin that promotes anabolism and lipid storage in adipose tissue [31]. In contrast, lipolysis is strongly stimulated by hormones that increase cAMP-levels and phosphorylation of enzymes and lipid droplet components like perilipins [33].

Lipoprotein lipase (LPL) The function of LPL was first discovered in 1943 by Hahn [34], during studies of red-blood cells in dogs receiving donor blood. One animal had apparent lipemia when a blood sample was taken, but after injection of donor blood cells that contained heparin as anticoagulant, the lipemia was abolished. The experiment was further validated in several other animal species, confirming that after an injection of heparin, lipids were cleared from the blood. During this time LPL was called “clearing factor”. An important additional discovery was that the clearing factor was unable to clear lipemic plasma in vitro when heparin was added to an already drawn blood sample. In 1955 Korn published a paper in J Biol Chem demonstrating that he extracted clearing factor activity from rat heart [35]. The extracted clearing factor could hydrolyze fat in chylomicron lipoproteins. Korn thereby proposed a new name for clearing factor, lipoprotein lipase.

LPL is produced in parenchymal cells of many tissues of the body, for example in adipocytes in adipose tissue, and myocytes in skeletal muscle and heart [6,7]. LPL

is synthesized in the endoplasmic reticulum with the help of the specific chaperones lipase maturation factor 1 (LMF-1) and Sel1L that helps the enzyme to fold into its active form (Figure 3). Folding is dependent on N-glycosylation at specific sites in the protein [36,37]. After further maturation in Golgi, LPL is transported in secretory vesicles to the cell membrane and secreted into the subendothelial space. LPL has strong affinity for negatively charged polyanions like heparin and for glycosaminoglycans found on cell surfaces and in connective tissue like heparan sulfate proteoglycans (HSPGs). It is likely that LPL travels between binding sites on these structures [38]. When LPL reaches the basolateral side of the capillary endothelium it binds to the endothelial transport protein GPIHBP1. GPIHBP1 is attached to the cell membrane by a GPI-anchor and is responsible for the translocation of LPL to the luminal side of the capillary endothelium. There the enzyme probably remains attached to GPIHBP1 while acting on triglycerides in TRLs, generating release of FA [39]. If GPIHBP1 is non- functional, LPL gets trapped in the subendothelial space, resulting in severe hypertriglyceridemia [40,41]. The condition is similar to that in patients suffering from total LPL deficiency [42]. A schematic illustration following LPL from its place of synthesis to its site of action at the endothelium is summarized in Figure 3.

For decades LPL has been described as a homodimer, with two identical monomers arranged in a head-to tail fashion by non-covalent bonds [43–45]. The N-terminal domain of the subunit monomer catalyzes hydrolysis of the ester bonds in triglycerides of plasma lipoproteins. The C-terminal domain of the LPL subunit is known to interact with lipoproteins, and also with GPIHBP1 [46,47]. Due to recent knowledge about the strong conformational stabilization of LPL by the interaction with GPIHBP1, it was possible to obtain a crystal structure for LPL in complex with the soluble form of GPIHBP1 [46]. This occurred 75 years after the discovery of clearing factor. For several decades the dogma has been that LPL is only active as a dimer, and that on dissociation to monomers the naked subunit becomes susceptible to unfolding and thereby becomes inactivated [48,49]. Again, with the use of the stabilizing effects on LPL by GPIHBP1, and also by the use of a well-known monoclonal anti-LPL antibody 5D2, recently published data propose that active LPL is actually a monomer. It follows that both spontaneous and induced inactivation of LPL occurs through minor unfolding of the active form of the monomer [50]. These new findings have impact on the view on how LPL folds and mature during synthesis, as well as on the mechanism behind inactivation of LPL by the ANGPTLs (see below).

Figure 3: Illustration of LPL and its regulatory proteins while the enzyme is involved in hydrolyzis of lipoprotein triglycerides. Black arrows demonstrate the synthesis pathway for LPL to its site of action at the luminal side of the capillary endothelium. By courtesy of Dr Oleg Kovrov.

Non-catalytic functions of LPL In addition to being a triglyceride lipase, LPL was shown to interact with lipoprotein receptors like LRP [51] and later with other receptors related to the LDL-receptor family like sortilin and SorLA [52,53]. Binding of LPL to receptors is independent of the enzymatic activity of LPL, but it is dependent on native folding of the LPL protein. By interaction on one side with lipoproteins and on the other with receptors, or with cell surface proteoglycans like HSPG, LPL can efficiently tether lipoproteins, like chylomicrons and ApoE-loaded remnants, to cell surfaces [54,55]. LPL may also be involved in intracellular sorting or transcellular transport of ligands by receptors that are mainly intracellular, like

SorLA [52]. In the brain, where there are no TRLs due to the blood brain barrier, LPL may be involved in transport of ApoE-containing cholesteryl-rich lipoprotein aggregates between cells, in clearance of lipid debris on neuronal injury or remodeling [56], and possible also in handling of Alzheimer-related amyloid precursor protein [57].

Proteins involved in regulation of LPL Within the last decades several important proteins that are involved in control of LPL activity by specific protein-protein interaction with the enzyme have been identified. Below, GPIHBP1 and ANGPTL4 are presented, and the related ANGPTL3 and ANGPTL8 are briefly mentioned.

GPIHBP1 Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is crucial for intravascular hydrolysis of lipoprotein triglycerides. GPIHBP1 was discovered about 10 years ago, and its discovery added a significant piece to the puzzle about LPL-mediated lipid hydrolysis. In 2007 the group of Stephen Young at UCLA described a new mouse model with hypertriglyceridemia, a Gpihbp1 knock-out mouse (Gpihbp1-/-) [40]. The mice had very high plasma triglyceride levels, even on a low-fat diet. The authors demonstrated that GPIHBP1 is expressed in endothelial cells and has the ability to bind LPL [40]. GPIHBP1 is only expressed in capillary endothelial cells and not in larger arteriols or venules [39,40,58]. In order for LPL to act on TRLs in the capillary lumen, LPL is captured by GPIHBP1 and transferred over the endothelium from the subendothelial space [39], see schematic illustration in Figure 3. The transfer is bidirectional [59]. The discovery of GPIHBP1 solved cases of hypertriglyceridemia in patients where severe gene mutations neither for LPL nor for ApoCII had been found. One example was a family in Northern Sweden where three siblings had hypertriglyceridemia and typical incidences of pancreatitis, but they showed normal LPL activity and mass in adipose tissue biopsies [41]. Only minor amounts of LPL activity was measurable in blood after heparin injection in the affected siblings, indicating that there was no LPL on their capillary endothelium. The parents were found to carry one discrete missense mutation each in the gene of GPIHBP1. Their children with both mutations had hypertriglyceridemia, while the heterozygote parents and sibling were essentially normolipidemic. The mutations affected two cysteine residues in the tightly folded Ly6 domain of GPIHBP1. Both mutations resulted in inability to bind to LPL, thereby leaving LPL stuck in the subendothelial space, just like in the Gpihbp1-/- mice [40,41]. Later, several more non-functional natural mutations have been found in GPIHBP1, many of which affect the Ly6 domain [46].

The C-terminal domain of GPIHBP1 attaches to the endothelial cell membrane via a glycosylphosphatidylinositol (GPI) anchor. Both the central Ly6 domain and the acidic N-terminal domain can interact with LPL [60,61]. The N-terminal domain of human GPIHBP1 carries 21 negative charges in a stretch of 25 residues and therefore interacts with LPL rather non-specifically, like many other polyanions, e.g. heparin and HSPGs. Besides facilitating transport of LPL from the subendothelial space to the luminal side of the capillaries and holding the enzyme in place there, interaction with GPIHBP1 also stabilizes the conformation of LPL and protects the enzyme from inhibition of the ANGPTLs [62,63]. GPIHBP1 helps with margination of TRLs from blood to the capillary endothelium [64], but this is likely to be mediated through LPL since no direct interaction between TRLs and GPIHBP1 has been demonstrated. Aside from mutations in the gene for GPIHBP1, autoantibodies against GPIHBP1 have been found to cause hypertriglyceridemia due to blockage of binding between the two proteins [65].

Before GPIHBP1 was discovered in 2003 [66] and its function described in 2007 [40], the binding sites of LPL at the endothelium was thought to be based on HSPGs that are always present on cell surfaces [67]. HSPGs have been shown to interact with LPL and hepatic lipase, as well as with lipoproteins [68]. LPL has high affinity for HSPGs that due to its negative charge stabilizes LPL and protects it from spontaneous inactivation [69]. HSPGs alone cannot protect LPL from inactivation by ANGPTLs, like GPIHBP1 can [48,62]. HSPGs probably helps in the transport of LPL from the synthesizing parenchymal cell to the abluminal side of the endothelium where high affinity attachment to GPIHBP1 can occur, probably first by interaction with the acidic N-terminal domain of GPIHBP1 [38]. HSPGs on endothelial cells are likely to support triglyceride hydrolysis by tethering TRLs of which both ApoB100 and ApoE have heparin-binding structures [54,55].

Angiopoietin-like proteins The angiopoietin-like protein (ANGPTL) family consists of a total of eight known proteins. All ANGPTLs contain an N-terminal coiled-coil domain and a fibrinogen-like C-terminal domain, except for ANGPTL8 that only contains the N-terminal domain [70]. The ANGPTLs involved in LPL activity regulation are ANGPTL3, ANGPTL4 and ANGPTL8. The other proteins in the family are involved in other areas such as angiogenesis, cancer and regulation of glucose metabolism. Podocyte-specific hyposialylated ANGPTL4 has been shown to be involved in regulation of proteinuria [71]. The involvement of ANGPTL3, 4 and 8 in regulation of blood triglyceride levels has been shown in mouse models that either overexpress one of the proteins, or where the expression has been knocked out. In genetically modified mice with overexpression, circulating triglyceride

levels are increased, while in the knock-outs the levels are much decreased compared to WT mice [72]. The expression of ANGPTL 3, 4 and 8 is differently regulated and the tissue specific expression also differ.

ANGPTL3 was discovered when a strain from an obese mouse model (normally exhibiting hyperinsulinemia, hyperglycemia and hyperlipidemia) had very low triglyceride levels [73]. A mutation in the gene encoding for Angptl3 was found and the hypolipidemia was reversed by injecting ANGPTL3, or by overexpressing Angptl3 in the mice. ANGPTL3 was the first of the ANGPTLs that was recognized as a modulator of triglyceride metabolism through effects on LPL activity [74]. ANGPTL3 is almost exclusively expressed in the liver. ANGPTL3 is a secreted protein that is found at relatively stable concentrations in blood. Humans lacking functional ANGPTL3 have decreased levels of all lipoprotein fractions, increased activity of LPL and increased insulin sensitivity, and therefore tend to stay healthy to old age [75,76]. Exactly how ANGPTL3 inhibits LPL is yet not entirely known. In recent years it has been demonstrated that ANGPTL3 needs to act together with ANGPTL8 to inactivate LPL. In order to form an LPL regulatory complex, the proteins need to be co-expressed [77]. ANGPTL8 originates mainly from liver and white adipose tissue (WAT) [78,79]. The expression of ANGPTL8 is induced in the fed state by insulin.

In this thesis the focus has been on ANGPTL4, because this protein is the most potent modulator of LPL activity and triglyceride hydrolysis [80,81]. It was known for quite some time that an unknown protein was involved in regulation of LPL activity in adipose tissue [82]. By treating ad libitum fed rats with actinomycin that blocks transcription, it was demonstrated that LPL activity was not down-regulated by fasting as in untreated rats. Furthermore, after administration of actinomycin to fasted rats, LPL activity in adipose tissue increased, as if the rats had been given food. This indicated that fasting induces transcription of a gene which protein product inhibits LPL activity [82]. This gene product was later found to be ANGPTL4, and it went under the name fasting- induced adipose factor [83]. ANGPTL4 is expressed in many tissues, most notably in different depots of adipose tissue and in liver, but also in placenta, kidney, heart and skeletal muscles [84]. The expression rate and protein levels of ANGPTL4 vary depending on the nutritional state of the subject. Fasting and hypoxia induces ANGPTL4 expression, probably to a large extent mediated by peroxisome proliferator activated receptors (PPARs) and by HIF1α [85], while ANGPTL8 that potentiates the effect of ANGPTL3 is induced in the fed state by insulin. ANGPTL4 is thought to be involved in the tissue-dependent regulation of LPL in order to shuttle FA according to tissue-specific needs [70,80]. Until recently, ANGPTL4 was thought to inactivate LPL through dissociation of the active LPL dimer into inactive monomers involving some minor conformational changes [48]. Studies published in the last couple of years implies that LPL, under

stable physiological conditions, may in fact be active as a monomer that can bind ANGPTL4 and thereby change to an inactive conformation [50]. Therefore the mechanism behind LPL inactivation by ANGPTL4 may be re-written. Important facts that still holds true is that the inactivation of LPL by ANGPTL4 is practically irreversible, and that ANGPTL4 is not consumed during inactivation, but detaches from LPL and can be re-cycled to inactivate new enzyme molecules in a catalytic manner [48].

Obesity and insulin resistance The intake of a diet high in saturated fats and sugar (often called a Western Diet), combined with a sedentary lifestyle, is a cornerstone in the global obesity epidemic [86]. Obesity develops when the energy balance is positive and caloric intake exceeds energy expenditure for a longer period of time. Obesity is strongly associated with metabolic disease, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes and cardiovascular disease [87]. Insulin resistance plays a major role in the development of these diseases. On a group level, it has been shown that insulin sensitivity declines with increasing body weight [88]. Impaired insulin sensitivity, or insulin resistance, means failure to respond properly to insulin. Insulin-dependent cells (e.g. muscle cells and adipocytes) in the body require a higher concentration of insulin for removal of a given amount of glucose from the blood, compared to normal insulin sensitivity. For a period of time, the body can compensate for the insulin resistance by increasing the secretion of insulin to maintain normoglycemia. When the insulin secretion becomes insufficient, glucose levels will rise and the individual will suffer from hyperglycemia and may be diagnosed with type-2 diabetes. There are several contributing factors in development of insulin resistance like inflammation, oxidative stress, mutations in the insulin receptor and mitochondrial dysfunction, but the underlying cellular mechanisms are still not fully understood [89,90]. Effects of insulin resistance on metabolic processes in liver, adipose and muscle tissues have been well described.

In adipose tissue, insulin normally inhibits intracellular lipolysis by blocking the action of ATGL and HSL [33]. In the case of insulin resistance, lipolysis cannot be sufficiently blocked, meaning that FA leak out rather than being stored as triglycerides in the adipose tissue [31]. A serious consequence of elevated levels of NEFA in blood is lipid accumulation in other tissues like liver, heart, and even in pancreas [91,92]. This usually causes functional disturbances of the tissues, and may cause severe cell damage and further impair insulin signaling, a phenomenon called lipotoxicity [93,94].

In skeletal muscle, insulin binds to the insulin receptor on the surface of the myocyte and a signaling cascade initiates translocation of the glucose transporter 4 (GLUT4) to the cell surface [95]. Via GLUT4, glucose is transported into the cell

and further oxidized to generate energy. In insulin resistance, glucose uptake via GLUT4 becomes reduced, with impact on both glycolysis and glycogenesis, leading to an elevation of blood glucose [96].

The liver is the main organ responsible for maintaining blood glucose levels during fasting. This is of uttermost importance since there are cells in the body that only utilize glucose for ATP production. During fasting, or in between meals, the body needs to increase glucose production by gluconeogenesis. When food is ingested, providing exogenous sources of glucose, insulin rises following increased glucose levels in blood, and gluconeogenesis in liver is thereby stopped. This is all governed by intricate sensing and signaling systems involving insulin and its receptor, and is to a large extent executed through control of activities of key enzymes [97]. During insulin resistance, when signaling chains from the insulin receptor are dysfunctional, the liver continues to produce glucose even though glucose levels in blood are already high, thereby aggravating the condition [98].

The increased outflow of FA from the adipose tissue during insulin resistance, causes increased uptake of NEFAs by the liver [99]. When NEFA influx exceeds the capacity of the liver for lipid oxidation and VLDL secretion, the surplus is stored as triglycerides in lipid droplets, causing steatosis [100]. When the TG stores exceed 5.6 % of the liver weight, corresponding to macroscopic steatosis in >15 % of hepatocytes, the condition is classified as NAFLD [101]. NAFLD is a very common condition and is also the most common cause of elevated liver transaminases [102]. Untreated, NAFLD may develop into liver steatohepatitis with and increased risk for liver cirrhosis and hepatocellular carcinoma. Obesity and type 2 diabetes are closely linked to NAFLD. Around 60 % of patients with type 2 diabetes suffers from this condition and up to 90 % of obese individuals [102]. NAFLD is also on its own a risk factor for cardiovascular disease [103].

Hyperlipidemia Hyperlipidemia refers to elevated levels of blood lipids. The elevation could be in cholesterol (hypercholesterolemia), in triglycerides (hypertriglyceridemia) or in both (mixed hyperlipemia). It is well known that hypercholesterolemia, due to elevated levels of LDL, is a strong risk factor for plaque formation in artery walls, and for development of atherosclerosis, resulting in cardiovascular disease with severe consequences like myocardial infarction and stroke [104]. It is now well accepted that also elevated levels of plasma triglycerides (in chylomicron remnants, VLDL and IDL) constitute an independent risk for development of atherosclerosis [105]. However, mixed forms of hyperlipemia are most common and are often connected to obesity, physical inactivity and/or insulin resistance. Hyperlipidemia is part of the so called metabolic syndrome which is characterized

by high plasma TG, elevated LDL-cholesterol, low HDL-cholesterol, abdominal obesity, high blood pressure, and elevated blood glucose [106]. Hypercholesterolemia can be due to genetic defects in the LDL receptor or in its important regulator PCSK9 [107]. Hypertriglyceridemia can be due to defects in adipose tissue function, renal disease, hypothyroidism, diabetes or be directly due to genetic defects in apolipoproteins (usually ApoCII or ApoAV), LPL, or LPL regulatory proteins [108].

There are conditions when a high level of plasma triglycerides is not associated with disease, like during pregnancy or a result of specific diets [108,109]. After a meal containing high amounts of fat, triglyceride levels in blood rise to a peak level usually after 2-3 hours, but return to fasting levels after 5-6 hours (in humans). Meals containing normal, more balanced, amounts of fat may not cause any significant, elevation of triglycerides in healthy, young and physically active individuals. It is likely that modest, intermittent hypertriglyceridemia or mixed hyperlipemia during the day can be well tolerated, and in the healthy subject the lipid levels normalize during fasting overnight. If this does not occur, due to a combination of reduced clearance rates from blood of ingested fat, dysfunctional chylomicron remnant removal and/or increased amounts of VLDL being secreted from the liver, pathological hyperlipidemia in plasma taken after fasting is a fact. Reduced clearance of triglycerides in chylomicrons and VLDL is probably due to dysfunction of the LPL system [108]. When the TRLs stays in the circulation for a prolonged time, smaller and denser LDL than the normal may be formed due to lipid exchange between lipoproteins of triglycerides and cholesteryl esters catalyzed by the protein cholesterylester transfer protein. The small, dense LDL are for several reasons thought to be more atherogenic and will increase the risk for cardiovascular disease [110].

Kidney disease and dyslipidemia Nephrotic syndrome, a symptom of kidney disease where protein leaks into the urine, is accompanied by dyslipidemia to varying degree. Both lipid and lipoprotein metabolism are altered, with or without chronic renal failure [111]. The severity of the dyslipidemia is correlated with the degree of proteinuria. Plasma concentrations of ApoB-containing particles like VLDL, IDL and LDL are elevated, and their composition of apolipoproteins may be altered. Many apolipoprotein increase, ApoB, ApoE and the ApoCIII/ApoCII ratio [112]. Circulating ANGPTL4 is also increased in glomerular disease [113]. The protein loss in nephrotic syndrome reduces the level of circulating albumin and thereby increases the ratio of free FA to albumin. This may promote the uptake of FA from blood, causing activation of nuclear receptors and stimulation of ANGPTL4 production [114]. The increase in ANGPTL4 levels, and typically also in the amounts of ApoCIII, may reduce the clearance of TRLs by inactivation and/or

inhibition of LPL, and inhibition of receptor-mediated uptake of remnants. The amount of GPIHBP1 is decreased in nephrotic syndrome, which may result in a smaller pool of active LPL at the capillary endothelium [115]. Another part in the reduced lipoprotein clearance in nephrotic syndrome is due to an increase in PCSK9, and thereby increased degradation of the LDLR [114]. The process of so called reversed cholesterol transport by HDL to the liver is also hampered in nephrotic syndrome, due to impaired maturation of HDL [114]. The dyslipidemia is further aggravated in chronic renal failure and is involved in accelerated atherosclerosis with the risk of premature death [116].

The role of LPL in different tissues Tissues like heart, skeletal muscle and adipose tissue have the highest levels of LPL activity since these tissues utilize a substantial amount of FAs. LPL activity is under tight control in order to direct FAs to specific tissues depending on exogenous and endogenous factors. The nutritional state is a major factor determining the flow of FAs from triglycerides in plasma lipoproteins. Fasting inhibits LPL activity in adipose tissues, while in the muscles, LPL activity stays the same or may be increased [72,117]. In the postprandial state the situation is reversed with high LPL activity in the adipose tissue and low in the muscle. This regulation of LPL activity enables the ingested fat to be stored in times of excess and to be used in working muscles when energy substrates are scarcer, or in brown adipose tissue when there is a need to generate heat. A summary of how LPL activity responds to different physiological conditions is found in Table 3.

Conditions White Brown Skeletal Heart Kidney influencing adipose adipose muscle LPL tissue tissue activity Fasting ↓ ↓ ↑ ↑ ↓ Feeding ↑↑ ↑ ↓ ↓/↔ ↑↑ Exercise ↓ ↑ Cold ↓ ↑↑↑ Obesity ↑/↓ ↓ ↑/ ↔ ↓

Table 3: A summary of conditions and factors influencing LPL activity.

Adipose tissue Adipose tissue serves as a reservoir for energy in the form of triglycerides. At stable conditions there is a balance between influx and efflux of FAs from these stores. LPL is a determining factor for FA influx. LPL activity in adipose tissue is correlated to the uptake of TG derived FAs from TRLs. The uptake of TG is high in the post-prandial state when LPL activity also is high, and low in the fasted state when LPL activity is low [118]. A meal high in carbohydrates stimulates

adipose tissue LPL to a greater extent, compared to a high-fat meal. Both glucose and insulin have a stimulating effect. Depending on the adipose tissue depot, LPL activity and the effect of insulin and glucocorticoids may vary [119].

Although LPL is important for the delivery and uptake of FA in adipose tissue, the enzyme is not crucial for building adipose tissue stores. This is demonstrated by the fact that LPL-deficient subjects have essentially normal adipose tissue distribution [23,120]. A clear sign of reduced FA uptake is, however, that the fatty acid composition in the stored lipid is different from normal, indicating increased local de-novo lipogenesis. In subjects with heterozygous LPL deficiency, plasma triglyceride levels are usually in the normal range, but the amounts of LPL activity in post-heparin plasma is lower than in non-carriers. Heterozygotes are prone to develop hypertriglyceridemia when exposed to metabolic challenges [121].

LPL expression and translation in WAT is usually stable with only slight variations [72,117,122]. The daily variation in nutritional status has little or no effect on LPL expression and protein mass [123,124]. Thus, the regulation of LPL activity is mostly posttranslational and was shown to be dependent on transcription of other proteins [82,125]. As discussed above, inactivation of LPL on fasting is due to ANGPTL4 [82,83,126]. In Angptl4-/- mice LPL activity in WAT remains at post-prandial levels during fasting [122]. ANGPTL4 transcription is thought to be mediated by binding of FA to PPARγ when NEFA levels, and thereby intracellular FA levels, are high [126–128].

The nutritional response of LPL activity in adipose tissue on food withdrawal, or fasting, was shown to be abolished with aging in rats [122,129]. Similar findings are reported from studies on humans [130]. This is explained by an increased insulin resistance in the tissue and a thereby blunted response of ANGPTL4 expression to insulin, resulting in high levels of ANGPTL4 and low activity of LPL also in the post-prandial state [122].

Skeletal muscle The nutritional regulation of LPL in skeletal muscle goes in the opposite direction to that in WAT. LPL activity in muscle is high in the fasted state in order to deliver FA for ATP production. In the post-prandial state the LPL activity in skeletal muscle is reduced, directing FA from TRLs to adipose tissue for storage [72]. Skeletal muscles can broadly be divided into two different fiber types [131]. Type I are so called slow twitch red fibers that have a high oxygen-binding capacity and rely mainly on oxidative metabolism. Type I fibers are used during low-intensity physical activity and can work for a long period of time. Type II, or fast twitch white fibers, are used during short but high-intensity physical activity. The fast twitch white fibers are mainly glycolytic and contains high levels of glycolytic

enzymes [132]. The muscles with the highest content of red oxidative muscle fibers also have the highest level of LPL activity and TG uptake [133]. The soleus muscle of the calf contains around 80% red fibers while the vastus muscle contain 57%, which is also reflected in the level of LPL activity [132,134]. The composition of muscle fibers varies between individuals and species. The rodent has a higher proportion of white fast-twitch glycolytic fibers because they are prey-animals. The mouse soleus muscle contains a high proportion of red oxidative fibers with high LPL activity (Paper II).

The difference between LPL activity in homogenates of skeletal muscle between the fasted and the fed state is not as prominent as what can be seen in homogenates from WAT. The LPL activity in skeletal muscle is often found slightly higher in the fasted state but not always (Paper II). The levels of LPL mRNA are usually quite stable in both the fasted and the fed state. It is likely that the most important regulation of LPL in skeletal muscle, like in the heart, concerns how much of the enzyme that is exposed on the luminal side of capillaries and there can act on lipoproteins. An estimate of this pool for the whole body is given in measures of LPL activity in blood after heparin injection, LPL activity in post-heparin plasma, sometimes named the functional pool of LPL [117]. To get measures for individual tissues, samples of the tissues have been incubated in heparin-containing buffers to extract the extracellular LPL. These measures are technically difficult and may explain why the overall picture of regulation of LPL in muscle is relatively unclear.

Transcription of ANGPTL4 in muscle is upregulated upon fasting, like in adipose tissue. This is probably due to the increased NEFA-levels that stimulate PPARs. Despite high levels of ANGPTL4 during fasting, LPL activity increases compared to the fed state, where ANGPTL4 levels are much lower (Paper II). This may indicate that LPL and ANGPTL4 are not in contact in the same cellular or tissue compartment, or that LPL is immediately stabilized after synthesis by interaction with other ligands or with GPIHBP1.

A potent stimulator of muscle LPL activity is exercise [135,136]. Longer training programs with specific exercises repeated for several weeks increases LPL activity compared to sedentary controls [137]. In acute exercise the effect on LPL activity has been contradictory [137]. An interesting aspect is that ANGPTL4 has been proposed to have a role in protecting inactive muscles from excessive FA uptake under conditions when other muscles in the body are working and rapidly consuming FA [135].

Heart The heart is the most mitochondrial-dense and oxygen-consuming organ in the body. Around 50-75% of the generated ATP comes from FAs [138]. However, in senescence and in metabolic diseases the substrate preference of cardiomyocytes can change, inducing so called metabolic shift or “metabolic remodeling”. In heart failure, FA oxidation is reduced and glycolysis is increased [139]. The metabolic change may induce cell damage and aggravated disease by an overload of FA [140]. Mice fed a HFD develop obesity and signs of metabolic syndrome, and may also develop diabetic cardiomyopathy [141]. This illustrates the need to carefully regulate the influx of FA to the heart.

The majority of FA utilized in the heart originates from TG transported by lipoproteins and LPL is needed for accumulation of lipid in the murine heart [142]. Like in skeletal muscle, LPL activity in the heart is usually slightly higher in the fasted state, when measured in whole tissue homogenates, while the levels of LPL mRNA are stable (Paper II). The functional pool of active LPL, determined as the amount of heparin-releasable LPL activity, is a small fraction of the total tissue LPL activity [143–145]. The functional pool of LPL increases during fasting together with an increase in uptake of TG from lipoproteins [72]. Like in skeletal muscle, this important modulation of the distribution of LPL in the tissue must involve other factors than ANGPTL4. No clear mechanistic picture for this has yet emerged [146,147].

The central nervous system LPL is present in several parts of the nervous system, in various regions of the brain, the spinal cord and in peripheral nerves [57]. The highest levels of LPL mRNA are found in the hippocampus [148,149]. LPL has been found intracellularly in neurons, but has also been seen associated with endothelial cells [150]. GPIHBP1 is not present in capillaries of the normal brain [39]. However, GPIHBP1 has been found in endothelial cells of gliomas where the protein was proposed to transfer LPL to the luminal side for capture of TRLs [151]. Normally, TRLs do not pass the blood-brain barrier, only HDL appears to have a ticket [152]. Lipoproteins found in the brain are therefore produced and used within the nervous system, and they are rich in ApoE [153]. ApoE is a well-known component of Alzheimer plaques, especially the E4 isoform which is a risk factor for early development of the disease [154,155]. In the peripheral circulation ApoE binds to the LDLR and LRP1, thereby mediating lipoprotein uptake and degradation of lipoprotein remnants, mostly in the liver [156]. Lipoprotein receptors are also present in the brain. They have signaling functions, but are also likely to be involved in metabolism of brain lipids and in uptake of amyloid precursor protein (APP) and amyloid-beta peptide (Aβ) [148]. Studies have shown that LPL can mediate uptake of Aβ in astrocytes [157].

Aims of the thesis

The overall aim was to study LPL in organs where the localization and function of the enzyme was less known than in the major metabolic organs like adipose tissue, heart and skeletal muscle. We focused on pancreas, kidney and placenta in mice. In order to understand more about the function of LPL in each case, we also aimed to compare the regulation of LPL activity and the impact of metabolic challenges to what was previously known from other tissues.

Paper I. Pancreas

 To develop an immunofluorescence protocol for detection of LPL using an affinity-purified polyclonal LPL antibody produced in-house  To localize LPL and study the effect on LPL distribution of obesity induced by leptin deficiency or HFD

Paper II. Kidney

 To localize LPL by immunofluorescence and in situ hybridization  To investigate the role of ANGPTL4 in regulation of LPL activity  To study the effect on regulation of LPL activity after long-term HFD  To study the role of LPL in uptake of lipids from chylomicrons

Paper III. Kidney

 To develop a protocol for investigation of uptake of glucose and long- chain fatty acid analogues using PET/CT  To investigate effects of HFD on energy substrate preference  To use the LPL knock-out model MCKL0 to support studies on location and function of LPL

Paper IV. Placenta

 To localize LPL by immunofluorescence and in situ hybridization  To use the Angptl4-tg+/- and Angptl4-/- mouse models to study the role of ANGPTL4 in regulation of LPL activity during pregnancy and potential effects on fetal growth

Materials and Methods

Mouse models with metabolic dysfunction To study human disease a powerful and common strategy is to use genetically modified mice. By using different techniques a human gene that is overexpressed can be introduced so that the mice will produce high amounts of the protein of interest. This is usually called a transgene model (tg) and if the mouse is homozygous for the human gene it will be labeled tg+/+. In our experiments the mice were heterozygous and therefore labeled tg+/-. In a knock-out mouse, the gene is missing on both alleles and labeled -/-.

For studies on energy metabolism, metabolic changes can be induced in mice by feeding specific diets. In this thesis both genetically modified mice, mice with a spontaneous mutation leading to metabolic dysfunction, and mice with diet- induced obesity were included. All models were on a C57Bl/6J background, one of the most common laboratory mouse strains. This was used as the reference mouse and is designated wildtype (WT). The mouse models used in each Paper are summarized in Table 4. For all experiments the mice had free access to food and water, except before blood sampling when animals where fasted. All animal experiments were approved by the Animal Review Board at the Court of Appeal for Northern Norrland in Umeå, Sweden (A54-11, A2-14, A35-17).

C57Bl/6J DIO Angptl4-/- Angptl4-tg+/- ob/ob MCKL0 Paper I M M F Paper II M/F M/F M Paper III M M M Paper IV F F F

Table 4: A summary of mouse models used in the present thesis. M = Male; F = Female.

Diet-induced obesity (DIO) To study obesity and metabolic disturbances induced by diet, we fed C57Bl/6J mice a high-fat diet ad libitum (Research Diets, D12492), with 60 kcal% from fat. Regular rodent chow diet contains around 9 kcal% fat and 69 kcal% from carbohydrates (801730, Special Diets Services). Body weight was followed to overlook obesity development and as a complement, body composition was measured using an Echo-MRI system (Echo Medical Systems, TX, USA). An adult mouse on regular diet weigh around 25-30 grams while a mouse fed a high-fat diet can be twice as heavy (50-60 grams, Figure 4).

Figure 4: C57Bl/6J mice with diet-induced obesity. Photo by Madelene Ericsson.

Ob/ob The ob/ob, or the obese/obese mouse, was discovered in Jackson Laboratories [158] and later bread on the C57Bl/6J background. At young age the mice start to gain weight rapidly. At 3 months they weigh twice as much as their healthy siblings [158]. The weight gain is caused by a mutation in the gene encoding leptin [159]. Leptin is a hormone produced in adipose tissue and involved in appetite regulation by binding to leptin receptors in the hypothalamus [160]. In homozygotic deficient mice, where leptin is completely absent, no signal on satiety is returned after a meal. The mice become hyperphagic, obese, hyperinsulinemic and hyperglycemic [161], making the ob/ob mouse a good model for some studies on metabolic disturbances.

Angptl4-tg+/- and Angptl4-/- In order to study functions of ANGPTL4, Köster and co-workers developed a mouse model overexpressing human ANGPTL4 in the liver, and a complete knock-out model [162]. To overexpress human ANGPTL4 cDNA they used a vector for liver-specific expression (using the ApoE promoter and its hepatic control region) in a similar manner as had been done for the hepatic lipase transgenic mice generated by Fan et al in 1994 [163]. Overexpression reduced LPL activity and increased levels of triglycerides and cholesterol in blood. The Angptl4-/- was generated using a targeting vector to delete the endogenous gene. In the absence of ANGPTL4, triglycerides in plasma of fasted animals were reduced by 65-90% compared to WT mice. The strong effects on the plasma lipid profile in these genetically modified mice makes them interesting models for studies of different aspects of lipid metabolism.

Implications of circadian rhythm, nutritional state and sex To study variations in LPL activity depending on nutritional state there are a couple of things to bear in mind. The activity of LPL follows a circadian rhythm,

meaning that the activity will vary depending on the hour of the day. The duration of both fasting and feeding time will also affect the level of LPL activity [122,164– 166]. For both kidney and adipose tissue the majority of LPL activity has dropped 6 hours after food withdrawal [164,166]. Prolonged fasting may induce unnecessary stress and may not affect the LPL activity much more. It is therefore important to consider the hour of the day at which the experiments are performed, as well as fasting and feeding time.

Rodents are nocturnal animals and are most active during the night. LPL activity in adipose tissue follows this rhythm, being highest during the night and early morning [165]. In the animal facilities rodents are kept on a 12:12 hour light-dark cycle. To be able to perform experiments during the mice active period, the dark period was set to 01:00-13:00. It is challenging to do well-controlled comparisons between the fed and the fasted state in rodents. Therefore, a protocol with refeeding after a period of fasting is often used to increase the probability that each individual animal is well fed at the time for the experiment. The refeeding period should preferably occur during the dark and active period. To ensure that food intake was fairly similar in the experiments in this thesis, the food was measured at the end of the refeeding period. Animals with low intake was excluded, because eating behavior and food intake will affect the level of LPL activity [166].

The C57Bl/6J strain, is susceptible to diet-induced obesity, especially the males [167]. Female mice appear to be more resistant to the HFD and obesity development [168], which is why both genders were included in Paper II.

To increase the rate of pregnancies in Paper IV, bedding from male mice was introduced to the cages of females to induce ovulation. After a couple of days, the respective male was introduced to the female in the afternoon. For the following 3 days the females were checked for a vaginal plug twice a day. When a vaginal plug was visible, females were transferred to a cage of her own for us to be able to measure individual food intake. If no plug was seen after 3 days the pair was separated and the procedure was repeated after 1.5-2 weeks.

Tissue handling and analyses Tissues for gene expression analyses were harvested and stored in RNAlater before RNA extraction. cDNA was prepared and analyzed by RT-qPCR. The level of mRNA was measured using gene expression assays from ThermoFischer. Relative expression was calculated using 2(-ΔΔCt) with an endogenous control (housekeeping gene). For protein analysis, tissues were snap frozen in liquid nitrogen and analyzed by western blots. Tissue samples for measurements of LPL activity were also snap frozen in liquid nitrogen and stored in -80 °C until homogenization in buffer with detergents and protease inhibitors. LPL activity

was measured as earlier described [124]. LPL activity is expressed as mU/g wet tissue or per mU/mg total protein, where one mU corresponds to release of one nmol fatty acid per minute during incubation at 25°C. The activity levels are dependent on the lipid substrate used, and can vary considerably between experiments, probably due to the properties of the lipid emulsion. Therefore care was taken to always analyze samples for comparison using the same batch of emulsion, and also using known standards. Total protein was measured using the modified Lowry method [169]. Double-radiolabeled chylomicrons were prepared in rats as described [170], and were injected in mice. At termination, remaining chylomicrons were removed from the vasculature by PBS perfusion through the heart. Tissues were harvested and snap frozen in liquid nitrogen. Lipid extraction was performed using chloroform-methanol [170] and the accumulated radioactivity levels were counted.

Visualization of LPL was done using immunofluorescence. Harvested tissue was fixed in freshly prepared 4 % PFA and frozen in OCT compound. By using primary antibodies that binds to specific epitopes together with secondary antibodies coupled to a fluorescent dye, the protein of interest can be visualized. In Paper I, the aim was to study the localization of LPL in mouse pancreas. A good and reliable LPL antibody for immune staining was therefore needed. To find new antibodies suitable for this method several LPL antibodies that had previously been produced in-house were evaluated. Chickens and rabbits were immunized with LPL originating from bovine milk. After immunization, antibodies in rabbit sera (IgG) or egg yolk (IgY) were affinity purified using LPL- sepharose columns [171]. The antibodies were initially tested using dot blots. IgY from thirteen immunized chickens where selected for immunofluorescence on mouse tissues. Five were affinity purified and the rest were used as the whole fraction of IgY. As controls IgY from four non-immunized chickens were used. The antibodies were then tested on frozen sections of mouse cardiac tissue, because of its high content of LPL protein. The most promising antibodies were also evaluated on pancreas and kidney. Affinity purified IgY from chicken 219 (affi 219) convincingly showed specific staining for LPL, together with low background staining. This antibody was used in Paper I-IV and has also been used by collaborators [39,52,172–174]. All other antibodies used were commercial, and referred to in the papers. Confocal microscopy was used to obtain images of immunostained tissue. To visualize the location of LPL mRNA, a digoxigenin-labeled probe was used for in situ hybridization (ISH). Images were obtained with a light microscope or a Pannoramic 250 Flash scanner from 3DHistech.

Blood biochemistry Glucose was measured on blood from the tail vein collected using a hand held Accu-Chek glucometer. Larger blood volumes were sampled from tail veins in EDTA-covered tubes. The blood was centrifuged (4-10,000 rpm) for 10 min at 4 °C and plasma was collected and frozen in -80 °C. Plasma was used for measurements of NEFA, insulin and TG by commercially available kits, refered to in the respective paper. Glucose and insulin levels were used to calculate HOMA-IR ((fasting glucose (mmol/l) x fasting insulin (mU/l))/22.5). IntraPeritoneal-Glucose Tolerance Test (IP-GTT) was performed to measure glucose handling. A 20%-glucose solution in saline was injected i.p. (10 µl/g body weight), and blood glucose was measured at several time points (5, 15, 30, 60, 90 and 120 min). To obtain a profile for TG in lipoprotein subclasses, a high- performance liquid chromatography system developed for mouse plasma, HPLC, was used [175].

Small-animal PET/CT In paper III a small animal PET/CT (nanoScan, Mediso imaging system, Hungary) was used to study energy substrate uptake from blood in vivo and ex vivo. PET stands for positron emission tomography. This technique can be used to study metabolic processes by the injection of radiolabeled tracers. For morphological orientation in the specimen a CT (computed tomography) scan is performed together with the PET. In paper III a [18F]-labeled glucose analogue and a [18F]-labeled FA analogue was used. The glucose analogue, FDG (2-deoxy- 2-[18F]fluoro-D-glucose), is a tracer commonly used in the clinic for metastatic diagnosis and follow-up. The FDG used in the study was produced locally at the University Hospital of Norrland, Umeå, Sweden. The FA tracer used was FTHA, 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid. FTHA is a long-chain FA that was first synthesized by DeGrado et al in 1991 [176] to be used in characterization of oxidative substrate metabolism in the myocardium. Synthesis of the FTHA used in paper III was modified to reduce the final amount of ethanol in the tracer, thereby avoiding administering a high dose of ethanol to the mouse.

Statistics Data presented in this thesis are either presented as mean ± standard error of mean (SEM) or mean ± standard deviation (SD). Group sizes were chosen based on power calculations and the impact of individual variation, especially when it came to measurements of LPL activity in refed mice. Depending on the number of groups, either t-test (paired or unpaired) or One-Way ANOVA was used. In the case of non-parametric distribution the non-parametric version was used to compare ranks. For studies of correlations between two variables, Pearson´s correlation was used.

Results and discussion

Paper I

Background Pancreas has major roles in metabolism. By producing a mixture of enzymes (pancreatic juice), which is secreted into duodenum, the pancreas assists in food digestion. Spread out in the exocrine glandular tissue thousands of small cell clusters, called islets of Langerhans are found. These islets houses four different cell types. All of them produce and secrete their unique hormone destined for different metabolic purposes. The most abundant cell type is the β-cell that secrete insulin to maintain normoglycemia. Increased glucose levels in blood leads, by a chain of intracellular events, to an increase in the β-cell ATP/ADP ratio. This triggers voltage-sensitive KATP channels in the β-cell membrane to close, and thereby a sudden influx of calcium. This causes fusion of insulin- containing granulae with the cell membrane and release of their insulin into local capillaries for further transport with blood to different organs to assist in glucose uptake [177]. Aside from glucose, FA can stimulate insulin secretion. An acute exposure to FA stimulate insulin secretion, while a prolonged exposure to FA blunts the β-cell response to glucose [92,178,179]. The exact downstream mechanism from FA binding/uptake to insulin secretion is in focus for an ongoing field of research [178,180]. There are FA receptors (G-protein coupled), present on β-cells that are involved in FA-stimulated insulin secretion [181]. The saturation and length of the FA has an impact on β-cell viability and insulin response. Long saturated FAs may induce cytotoxicity, while shorter unsaturated FAs are not harmful for the β-cell [92].

FA affecting insulin secretion can either originate from circulating NEFAs or be locally released from circulating plasma lipoproteins (TRLs). Our aim was to investigate the cellular distribution of LPL in pancreas by immunofluorescence, as LPL in other tissues provides FA from TG in plasma lipoproteins. LPL has previously been found in islets of Langerhans and in β-cells [182,183]. LPL activity has been shown to be stimulated by high glucose levels in INS-1 cells [184]. Mice with β-cell specific overexpression or absence of LPL were both reported with decreased insulin secretion, implying that a balanced amount of LPL is needed for normal insulin secretion [183].

Results We found that LPL is present both in the glucagon-producing α-cells, and insulin- secreting β-cells, but LPL is absent in the other islet cell types, the pancreatic polypeptide cells and in the somatostatin producing δ-cells. LPL in α- and β-cells appeared to be mostly intracellular, since staining was unaffected by injection of heparin, which is known to release LPL from cell surfaces. In contrast, in the exocrine pancreas, where LPL was localized to capillary endothelial cells, the staining for LPL disappeared after heparin injection. This indicated that in the exocrine pancreas, LPL was positioned on the endothelium inside the capillary lumen, like in other tissues. In this position LPL is probably able to act on plasma TRLs to provide the surrounding tissue with FAs and monoglycerides. In support of this, LPL seemed to colocalize with the trans-endothelial transporter protein GPIHBP1 in the exocrine pancreas. In other tissues GPIHBP1 transports LPL to its endothelial sites, holds it in place on the endothelial cell membrane and protects the enzyme from unfolding and inactivation. LPL was not found in the acinar cells of the exocrine pancreas, suggesting that LPL on the capillaries might be produced elsewhere.

Next we investigated the distribution pattern of LPL in pancreas of the leptin deficient ob/ob mouse, which is both obese and insulin resistant [159]. A very clear finding was that the immunostaining for LPL in β-cells of these mice was abolished compared to WT, while staining in the α-cells and in the exocrine pancreas was intact. The mice ranged from 5 weeks to 9 months of age. The absence of LPL in β-cells was apparent already after 5 weeks, and in older ages LPL was still absent. We investigated the levels of LPL mRNA in islets from ob/ob mice at different ages (1-4, 8 and 11 months). The LPL expression was found to be similar in all age-groups. The leptin deficient ob/ob mice are known to be “cured” by daily injections of leptin. We therefore administered leptin, and already after 12 days the mice were normoglycemic and had lowered their food intake. Interestingly, LPL reappeared in the β-cells. At the time of analysis some islets had completely recovered so that LPL staining was similar to that in WT mice, even though some islets only had a few LPL-positive β-cells. Expression of Lpl mRNA was decreased by 50% in ob/ob islets compared to WT and after leptin treatment, Lpl expression increased by ~25% (p>0.05). If the leptin treatment had been prolonged, it is possible that more islets had recovered and the Lpl expression measured by RT-qPCR might have been normalized. To investigate if a diet-induced obese mouse model with hyperglycemia would exhibit a similar loss of LPL in their β-cells, we investigated pancreas from mice fed a HFD for 12 weeks [174]. In islets from the HFD-fed mice, LPL was still present in β-cells, but the staining appeared slightly weaker.

Conclusions The expression of LPL in β-cells of mice seems to be dependent on leptin. The majority of LPL is intracellular in the β-cells, suggesting that at this site involvement in TG hydrolysis is not the major function of the enzyme. It is possible that LPL in the β-cells is involved in endocytotic uptake of lipoproteins via the LDLR or some other receptor, as the LPL was previously shown to have non-catalytic functions in receptor-mediated uptake of lipoproteins [51]. In contrast, in the exocrine pancreas LPL is localized on capillaries and is most likely involved in TG hydrolysis in plasma lipoproteins. The released FAs may be used for energy production in the tissue, and/or they may potentiate insulin secretion from β-cells when glucose levels are elevated. However, the origin of LPL in the exocrine pancreas is not clear. In the untreated ob/ob mice, LPL was still present in the exocrine pancreas despite that LPL was absent in the majority of the islet β-cells.

Paper II and III

Background The human kidney contains several lobes, whereas the mouse kidney is unilobular (Figure 5). Kidneys from both mice and men consists of cortex and medulla. The medulla can be further divided into the outer and inner medulla (Figure 5). Despite differences in size and in number of lobules, human and mouse kidneys contain the same functional unit, the nephron. The kidneys have several functions. They work as a filter to remove waste products and excess water and make sure that vital nutrients and electrolytes are reabsorbed. The majority of the reabsorption takes place in the proximal tubules (PT), and is an energy- requiring process [185]. The main source of energy comes from oxidation of FAs, but the kidney can also utilize glucose, lactate, ketone bodies and glutamine for energy production [186]. The highly oxygenated and mitochondrial-dense cells in the cortex favors FA. Tubular cells in the medulla contain fewer mitochondria, and the medulla is less oxygenated, making glucose the preferred substrate in this part of the kidney [187]. In states of metabolic dysfunction, such as obesity, insulin resistance and early stages of type-2-diabetes, glucose metabolism in the cortex increases as well as FA oxidation [188]. If the balance between uptake of energy substrate finally exceeds expenditure, intracellular lipid stores may increase. This may lead cell damage through lipotoxicity, and in the end to that tissue functions are lost [93].

Figure 5: A schematic drawing of a mouse kidney containing a nephron, from Paper II. OMOS = Outer medulla outer stripe OMIS = Outer medulla inner stripe PT = Proximal tubule DT = Distal tubule CT = Collecting tubule

The location and specific functions of LPL in kidney was relatively unknown. LPL protein is present in the kidneys of several species, but with a great variation in activity level. In kidneys of mouse, mink, and Chinese hamster LPL activity is high, while in rat and guinea pig the activity is low [164]. LPL is present in human kidney [189], but the level of LPL activity and its possible nutritional response is not known. LPL mass in mouse kidney is not affected by the circadian rhythm, but some variations in LPL activity were detected [164]. Level of mRNA was relatively stable indicating post-translational regulation, similar to that in adipose tissue [164]. ANGPTL4 regulates LPL activity in WAT [80] and during fasting Angptl4 mRNA is increased severalfold in adipose tissue [122,127,128]. The role of ANGPTL4 for metabolic regulation in mouse kidneys was not known.

Results

LPL is localized to proximal tubules Staining for LPL was mainly found in the outer medulla outer stripe (OMOS), but some LPL was also present in the cortex (Paper II). Starting at the beginning of the nephron in the glomeruli, only few cells contained LPL. From the literature, these cells are likely mesangial cells [190]. Moving further into the first part of the renal tubule, the proximal tubule (PT), neither Lpl mRNA nor intracellular LPL is present. Interestingly, in the later segments of the PT, Lpl mRNA is expressed and strong staining is found for LPL protein. After heparin treatment, LPL still remained in PT cells, suggesting an intracellular location. We verified the localization of LPL to PTs by co-staining with an antibody to megalin, which is PT specific [191]. Megalin, also called LRP-2, is a member of the LDL-receptor family involved in receptor-mediated endocytosis, and it may also bind to LPL [192]. We speculated that due to the intracellular location of LPL in PTs, LPL could be involved in potentiating receptor-mediated uptake of filtered low- molecular weight proteins. However, no colocalization was found between

megalin and LPL, making this hypothesis unlikely. We also studied the distal tubules (DT) using an antibody to E-cadherin as marker [193]. No costaining with LPL was found, indicating that LPL is present only in PTs. LPL was also absent further down along the nephron, in the collecting tubules, and the inner medulla. To verify the specificity of the antibody we investigated kidneys from the LPL knock-out mouse model MCKL0. There was no LPL staining in kidneys of these mice with our antibody (Paper III).

To investigate if the nutritional state could have an impact on the localization of LPL, we studied both fasted and refed animals. Interestingly, we found a difference in staining intensity between the fasted and the refed state. In mice in the refed state, LPL staining was intensified in the PT cells and in intertubular vessels. By western blots we found increased levels of LPL protein. In the refed state, LPL colocalized with GPIHBP1, suggesting that LPL was then to some extent translocated from its intracellular location in tubular cells to the luminal side of the inter-tubular capillary endothelium. It is therefore likely that in the fed state LPL can act on TG in the TRLs in blood (Paper II). Heparin treatment also showed that LPL in intertubular vessels could be removed, supporting a luminal location on endothelial cell membranes.

LPL is not involved in triglyceride uptake and accumulation To investigate the role of LPL in uptake of TG from TRLs in blood, we administered radiolabeled chylomicrons intravenously (Paper II). Despite high LPL activity in the mouse kidneys, uptake of chylomicron-derived TG was low and the uptake was the same in both fasted and refed mice. For comparison we quantified uptake in adipose tissue and, as expected, uptake of chylomicron- derived TG label was high in adipose tissue of refed mice. This clearly demonstrated that the level of LPL activity in kidneys did not matter for TG uptake from TRLs. A similar result was previously reported from studies on mink, who, like mice, have high LPL activity in their kidneys [194,195].

During fasting, TG accumulates in the kidney, probably due to massive inflow of NEFAs [196]. To finally exclude the possibility that LPL could have a role for uptake of FAs from TRLs, contributing to the TG accumulation, we used the MCKL0 model. In paper III we could show that despite the total absence of LPL, TG levels were not affected. LPL knock-out mice had accumulated the same amount of TG as WT mice.

LPL activity in kidney is regulated by ANGPTL4 In paper II we found that fasting stimulated a severalfold increase in Angptl4 mRNA levels in kidney, and an increase in the amounts of ANGPTL4 protein. In Angptl4-/- mice LPL activity in kidneys remained high in fasted mice. We

therefore concluded that LPL activity in mouse kidney is controlled by ANGPTL4, similar to LPL in adipose and other tissues.

The ANGPTL4 protein in kidney appeared to be slightly smaller than ANGPTL4 in other tissues. On western blots, kidney ANGPTL4 was found at a position corresponding to a molecular mass of 53-56 kDa, while in heart and adipose tissue the migration corresponded to 58 kDa (Paper II). Treatment with the deglycosylation enzyme PNGase F revealed protein bands corresponding to 45 kDa and to 50 kDa. In heart, PNGase F treatment gave only one band at 50 kDa. We concluded that mouse kidney appears to have a form of ANGPTL4 that is different in molecular size and/or glycosylation from ANGPTL4 in other tissues. The smaller ANGPTL4 could have been produced by podocytes, and has been described by others [71].

Effects of obesity on LPL The nutritional regulation of LPL in adipose tissues is hampered in cases of obesity and insulin resistance [119,122]. We therefore wanted to study the effect insulin resistance and obesity might have on LPL in kidneys. Obesity and insulin resistance was induced by feeding WT mice a HFD for ~20 weeks. We noticed that male mice became more obese and more insulin resistant compared to female mice. For all mice, the nutritional regulation of LPL activity was lost in adipose tissue. Also the expression of Angptl4 mRNA and ANGPTL4 protein levels had lost their dynamic response to the nutritional status. LPL activity in kidneys of male mice was dysregulated in response to fasting, while the regulation was preserved in female mice (Paper II). The explanation may be that in female mice, the expression of Angptl4 in kidney still responded to fasting. These data suggest that female mice are more resistant to HFD, and that the level of obesity and insulin resistance has impact on regulation of LPL activity. Insulin resistance may severely hamper the role of ANGPTL4 as a controller of LPL activity also in mouse kidney. That female mice are more resistant to metabolic dysfunction with regard to insulin resistance after HFD feeding has previously been demonstrated by others [167,168].

Obesity increases FDG and FTHA uptake The kidney can utilize several types of substrates for ATP production, and the different morphological structures differ with regard to substrate selection [186,187]. It is well known that obesity and insulin resistance changes the substrate preference in heart [197–199]. In diabetic kidneys, both glucose oxidation and FA utilization are known to increase [188]. Cardiac metabolism and the response to diet-induced metabolic dysfunction in the heart has been thoroughly studied, but less is known for the kidneys [141,200].

In paper III, male mice were used for studies by PET/CT due to their increased susceptibility to HFD, as shown in paper II. Just like the male mice in paper II, the male mice in paper III lost their ability to down-regulate LPL activity after food removal, both in pgWAT and in kidneys, after ~20 weeks of HFD. PET demonstrates the actual uptake of a specific radiolabeled metabolite in a certain volume of the body. Therefore it is important that the signal measured comes from that organ, and not from unspecific uptake in adjacent structures. The standard procedure for administrating the tracer is by intravenous injection. Excess tracer will then be found in the blood, and some is excreted via urine. Measurements of uptake in kidneys will therefore be influenced by signals coming from both blood and urine. Due to similar Hounsfield CT values in the kidney and its surrounding tissues, the kidney border is difficult to accurately delineate. We developed a protocol to reduce the impact of these hampering factors that firstly included leaving the mice awake and freely moving for 3 hours after injection to reduce the tracer content in urine. Secondly, we performed perfusion with PBS before scanning to remove as much as possible of blood. Thirdly, scanning of the kidneys was performed after dissection ex vivo, with all surrounding pararenal adipose tissue removed.

In paper III we found, by using the glucose analogue FDG and the FA analogue FTHA, an increased uptake of both tracers in kidneys of obese mice fed HFD compared to that in kidneys from healthy, young mice. The FDG uptake in kidneys in a group of old mice was similar to young mice. Both the HFD mice and the old mice on normal chow were obese, but the chow fed old mice were normoglycemic in contrast to the insulin resistant HFD-fed mice. The total uptake of the tracers in kidneys was low compared to heart. We noted that FDG seemed to be concentrated in the medullary part of the kidney, whereas FTHA was more evenly distributed. The medullary accumulation of FDG could be due to dependency of more energy production from glucose in this part of the kidney. However, the sodium-glucose transporter 2 (SGLT2) has low affinity for FDG which can contribute to the relative FDG accumulation [201,202]. In a recent study PET/CT was performed on FDG uptake in rat kidneys. Those authors described technical difficulties with the small size of mouse kidneys [203]. By using the protocol described in paper III, it is possible to investigate mouse kidneys by PET/CT.

Conclusions In mouse kidney LPL appears to be mainly intracellular in PTs. In the refed state LPL is also present associated with intertubular vessels and GPIHBP1. LPL in kidney responds to the nutritional status and its activity is regulated by ANGPTL4. Uptake of chylomicron-derived TG in kidney is independent of the level of LPL activity, and TG is accumulated in kidneys of LPL knock-out mice to the same extent as in WT mice. Diet-induced obesity abolish the nutritional

regulation of LPL activity in male mice. The insulin resistant HFD fed mice have increased uptake of FDG and FTHA, suggesting an increased need for ATP imposed by HFD and obesity.

Paper IV Background In contrast to the female human reproductive anatomy, female mice do not have a uterine body. Mice have two long tubular structures, uterine horns, which joins at the cervix in the pelvic cavity. Each uterine horn has its separate cervical canal. At the implantation site, each fetus develops a placenta and has its own sac of fetal membranes. The laboratory mouse, C57Bl/6J, has a gestational length of ~18.5 days. After mating, a copulation plug (vaginal plug) is often seen the day after, counted as embryonic day 0.5 (E0.5). At the end of gestation, the fetuses are lined up along the uterine horns, resembling a pearl necklace. The mouse placenta is divided into four layers (Figure 6). Closest to the fetus is the chorionic plate where the chorionic vessels connect the fetal capillaries of the labyrinth zone to the vessels of the umbilical cord. In the labyrinth zone fetal capillaries are in contact with maternal blood for nutrient exchange. The junctional zone consists of different trophoblast cells, one of them being the glycogen trophoblasts. The decidua is closest to the uterine wall and contain maternal arterial and venous vasculature [204].

During pregnancy the metabolism adapts to ensure sufficient energy supply to the growing fetus. Glucose is the major nutrient connected to fetal growth and size [205]. FAs are also transferred to the fetus and utilized as an energy source, and essential FAs are crucial for organ development [206]. FAs can originate from maternal NEFAs or from TRLs. During the three trimesters of pregnancy, plasma levels of TG steadily increase [109]. TRLs, the large lipoproteins, cannot readily pass the placenta. Therefore the TGs needs to be hydrolyzed. LPL is known to be present in mouse and human placenta [207–209]. In human placenta LPL is present in trophoblast and syncytiotrophoblasts [208]. The location of LPL in the mouse placenta was not described. The level of LPL activity in human placenta is low, and the majority of the LPL protein appears to be inactive [208]. Associated with the increase in TG during pregnancy, there is an increase in plasma of the LPL inhibitor ANGPTL4 [210–212]. High expression levels of Angptl4 is found in the placenta [213], and shortly after birth, ANGPTL4 levels in blood returns to pre-pregnancy levels (unpublished data from our group).

Figure 6: H&E staining of one placenta from a C57Bl/6J mouse at E17.5. The four placental layers; decidua (dec); junctional zone (jz); labyrinth zone (lab); chorionic plate (cp). Asterisks marks finger-like projections originating from the junctional zone.

Results We wanted to investigate the role of ANGPTL4 in placenta for pregnancy in mice and for growth and development of the pups. Therefore we used pregnant mouse models without or with overexpression of ANGPTL4 and compared them to WT mice. Pregnant Angptl4-tg+/- mice gained weight and fat mass similar to pregnant WT mice. The Angptl4-/- mice gained more weight and accumulated more fat in the scWAT depot, and they also had a higher food intake than the other groups. All mice carried a similar number of fetuses per litter. Notably, fetuses from Angptl4-tg+/- mice were smaller and those from the Angptl4-/- were larger than fetuses from WT females. No changes in glucose handling was found between the different genotypes. All were normoglycemic. Before pregnancy, the plasma lipoprotein profile for the Angptl4-tg+/- and the Angptl4-/- were at opposite ends of the spectrum. VLDL and total plasma TG was unchanged in Angptl4-tg+/- during pregnancy, while in the Angptl4-/- mice there was an increase of both total TG and VLDL-TG from very low levels. At mid-pregnancy the WT mice had increased plasma TG and VLDL-TG to levels similar to those in Angptl4-tg+/-. Despite differences in total TG-levels, the NEFA levels did not differ between the groups. We investigated if the increase in weight of the pups in the Angptl4-/- dams could be due to increased placental LPL activity, but found that all pregnant groups of mice had similar levels. The difficulties in preserving the LPL activity before measurements could have had an impact on our results. Unfortunately, we did not collect cord blood to measure the actual transfer of NEFAs to the fetus. In WT mice, high expression level of Angptl4 was correlated with both reduced LPL activity and a smaller fetus. During pregnancy, Angptl4 expression increased also in scWAT and liver of the dams and could probably contribute to ANGPTL4 in blood. Unfortunately, no good antibodies were available for quantification of the ANGPTL4 protein in mouse plasma and tissues.

By immunofluorescence on placenta we localized LPL to the zone where nutrient exchange occur between the maternal circulation and that of the fetus, the labyrinth zone. Staining for LPL was found associated with CD31-positive cells, a marker for endothelial cells. This suggested that LPL was mainly localized to fetal endothelial cells, because the cells lining the maternal sinusoids are trophoblast cells. The endothelial membrane protein GPIHBP1 was also found in the labyrinth zone, together with LPL.

Conclusion The presence of maternal ANGPTL4 may influence fetal weight due to general effects on maternal plasma triglyceride levels executed through control of LPL activity. We found, however, that all genotypes carried the same number of fetuses to term. Angptl4-tg+/- dams carried the smallest fetuses. In WT mice, placentas with high expression of Angptl4 correlated with smaller fetuses. No gross morphological differences were found in placenta histology between WT, Angptl4-/- or Angptl4-tg+/- mice, demonstrating that although the ANGPTL4 protein has the potential to influence both angiogenesis and tissue development, there were no such obvious effects in placentas.

LPL is present in the mouse placenta and to a large extent in the labyrinth zone where nutrient exchange occur. LPL in the labyrinth zone is present together with GPIHBP1. This means that GPIHBP1 can translocate LPL and also protect it from inactivation by ANGPTL4. LPL activity in placenta did not differ between WT, Angptl4-tg+/- and Angptl4-/- suggesting that ANGPTL4 does not have a strong effect on LPL activity locally in the placenta. On the other hand, ANGPTL4 appears to affect blood triglycerides during gestation in mice, and despite high levels of TG in Angptl4-tg+/- their pups were smaller, suggesting that a local hydrolysis of TG in placenta is beneficial for fetal growth.

Major findings

Pancreas

 LPL is expressed in α- and β-cells in the islets of Langerhans, the LPL protein is intracellular in both cell types and the expression in β-cells is dependent on leptin  In the exocrine pancreas LPL is located on the luminal side of the endothelium of capillaries, together with GPIHBP1  Insulin resistance and obesity induced by HFD does not affect the cellular distribution of pancreatic LPL

Kidney

 LPL is present in the later segments of the proximal tubules of the nephrons  LPL is mainly intracellular in the epithelial cells of the proximal tubules, but is also present on the luminal side of the intertubular capillaries together with GPIHBP1  LPL activity in mouse kidney is reduced on fasting by increased expression of ANGPTL4, indicating a metabolic role of kidney LPL  The nutritional response of LPL activity is dysregulated in obese male mice, but intact in females  LPL is neither involved in uptake of chylomicron-derived TG nor in general TG accumulation in kidneys  Diet-induced obesity led to increased uptake of glucose and FA analogues as shown by PET/CT

Placenta

 LPL protein is located to the labyrinth zone of mouse placenta where the exchange between maternal and fetal blood occurs. LPL is present in fetal endothelial cells together with GPIHBP1  Total placental LPL activity was not affected by the extent of ANGPTL4 expression  Maternal overexpression of Angptl4 resulted in smaller pups  Lower placental LPL activity was correlated with smaller fetuses in WT mice  ANGPTL4 expression increases both in placenta and in maternal tissues during pregnancy, making it difficult to judge which source contributes most to the increased levels of ANGPTL4 and triglycerides in blood

Perspectives

LPL is present in the three organs we have studied, but the presence of the enzyme is not crucial in any of them. This is known from both humans and mice that survive with total LPL deficiency without any significant symptoms from any of these organs. Overall, direct effects of the lack of LPL have been difficult to demonstrate, apart from severe increase in plasma triglycerides and risk for acute pancreatitis. There are scattered, but no systematic, reports in the literature, and also unpublished rumors, on problems relating to glucose handling and to cognitive functions due to LPL deficiency.

In placenta, LPL seems to be logically positioned on endothelial cells to participate in delivery of energy substrates to the fetal circulation. There are also indications that the level of LPL activity in placenta matters for fetal growth. ANGPTL4 participates in regulation of LPL activity in maternal tissues, but the possible role of ANGPTL4 for local regulation of LPL in placenta needs more understanding.

In the endocrine pancreas and in kidney nephrons, LPL is mostly intracellular in endocrine cells, and in tubular epithelial cells, respectively. LPL cannot have its normal role for hydrolysis of triglycerides in plasma lipoproteins in any of these sites, while in capillaries in exocrine pancreas and in intertubular areas of kidneys LPL may contribute to FA uptake. In β-cells the expression of LPL is dependent on leptin. It will be interesting to investigate whether this is true in any other cells, e.g. in the brain.

LPL is described as a crucial enzyme for plasma lipid metabolism through its ability to hydrolyze triglycerides in lipoproteins that carry the cofactor ApoCII. This promotes local uptake of FA and is considered to be the major function of LPL in adipose and muscle tissues. In endocrine pancreas, kidney and also in the brain, (that for reasons of time was not studied here), LPL may have other functions that may not be dependent on the enzymatic activity. The intracellular location of LPL in these sites may indicate intracellular roles e.g. in trafficking of ligands back and forth in the cells. Future studies on LPL in these tissues should increase our understanding of other roles of the potent LPL molecule. Much new knowledge about the LPL system has been unraveled in the last two decades but still important questions remain to be solved.

Acknowledgements

Först och främst vill jag tacka min handledare Gunilla för att hon gett mig den här möjligheten samt hennes tålamod genom åren. Tack för att man alltid känt sig välkommen trots alla avbrott under forskarstudierna och för alla skratt. Tack för den utbildning jag fått, jag har lärt mig otroligt mycket och du har förberett mig för att kunna stå på egna forskarben framöver.

Madde, min bi-handledare och vän, utan dig hade de sista åren inte alls varit lika roliga. Tack för allt stöd, pepp och alla diskussioner, allt från mouse-match till ligister och galna kattvänner. Det har varit ovärderligt, och tack för allt du lärt mig om de där små svarta fyrbeningarna med runda öron.

Sedan vill jag tacka alla medarbetare på Fysiologisk Kemi för många fina år med hårt arbete, diskussioner, både forskningsmässiga liksom allt annat mellan himmel och jord. Solveig, tack för allt stöd på labbet, för kaffesällskapet bland te-drickarna och för att jag fått lära mig den (ö)kända LPL-aktiviteten. Elena, tack för all hjälp med western blots och för att du hållit ordning på oss på labbet. Lasse, tack för alla trevliga diskussioner och att du alltid verkar ha svar på mina frågor, oavsett ämne, och för att du fixade så jag fick mina 5 minuter i tv-rutan. Thomas, en sann förebild. Stefan, det är aldrig tråkigt när du är med, alltid något spännande projekt på gång. Mikael, rums- och byggarkompis, varje gång jag dödar ett äpple tänker jag på dig. Evelina, det var många fina år vi fick med labb, babbel och träning på IKSU. Oleg, a true scientist, cyclist and a thousand thanks for all your help with illustrations. Fredrick A, du vet att du är nummer 1, F1. Niklas och Fredrik L, det var synd att vi förlorade sådana eminenta forskare till andra lyckligt lottade. Sandhya, Massi and Valeria thank you for all your help and lovely company. Elda and Slava, thank you for all the laughs at the fika table.

Tack till ”barnen”, Lotta, Carina, Sussi, Anna, Yvonne, Catarina och Kotryna för alla trevliga fikastunder i våran del av korridoren. Urban, tack för att jag fått invadera både ditt kontor och labb. Ser fram emot många spännande samarbeten. Tack till Jonas och Lee-Ann för trevligt kaffe- och bubbelsällskap.

I truly enjoyed getting to know new collagues and lab mates from the group of Franscesca Aguilo and of course the group leader herself. I tend to go into your lab still even though I have moved my office.

I am so greatful for getting to know so many amazing people through collaborations: Krissi, Liz, Caroline, Marcia, Nagila and Luciane.

Ett stort tack till hjältarna på våning två, Åsa och Clas, alltid redo och utan er hade inte ett enda försök gått att genomföra. Terry, Carina och Ida, utan er hade jag varit helt vilse. Extra tack till Terry för ditt peppande, speciellt nu på slutet. Clara, tusen tack för du gör så att allas arbete flyter på så smidigt och för alla buffertar.

Tack till alla nuvarande och tidigare doktorandkollegor och alla andra kollegor på 6M för spännande seminarium och sällskap i lunchrummet. Ett extra tack till Emma för träning, kaffe, AT-plugg och ältande. Jag slog ditt rekord!

Tack också till alla hjälpsamma medarbetare på UCCM och särskilt till Helena Edlunds grupp för introduktion till bl a immunofluorescens, konfokalmikroskopi och stöttning under åren. Tack Magnus och Monica för den extra stöttningen och hjälpen med att få ihop allt nu på slutet.

Mina vänner, ni vet vilka ni är, även om vi inte ses ofta så är det guld när vi ses. Benjamin, du ska veta att utan din pedagogiska photoshop-support hade jag nog blivit mentalt ärrad för livet och så tusen tack för all hjälp med illustrationer, hade aldrig kunnat göra det själv.

Min familj, det finns inte utrymme för att beskriva min beundran, respekt och tacksamhet. Ralf, min storebror, redan innan jag kunde gå så fick du mig att skratta, du kan verkligen alltid få mig på bra humör och är otrolig på att peppa. Min förlängda familj, Åsa och familjen Persson/Anundsson. Tack Barbro för att du är den bästa på att pyssla om oss. Ingemar, nu är jag klar med [rötterN]. Mårten, min livskamrat, min klippa och bästa vän. Tack för att du tar hand om mig. Beatrice, älskade dotter, du är det bästa med livet och jag är så stolt över att få vara din mamma. Pappa och mamma, de bästa utav förebilder, tack för ert outtömliga stöd i allt. Ett extra tack till min älskade mor som introducerade mig till forskningen. Tack för alla samtal och diskussioner, du har alltid lyft mig när jag bara vill ge upp, du är fantastisk.

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