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Background and purpose: Many drugs and environmental contaminants induce hypercholesterolemia and promote the risk of atherosclerotic cardiovascular disease. We tested the hypothesis that pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, regulates the level of circulating atherogenic lipids in humans and utilized mouse experiments to identify the mechanisms involved. Experimental approach: We performed serum NMR metabolomics in healthy volunteers administered rifampicin, a prototypical human PXR ligand or placebo in a crossover setting. We used high-fat diet fed wild-type and PXR knockout mice to investigate the mechanisms mediating the PXR-induced alterations in cholesterol homeostasis. Key results: Activation of PXR induced cholesterogenesis both in pre-clinical and clinical settings. In human volunteers, rifampicin increased intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and total cholesterol and lathosterol–cholesterol ratio, a marker of cholesterol synthesis, suggesting increased cholesterol synthesis. Experiments in mice indicated that PXR activation causes widespread induction of the cholesterol synthesis genes including the rate-limiting Hmgcr and upregulates the intermediates in the Kandutsch–Russell cholesterol synthesis pathway in the liver. Additionally, PXR activation induced plasma proprotein convertase subtilisin/kexin type 9 (PCSK9), a negative regulator of hepatic LDL uptake, in both mice and humans. We propose that these effects were mediated through increased proteolytic activation of sterol regulatory element-binding protein 2 (SREBP2) in response to PXR activation. Conclusions and implications: PXR activation induces cholesterol synthesis, elevating LDL and total cholesterol in humans. The PXR–SREBP2 pathway is a novel regulator of the cholesterol and PCSK9 synthesis and a molecular mechanism for drug- and chemical-induced hypercholesterolemia.

The prevalence of metabolic diseases is paralleled by increases in exposure to foreign chemicals, e.g. drugs and environmental contaminants. Rather common for these chemicals is their ability to activate Pregnane X receptor (PXR), a master regulator of drug metabolism. PXR has been also implicated to have roles in glucose and lipid metabolism as its activation impairs glucose tolerance, increases liver fat, and modulates cholesterol metabolism. Therefore, PXR activation may in part explain the association of chemical exposure and metabolic diseases. This thesis studies the function of PXR in different metabolic states. This thesis demonstrates that fasting mitigates transcriptomic PXR activation response and modulates PXR function in bile acid metabolism. In obese and metabolically dysfunctional mice, PXR activation aggravates liver fat accumulation and hepatic insulin resistance and increases cholesterol synthesis. However, these metabolic defects do not reflect to glucose metabolism due to increased hepatic glucose uptake and suppressed fasting response, which together lead to pseudo-improvement of glucose tolerance. Induction of cholesterol synthesis was due to activation of SREBP2, the main regulator of cholesterol synthesis. Similarly, one-week dosing of rifampicin, a human PXR activator, increased hepatic cholesterol synthesis and circulating atherogenic lipids in humans. Importantly, PCSK9, a drug target to lower LDL, was increased by PXR activation. This thesis reveals that metabolic status regulates PXR function. In obese mice, PXR activation elicits different and more severe response on glucose and lipid metabolism than in lean mice. Therefore, obesity may sensitize to harmful metabolic effects of PXR activating drugs and environmental chemicals. Further, the thesis defines PXR activation as a possible risk for cardiovascular health as it increases circulating atherogenic lipids and PCSK9., Tiivistelmä Lihavuuden ja metabolisten sairauksien ilmaantuvuuden on todettu liittyvän vierasaineille, kuten lääkkeille ja ympäristökemikaaleille, altistumiseen. Monet näistä vierasaineista aktivoivat pregnaani X -reseptorin (PXR) johtaen vierasaineiden nopeampaan eliminaatioon. PXR:n aktivaatio vaikuttaa metaboliseen terveyteen haitallisesti: se heikentää sokerinsietoa, rasvoittaa maksaa, ja vaikuttaa kolesterolimetaboliaan. PXR:n aktivaatio saattaakin osaltaan selittää kemikaalialtistuksen ja metabolisten sairauksien yhteyttä. Tässä väitöstyössä tutkittiin PXR:n toimintaa erilaisissa metabolisissa tiloissa. Työssä havaittiin paaston heikentävän PXR-aktivaation vastetta ja vaikuttavan PXR:n toimintaan sappihappimetabolian säätelyssä. Lihavuuden aiheuttama metabolinen häiriötila taas herkisti hiiriä PXR-välitteiselle maksan rasvoittumiselle, heikensi maksan insuliiniherkkyyttä ja lisäsi kolesterolin synteesiä. Nämä häiriöt eivät kuitenkaan heikentäneet sokerimetaboliaa, sillä PXR:n aktivaatio lisäsi maksassa sokerin soluunottoa ja heikensi paastovastetta. PXR:n aikaansaama kolesterolisynteesin kiihtyminen johtui SREBP2:n, keskeisen kolesterolisynteesin säätelijän, aktivaatiosta. Sama synteesin kiihtyminen havaittiin terveissä vapaaehtoisissa PXR-aktivaattori rifampisiinin annon jälkeen. Rifampisiini kiihdytti PXR-välitteisesti kolesterolisynteesiä ja lisäsi valtimonkovettumatautia aiheuttavia lipidejä. Lisäksi PXR:n aktivaatio nosti plasman PCSK9-pitoisuutta, mikä lisää LDL-kolesterolin määrää. Väitöskirja osoittaa aineenvaihdunnallisen tilan, tarkoittakoon se paastoa tai aineenvaihdunnallisia sairauksia, säätelevän PXR:n toimintaa. Lihavissa hiirissä PXR on voimakas maksan sokeri- ja lipidiaineenvaihdunnan säätelijä. Lihavuus saattaakin herkistää PXR:n aktivaattorien, kuten monien lääkkeiden ja ympäristökemikaalien, metabolisille haitoille. Lisäksi väitöstyön osoittama kolesterolisynteesin ja aterogeenisten lipidien ja PCSK9:n lisääntyminen viittaa PXR:n aktivaatio

Natural Abs are produced by B lymphocytes in the absence of external Ag stimulation. They recognise self, altered self and foreign Ags, comprising an important first-line defence against invading pathogens and serving as innate recognition receptors for tissue homeostasis. Natural IgG Abs have been found in newborns and uninfected individuals. Yet, their physiological role remains unclear. Previously, no natural IgG Abs to oxidation-specific epitopes have been reported. Here, we show the cloning and characterisation of mouse IgG mAbs against malondialdehyde acetaldehyde (MAA)-modified low-density lipoprotein. Sequence analysis reveals high homology with germline genes, suggesting that they are natural. Further investigation shows that the MAA-specific natural IgG Abs cross-react with the major periodontal pathogen Porphyromonas gingivalis and recognise its principle virulence factors gingipain Kgp and long fimbriae. The study provides evidence that natural IgGs may play an important role in innate immune defence and in regulation of tissue homeostasis by recognising and removing invading pathogens and/or modified self-Ags, thus being involved in the development of periodontitis and atherosclerosis.

Low plasma level of 25‐hydroxyvitamin D (25‐OH‐D), namely vitamin D deficiency, is associated with obesity and weight loss improves 25‐OH‐D status. However, the mechanism behind obesity‐induced vitamin D deficiency remains unclear. Here, we report that obesity suppresses the expression of cytochrome P450 (CYP) 2R1, the main vitamin D 25‐hydroxylase, in both mice and humans. In humans, weight loss induced by gastric bypass surgery increased the expression of CYP2R1 in the s.c. adipose tissue suggesting recovery after the obesity‐induced suppression. At the same time, CYP27B1, the vitamin D 1α‐hydroxylase, was repressed by the weight loss. In a mouse (C57BL/6N) model of diet‐induced obesity, the plasma 25‐OH‐D was decreased. In accordance, the CYP2R1 expression was strongly repressed in the liver. Moreover, obesity repressed the expression of CYP2R1 in several extrahepatic tissues, the kidney, brown adipose tissue, and testis, but not in the white adipose tissue. Obesity had a similar effect in both male and female mice. In mice, obesity repressed expression of the vitamin D receptor in brown adipose tissue. Obesity also upregulated the expression of the vitamin D receptor and CYP24A1 in the s.c. adipose tissue of a subset of mice; however, no effect was observed in the human s.c. adipose tissue. In summary, we show that obesity affects CYP2R1 expression both in the mouse and human tissues. We suggest that in mouse the CYP2R1 repression in the liver plays an important role in obesity‐induced vitamin D deficiency. Currently, it is unclear whether the CYP2R1 downregulation in extrahepatic tissues could contribute to the obesity‐induced low plasma 25‐OH‐D, however, this phenomenon may affect at least the local 25‐OH‐D concentrations.

Low 25-hydroxyvitamin D levels correlate with the prevalence of diabetes; however, the mechanisms remain uncertain. Here, we show that nutritional deprivation–responsive mechanisms regulate vitamin D metabolism. Both fasting and diabetes suppressed hepatic cytochrome P450 (CYP) 2R1, the main vitamin D 25-hydroxylase responsible for the first bioactivation step. Overexpression of coactivator peroxisome proliferator–activated receptor γ coactivator 1-α (PGC-1α), induced physiologically by fasting and pathologically in diabetes, resulted in dramatic downregulation of CYP2R1 in mouse hepatocytes in an estrogen-related receptor α (ERRα)–dependent manner. However, PGC-1α knockout did not prevent fasting-induced suppression of CYP2R1 in the liver, indicating that additional factors contribute to the CYP2R1 repression. Furthermore, glucocorticoid receptor (GR) activation repressed the liver CYP2R1, suggesting GR involvement in the regulation of CYP2R1. GR antagonist mifepristone partially prevented CYP2R1 repression during fasting, suggesting that glucocorticoids and GR contribute to the CYP2R1 repression during fasting. Moreover, fasting upregulated the vitamin D catabolizing CYP24A1 in the kidney through the PGC-1α-ERRα pathway. Our study uncovers a molecular mechanism for vitamin D deficiency in diabetes and reveals a novel negative feedback mechanism that controls crosstalk between energy homeostasis and the vitamin D pathway.

Pregnane X receptor (PXR) regulates glucose and lipid metabolism, but little is known of the nutritional regulation of PXR function. We investigated the genome wide effects of the nutritional status on the PXR mediated gene regulation in the liver. Mice were treated with a PXR ligand pregnenolone 16α-carbonitrile (PCN) for 4 days and subsequently either fasted for 5 hours or after 4-hour fast treated with intragastric glucose 1 hour before sample collection. Gene expression microarray study indicated that PCN both induced and repressed much higher number of genes in the glucose fed mice and the induction of multiple well-established PXR target genes was potentiated by glucose. A subset of genes, including bile acid synthesis gene Cyp8b1, responded in an opposite direction during fasting and after glucose feeding. PXR knockout abolished these effects. In agreement with the Cyp8b1 regulation, PCN also modified the bile acid composition in the glucose fed mice. Contribution of glucose, insulin and glucagon on the observed nutritional effects was investigated in primary hepatocytes. However, only mild impact on PXR function was observed. These results show that nutritional status modifies the PXR regulated transcriptome both qualitatively and quantitatively and reveal a complex crosstalk between PXR and energy homeostasis.

Treatment of periodontitis has beneficial effects on systemic inflammation markers that relate to progression of atherosclerosis. We aimed to investigate whether immunization with A hemagglutinin domain (Rgp44) of Porphyromonas gingivalis (Pg), a major etiologic agent of periodontitis, would lead to an antibody response cross-reacting with oxidized low-density lipoprotein (OxLDL) and how it would affect the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLR-/-) mice. The data revealed a prominent IgM but not IgG response to malondialdehyde-acetaldehyde modified LDL (MAA-LDL) after Rgp44 and Pg immunizations, implying that Rgp44/Pg and MAA adducts may share cross-reactive epitopes that prompt IgM antibody production and consequently confer atheroprotection. A significant negative association was observed between atherosclerotic lesion and plasma IgA to Rgp44 in Rgp44 immunized mice, supporting further the anti-atherogenic effect of Rgp44 immunization. Plasma IgA levels to Rgp44 and toPg in both Rgp44- and Pg-immunized mice were significantly higher than those in saline control, suggesting that IgA to Rgp44 could be a surrogate marker of immunization in Pg-immunized mice. Distinct antibody responses in plasma IgA levels to MAA-LDL, to Pg lipopolysaccharides (Pg-LPS), and to phosphocholine (PCho) were observed after Rgp44 and Pg immunizations, indicating that different immunogenic components between Rpg44 and Pg may behave differently in regard of their roles in the development of atherosclerosis. Immunization with Rgp44 also displayed atheroprotective features in modulation of plaque size through association with plasma levels of IL-1α whereas whole Pg bacteria achieved through regulation of anti-inflammatory cytokine levels of IL-5 and IL-10. The present study may contribute to refining therapeutic approaches aiming to modulate immune responses and inflammatory/anti-inflammatory processes in atherosclerosis.

Pregnane X receptor (PXR) is a nuclear receptor that senses chemical environment and is activated by numerous clinically used drugs and environmental contaminants. Previous studies have indicated that several drugs known to activate PXR appear to induce glucose intolerance. We now aimed to reveal the role of PXR in drug-induced glucose intolerance and characterize the mechanisms involved. We used PXR knockout mice model to investigate the significance of this nuclear receptor in the regulation of glucose tolerance. PXR ligand pregnenolone-16ɑ-carbonitrile (PCN) impaired glucose tolerance in the wildtype mice but not in the PXR knockout mice. Furthermore, DNA microarray and bioinformatics analysis of differentially expressed genes and glucose metabolism relevant pathways in PCN treated primary hepatocytes indicated that PXR regulates genes involved in glucose uptake. PCN decreased the expression of glucose transporter 2 (GLUT2) in mouse liver and in the wildtype mouse hepatocytes but not in the PXR knockout cells. Data mining of published chromatin immunoprecipitation-sequencing results indicate that Glut2 gene is a direct PXR target. Furthermore, PCN induced internalization of GLUT2 protein from the plasma membrane to the cytosol in the liver in vivo and repressed glucose uptake in the primary hepatocytes. Our results indicate that the activation of PXR impairs glucose tolerance and thus PXR represents a novel diabetogenic pathway. PXR activation dysregulates GLUT2 function by two different mechanisms. These findings may partly explain the diabetogenic effects of medications and environmental contaminants.

Carbamylation of proteins in vivo occurs by cyanate, non-enzymatically when urea is dissociated, and by myeloperoxidase-catalyzed oxidation from thiocyanate. Carbamylation of low-density lipoprotein is suggested to enhance atherogenesis in patients with with chronic kidney disease and uremia. This thesis study assessed the questions of whether healthy humans or uremic patients under enhanced carbamylation have antibodies recognizing carbamyl-epitopes in plasma, and what is their role in vivo. Also, humoral immune response to carbamyl-LDL immunization and its impact on atherogenesis in LDLR-/- mice was investigated. In this thesis study, plasma antibodies to carbamylated proteins were detected in humans, and IgG antibodies to carbamylated proteins were associated with uremia and smoking, conditions with enhanced carbamylation. The human IgG and IgM antibodies binding to carbamyl-epitopes were associated with oxidation-specific epitopes in plasma. Monoclonal Fab antibodies with characteristics of a natural antibody and ability to bind both carbamyl- and malondialdehyde-derived epitopes were cloned from healthy humans. An investigated Fab antibody was able to bind epitopes found in atherosclerotic lesions and inhibit the uptake of modified LDL by macrophages. Human plasma antibodies and the monoclonal Fab bound to epitopes found on apoptotic cells. Human B-cells secreted antibodies with similar cross-reactive binding properties between carbamyl- and malondialdehyde adducts and apoptotic cells in vitro. Immunization with mouse carbamyl-LDL without adjuvant resulted in specific IgG immune response in LDLR-/- mice, but also a cross-reaction with malondialdehyde-adducts was observed. Carbamyl-LDL immunized mice had enhanced plasma antibody binding to apoptotic cells. Carbamyl-LDL immunization did not affect atherogenesis in mice. This thesis demonstrates that IgG antibodies to carbamyl-epitope might serve as a novel indicator of carbamylation in vivo in uremic pat, Tiivistelmä Proteiinien karbamylaatiota tapahtuu syanaatin vaikutuksesta. Sitä muodostuu urean hajotessa tai myeloperoksidaasin katalysoimana tiosyanaatin hapettuessa. Low-density lipoproteiinin eli LDL:n karbamylaation on esitetty edistävän valtimonkovettumataudin eli ateroskleroosin kehittymistä munuaisten vajaatoimintaa sairastavilla ureemisilla potilailla. Väitöskirjatyössä tutkittiin, onko terveillä ihmisillä ja ureemisilla potilailla karbamyyli-epitooppeja tunnistavia vasta-aineita, ja mikä niiden merkitys on elimistössä. Humoraalista immuunivastetta karbamyyli-LDL-immunisaation jälkeen sekä sen vaikutusta ateroskleroosin kehittymiseen tutkittiin LDL-reseptoripuutteellisilla hiirillä. Tutkimuksessa osoitettiin, että ihmisillä on plasmassa karbamyloituja proteiineja tunnistavia vasta-aineita. IgG-luokan vasta-aineet ovat yhteydessä uremiaan ja tupakointiin, joissa karbamylaatio on lisääntynyt. Karbamyyli- ja hapettuneita epitooppeja tunnistavien plasman IgG- ja IgM-vasta-aineiden välillä havaittiin olevan yhteys. Työssä kloonattiin terveistä ihmisistä monoklonaalisia Fab-vasta-aineita, joilla on luonnollisten vasta-aineiden kaltaisia ominaisuuksia ja kyky sitoutua sekä karbamyyli- että malonidialdehydi-epitooppeihin. Yksi tutkittu Fab-vasta-aine sitoutui valtimonkovettumataudin ateroomissa oleviin epitooppeihin ja esti muuntuneen LDL:n sisäänoton makrofagi-soluihin. Ihmisen plasman vasta-aineet ja monoklonaalinen Fab-vasta-aine sitoutuivat apoptoottisten solujen pinnalla oleviin rakenteisiin. Soluviljelyolosuhteissa ihmisen B-solut tuottivat vasta-aineita, joilla oli samanlaisia ristireaktio-ominaisuuksia karbamyyli- ja malonidialdehydi-epitooppeja sekä apoptoottisia soluja kohtaan. Karbamyyli-LDL-immunisaatio sai aikaan IgG-immuunivasteen hiirillä karbamyyli-LDL:a kohtaan, mutta myös ristireaktio malonidialdehydi-rakenteita sekä apoptoottisia soluja kohtaan havaittiin. Karbamyyli-LDL-immunisaatio ei vaikuttanut ateroskleroosin kehittymiseen hiirillä. Tutkimus