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It is known that high-fat diet (HFD) and/or diabetes may influence substrate preferences and energy demands in the heart preceding diabetic cardiomyopathy. They may also induce structural glomerular changes causing diabetic nephropathy. PET/CT has been utilized to examine uptake of energy substrates, and to study metabolic changes or shifts before onset of metabolic disorders. However, conventional PET/CT scanning of organs with relatively low uptake, such as the kidney, in small animals in vivo may render technical difficulties. To address this issue, we developed a PET/CT ex vivo protocol with radiolabeled glucose and fatty acid analouges, [18F]FDG and [18F]FTHA,to study substrate uptake in mouse kidneys. We also aimed to detect a possible energy substrate shift before onset of diabetic nephropathy. The ex vivo protocol reduced interfering background as well as interindividual variances. We found increased uptake of [18F]FDG and [18F]FTHA in kidneys after HFD, compared to kidneys from young mice on standard chow. Levels of kidney triglycerides also increased on HFD. Lipoprotein lipase (LPL) activity, the enzyme responsible for release of fatty acids from circulating lipoproteins, is normally increased in postprandial mice kidneys. After long-term HFD, we found that LPL activity was suppressed, and could therefore not explain the increased levels of stored triglycerides. Suppressed LPL activity was associated with increased expression of angiopoietin-like protein4, an inhibitor of LPL. HFD did not alter the transcriptional control of some common glucose and fatty acid transporters that may mediate uptake of [18F]FDG and [18F]FTHA. Performing PET/CT ex vivo reduced interfering background and interindividual variances. Obesity and insulin resistance induced by HFD increased the uptake of [18F]FDG and [18F]FTHA and triglyceride accumulation in mouse kidneys. Increased levels of [18F]FDG and [18F]FTHA in obese insulin resistant mice could be used clinically as an indicator of poor metabolic control, and a complementary test for incipient diabetic nephropathy.

Age is associated with progressively impaired, metabolic, cardiac and vascular function, as well as reduced work/exercise capacity, mobility, and hence quality of life. Exercise exhibit positive effects on age-related dysfunctions and diseases. However, for a variety of reasons many aged individuals are unable to engage in regular physical activity, making the development of pharmacological treatments that mimics the beneficial effects of exercise highly desirable. Here we show that the pan-AMPK activator O304, which is well tolerated in humans, prevented and reverted age-associated hyperinsulinemia and insulin resistance, and improved cardiac function and exercise capacity in aged mice. These results provide preclinical evidence that O304 mimics the beneficial effects of exercise. Thus, as an exercise mimetic in clinical development, AMPK activator O304 holds great potential to mitigate metabolic dysfunction, and to improve cardiac function and exercise capacity, and hence quality of life in aged individuals.

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.

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.

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 βcellerna, 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 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.

Activity of lipoprotein lipase (LPL) is high in mouse kidney, but the reason is poorly understood. The aim was to characterize localization, regulation, and function of LPL in kidney of C57BL/6J mice. We found LPL mainly in proximal tubules, localized inside the tubular epithelial cells, under all conditions studied. In fed mice, some LPL, colocalized with the endothelial markers CD31 and GPIHBP1 and could be removed by perfusion with heparin, indicating a vascular location. The role of angiopoietin-like protein 4 (ANGPTL4) for nutritional modulation of LPL activity was studied in wild-type and Angptl4^-/- mice. In Angptl4^-/- mice, kidney LPL activity remained high in fasted animals, indicating that ANGPTL4 is involved in suppression of LPL activity on fasting, like in adipose tissue. The amount of ANGPTL4 protein in kidney was low, and the protein appeared smaller in size, compared with ANGPTL4 in heart and adipose tissue. To study the influence of obesity, mice were challenged with high-fat diet for 22 wk, and LPL was studied after an overnight fast compared with fasted mice given food for 3 h. High-fat diet caused blunting of the normal adaptation of LPL activity to feeding/fasting in adipose tissue, but in kidneys this adaptation was lost only in male mice. LPL activity increases to high levels in mouse kidney after feeding, but as no difference in uptake of chylomicron triglycerides in kidneys is found between fasted and fed states, our data confirm that LPL appears to have a minor role for lipid uptake in this organ.

Objective: Tissue macrophages induce and perpetuate proinflammatory responses, thereby promoting metabolic and cardiovascular disease. Lipoprotein lipase (LpL), the rate-limiting enzyme in blood triglyceride catabolism, is expressed by macrophages in atherosclerotic plaques. We questioned whether LpL, which is also expressed in the bone marrow (BM), affects circulating white blood cells and BM proliferation and modulates macrophage retention within the artery.

Approach and Results: We characterized blood and tissue leukocytes and inflammatory molecules in transgenic LpL knockout mice rescued from lethal hypertriglyceridemia within 18 hours of life by muscle-specific LpL expression (MCKL0 mice). LpL-deficient mice had ≈40% reduction in blood white blood cell, neutrophils, and total and inflammatory monocytes (Ly6C/Ghi). LpL deficiency also significantly decreased expression of BM macrophage-associated markers (F4/80 and TNF-α [tumor necrosis factor α]), master transcription factors (PU.1 and C/EBPα), and colony-stimulating factors (CSFs) and their receptors, which are required for monocyte and monocyte precursor proliferation and differentiation. As a result, differentiation of macrophages from BM-derived monocyte progenitors and monocytes was decreased in MCKL0 mice. Furthermore, although LpL deficiency was associated with reduced BM uptake and accumulation of triglyceride-rich particles and macrophage CSF–macrophage CSF receptor binding, triglyceride lipolysis products (eg, linoleic acid) stimulated expression of macrophage CSF and macrophage CSF receptor in BM-derived macrophage precursor cells. Arterial macrophage numbers decreased after heparin-mediated LpL cell dissociation and by genetic knockout of arterial LpL. Reconstitution of LpL-expressing BM replenished aortic macrophage density.

Conclusions: LpL regulates peripheral leukocyte levels and affects BM monocyte progenitor differentiation and aortic macrophage accumulation.

BACKGROUND: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins and enables uptake of lipolysis products for energy production or storage in tissues. Our aim was to study the localization of LPL and its endothelial anchoring protein glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in mouse pancreas, and effects of diet and leptin deficiency on their expression patterns. For this, immunofluorescence microscopy was used on pancreatic tissue from C57BL/6 mouse embryos (E18), adult mice on normal or high-fat diet, and adult ob/ob-mice treated or not with leptin. The distribution of LPL and GPIHBP1 was compared to insulin, glucagon and CD31. Heparin injections were used to discriminate between intracellular and extracellular LPL.

RESULTS: In the exocrine pancreas LPL was found in capillaries, and was mostly co-localized with GPIHBP1. LPL was releasable by heparin, indicating localization on cell surfaces. Within the islets, most of the LPL was associated with beta cells and could not be released by heparin, indicating that the enzyme remained mostly within cells. Staining for LPL was found also in the glucagon-producing alpha cells, both in embryos (E18) and in adult mice. Only small amounts of LPL were found together with GPIHBP1 within the capillaries of islets. Neither a high fat diet nor fasting/re-feeding markedly altered the distribution pattern of LPL or GPIHBP1 in mouse pancreas. Islets from ob/ob mice appeared completely deficient of LPL in the beta cells, while LPL-staining was normal in alpha cells and in the exocrine pancreas. Leptin treatment of ob/ob mice for 12 days reversed this pattern, so that most of the islets expressed LPL in beta cells.

CONCLUSIONS: We conclude that both LPL and GPIHBP1 are present in mouse pancreas, and that LPL expression in beta cells is dependent on leptin.

The lipolytic processing of triglyceride-rich lipoproteins by lipoprotein lipase (LPL) is the central event in plasma lipid metabolism, providing lipids for storage in adipose tissue and fuel for vital organs such as the heart. LPL is synthesized and secreted by myocytes and adipocytes, but then finds its way into the lumen of capillaries, where it hydrolyzes lipoprotein triglycerides. The mechanism by which LPL reaches the lumen of capillaries has remained an unresolved problem of plasma lipid metabolism. Here, we show that GPIHBP1 is responsible for the transport of LPL into capillaries. In Gpihbp1-deficient mice, LPL is mislocalized to the interstitial spaces surrounding myocytes and adipocytes. Also, we show that GPIHBP1 is located at the basolateral surface of capillary endothelial cells and actively transports LPL across endothelial cells. Our experiments define the function of GPIHBP1 in triglyceride metabolism and provide a mechanism for the transport of LPL into capillaries.