Le récepteur hépatique aux lipoprotéines LSR est impliqué dans la clairance des lipoprotéines riches en triglycérides telles que les résidus de chylomicrons pendant la phase postprandiale. La réduction de l’expression du LSR chez la souris (LSR+/-) est associée à une dyslipidémie et une lipémie postprandiale élevée. Afin de mieux comprendre la régulation de la distribution des lipides alimentaires, nous avons cherché quels étaient les facteurs pouvant affecter le niveau protéique de LSR. La leptine, hormone sécrétée par le tissu adipeux et connue pour son action d’hormone de satiété au niveau du système nerveux central, a été démontrée dans cette thèse comme modulant l’expression de LSR par la régulation de la transcription du gène lsr. La leptine est impliquée dans la régulation de la lipogénèse à travers SREBP-1. Grâce à l’utilisation d’un extrait de Garcinia cambogia contenant un inhibiteur de l’ATP citrate lyase, nous avons démontré une interaction importante entre les enzymes lipogéniques, l’expression de LSR et d’autres récepteurs lipoprotéiques, afin de maintenir un équilibre entre la synthèse de lipides endogènes et l’apport alimentaire de lipides exogènes. Soumises à un régime hyperlipidique, les souris sauvages montrent une diminution de l’expression des enzymes lipogéniques hépatiques, aggravée chez les souris LSR+/-. Ces résultats indiquent qu’il existe un mécanisme de maintien de l’équilibre entre la lipogénèse (synthèse endogène de lipides), la lipolyse (utilisation lipidique comme substrat énergétique) et le stockage de lipides à travers une forte interaction entre les enzymes lipogéniques et LSR. / The hepatic lipoprotein receptor LSR is involved in the clearance of triglyceride-rich lipoproteins including chylomicrons remnants during the post-prandial phase. Reduced LSR protein expression in mice (LSR+/-) is associated with dyslipidemia and increased postprandial lipemia; these mice exhibit increased weight gain with aging or when placed under a high-fat diet. In order to better understand the regulation of the distribution of dietary lipids, we looked for factors that could regulate LSR protein levels. Leptin is a hormone secreted by the adipose tissue that is a centrally-acting satiety factor, and was demonstrated to modulate LSR mRNA and protein expression through the modulation of transcription of the gene lsr. Leptin has been reported be involved in the control of lipogenesis through SREBP-1c. Using Garcinia cambogia extract containing an inhibitor of ATP citrate lyase, we demonstrated that there is an important link between lipogenic enzymes and LSR protein levels and with other lipoprotein receptors that provides the means to maintain a balance between endogenous lipid synthesis and dietary intake of exogenous lipids. When exogenous lipid intake is increased in the form of a high-fat diet, mice exhibited a decrease in hepatic lipogenic enzymes expression, but a deficiency of LSR led to increased lipid content in the peripheral tissues. These results suggest the presence of mechanisms for the maintenance for the balance between lipogenesis (de novo endogenous lipid synthesis), lipolysis (lipids used as energy substrate), and lipid storage through an important link between lipogenic enzymes and LSR.

Camundongos Swiss, quando submetidos à dieta hiperlipídica (HL), apresentam considerável ganho ponderal e de depósitos adiposos, tornando-se obesos e resistentes à insulina. O objetivo deste trabalho foi avaliar o efeito da dieta HL por 8 semanas no perfil inflamatório, síntese de triacilglicerol (TAG) com ênfase na vias de geração de glicerol-3-fosfato (G3P) e lipólise nos tecidos adiposos brancos (TAB) retroperitoneal (RETRO) e epididimal (EPI) de camundongos. Camundongos Swiss foram alimentados com as dietas: controle (CT) - dieta purificada (AIN-93G); ou HL - dieta AIN-93G modificada contendo 35% de lipídeos (4% de óleo de soja e 31% de gordura suína). Os camundongos alimentados com a dieta HL apresentaram uma maior massa corporal, acompanhada pelo aumento nos tecidos RETRO e EPI, além de desenvolverem resistência à insulina constatada no teste de tolerância à glicose (TTG), hiperglicemia e hiperinsulinemia. O conteúdo protéico da pAKT, avaliado por western blot (WB), e a adiponectina, dosada em homogenados dos tecidos adiposos, estão reduzidos apenas no EPI. Houve aumento na expressão gênica de MCP-1 e PAI-1, e foi observada menor área dos adipócitos no EPI, sem alteração no RETRO dos animais HL. A síntese de novo de ácidos graxos (AG), avaliada pela incorporação de 3H de 3H2O em AG foi maior em ambos os TAB, porém a captação de AG das lipoproteínas circulantes avaliada pela atividade e expressão da lipase lipoproteica (LPL) aumentou no EPI e reduziu no RETRO. A dieta HL induziu aumento na fosforilação do glicerol, avaliada pela atividade e conteúdo da GK que aumentaram nos dois TAB, e maior incorporação de 1-14C-glicerol em TAG no EPI. A captação de glicose in vitro e conteúdo do GLUT- 4, que indicam atividade da via glicolítica foram reduzidos no EPI e RETRO, assim como a gliceroneogênese avaliada pela incorporação de 1-14C-piruvato em TAG, sem alterações na atividade e conteúdo da fosfoenolpiruvato carboxiquinase (PEPCK). A atividade lipolítica basal foi avaliada in vitro pela liberação de glicerol por adipócitos isolados, e não foi alterada pela ingestão de dieta HL, porém quando estimulada por noradrenalina a liberação de glicerol foi menor nos animais HL, assim como as fosforilações da ATGL e HSL e conteúdo do receptor adrenérgico ?3. A dieta HL levou a uma redução no conteúdo de PPAR? e aumento de ATF3 em ambos os tecidos. No EPI houve aumento de pCREB, pSTAT3 e RGS2 em relação aos controles enquanto no RETRO a única diferença encontrada foi a menor pSTAT3. Nossos resultados demonstram que o aumento nos TAB é resultado de maior síntese e captação de AG, e que o G3P necessário para a esterificação a TAG é proveniente principalmente da fosforilação direta do glicerol pela GK; além disso, a reduzida lipólise também parece contribuir para esse quadro. Nos animais HL, o EPI parece ser mais propenso aos efeitos da dieta do que o RETRO / Swiss mice when subjected to high fat diet (HFD), shown considerable weight gain and adipose depots, becoming obese and insulin resistant. The aim of this study was to evaluate the effect of HFD diet for 8 weeks in the inflammatory profile, triacylglycerol (TAG) synthesis with emphasis in glycerol-3-phosphate (G3P) generation pathways and lipolysis in retroperitoneal (RETRO) and epididymal (EPI) white adipose tissue (WAT) of mice. Swiss mice were fed with diets: control (CT) - purified diet (AIN-93G); or HFD - purified diet (AIN-93G) plus 35% of fat (4% soybean oil and 31% of lard). Mice fed a HFD diet had a higher body mass, accompanied by an increase in RETRO and EPI tissues, in addition to developing insulin resistance, evidenced by glucose tolerance test (GTT), hyperglycemia and hyperinsulinemia. The protein content of pAKT, accessed by western blot, and adiponectin, measured in WAT homogenates, are reduced only in EPI. There was an increase in gene expression of MCP-1 and PAI-1, and was observed smaller area of adipocytes in EPI, with no change in RETRO of HFD fed animals. De novo synthesis of fatty acids (FA), evaluated by incorporation of 3H from 3H2O in FA was higher in both TAB, but the uptake of FA, from blood lipoproteins, evaluated by the activity and expression of lipoprotein lipase (LPL) was increased in EPI and reduced in RETRO. HFD induced increase in phosphorylation of glycerol, evaluated by the activity and content of glycerolkinase (GyK) which increased in both TAB and greater incorporation of 1-14C-glycerol in the TAG only in EPI. The in vitro glucose uptake and GLUT-4 content, which indicates the activity of the glycolytic pathway were reduced in EPI and RETRO, as well as glyceroneogenesis assessed by the incorporation of 1-14C- pyruvate into TAG without changes in the activity and contents of phosphoenolpyruvate carboxykinase (PEPCK). The basal lipolytic activity was evaluated in vitro by glycerol releasing from isolated adipocytes, and was not altered by HFD intake, but when stimulated by noradrenaline glycerol release was lower in HFD animals as well as the phosphorylation of ATGL and HSL and ?3 adrenergic receptor content. HFD led to a reduction in the content of PPAR gamma and an increase in ATF3 in both tissues. In EPI there was an increase in pCREB, pSTAT3 and RGS2 while in RETRO the only difference was reduced pSTAT3. Our results shown that TAB increase is result of increased FA synthesis and uptake, and G3P required for esterification TAG comes mainly from direct phosphorylation of glycerol by GyK; Furthermore, reduced lipolysis also seems to contribute to this scenario. HFD effects seem to be more prominent in EPI than in RETRO

Thesis (MSc. (Physiology)) -- University of Limpopo, 2016 / Relevance: An increase in the number of HIV positive patients receiving HAART raises important concerns about the metabolic impact of these regimens. The treatment effectively reduces viral load and increase CD4+ count; unfortunately it seems to disrupt carbohydrate and lipid metabolic pathways thereby increasing the risk for CDL by placing an already chronically ill HIV population at risk of more chronic diseases. As a developing country, accessibility to safer regimens of HAART is limited thus patients exposed to toxicities from long term exposure to sub-optimal regimens are even at greater risk. The aim of this study was to assess the long term effects of HAART on biochemical parameters and body composition as an indication of carbohydrate and lipid metabolism. Methods: A prospective cohort of 87 patients receiving HAART for 12 months or more was conducted at baseline and follow-up. Venous blood was collected after an overnight fast. An automated enzymatic colorimetric test was used to analyse plasma glucose and serum TC, HDL-C and TG. The LDL-C levels were calculated from TC and HDL-C. Leptin levels were analysed using human leptin radioimmunoassay kit. Insulin was analysed using an automated access ultrasensitive insulin assay. Anthropometric measurements were taken for the determination of body fat distribution and BMI. All statistical analyses were performed using SPSS version 23. Results: Total cholesterol, LDL-C, and waist circumference significantly decreased from baseline to follow-up (p<0.05). Triglycerides and LDL-C levels were significantly affected by durations between 24–47 and 49–72 months respectively. There were no significant changes in the mean levels of leptin observed within the two lines of regime. Mean leptin levels were 11.36±8.52 ng/ml and 9.67±6.42 ng/ml at baseline and follow-up respectively. Furthermore, the duration of HAART significantly affected BMI and WC at 49–72 months. Patients that met the criteria for diagnosis of DM were only found in PI containing regimens at 6.3% and 5.9% baseline and follow-up respectively. In the first line regimen, the prevalence of DM was only found at follow-up. Conclusion: The present study demonstrated that longer duration between months 49–72 has significant negative effects on the glucose and lipid metabolism of HIV positive patients. The study also highlighted that patients on combinations containing PIs and NRTIs such as stavudine and zidovudine are at higher risk of developing metabolic diseases. / University of Limpopo

Wie für viele andere Zivilisationskrankheiten werden auch für Atherosklerose und dadurch verursachte Erkrankungen wie Herzinfarkt oder Schlaganfall die Weichen mitunter schon vor der Geburt gestellt. Pränatale oder fetale Programmierung heißt der Mechanismus, durch den negative Umweltbedingungen während der Schwangerschaft, allen voran der Einfluss von Stresshormonen, auf die Entwicklung des Fetus wirken und die Prädisposition für spätere Erkrankungen schaffen können (SCHWAB 2009, SECKL 2001). Ziel der vorliegenden Arbeit war es, die Auswirkungen von Stress während der Schwangerschaft auf den Fettstoffwechsel der Nachkommen unter besonderer Berücksichtigung bekannter Herz-Kreislauf-Risikofaktoren zu untersuchen. Zu diesem Zweck wurde 28 Weißbüschelaffen (F0) während der Trächtigkeit eine Woche lang täglich Dexamethason (DEX) – ein synthetisches Glucocorticoid (GC), welches die Plazentaschranke passieren kann (TEGETHOFF et al. 2009) – oral verabreicht (BEINDORFF et al. 2006, EINSPANIER et al. 2006c). Die drei weiblichen Folgegenerationen DEX F1 (n = 5), DEX F2 (n = 6) und DEX F3 (n = 3) dieser Tiere wurden untersucht, wobei sich die Medikamentengabe auf die F0-Generation beschränkte und alle weiteren Trächtigkeiten ungestört verliefen. Im Alter von 3,3 bis 5,6 Jahren (DEX F1) bzw. von Geburt an bis 1,5 Jahre (DEX F2, DEX F3) wurden die Tiere wöchentlich gewogen. In Blutproben wurden einerseits Fettsäuren (FS), andererseits Cholesterol (CHOL), Triglyceride (TG) und Lipoproteine gemessen, wobei zwei Methoden – enzymatische Analyse nach Ultrazentrifugation und direkter Assay – zum Einsatz kamen. Alle Resultate wurden denen gesunder Kontrolltiere ähnlichen Alters (n = 12) gegenübergestellt. Die Körpermasse unterschied sich zu keinem Zeitpunkt signifikant zwischen den Nachkommen der mit DEX behandelten Tiere und den Kontrollgruppen. Entweder gab es keinen programmierten Effekt auf das Gewicht oder er wurde durch individuelle Schwankungen, möglicherweise verstärkt durch erhöhte Stressempfindlichkeit oder Hyperaktivität der DEX-Nachkommen (FRENCH et al. 2004, SCHWAB 2009) und damit einhergehende Tendenz zur Gewichtsabnahme (KAPLAN und SHELMIDINE 2010) maskiert. Beide Methoden zur Untersuchung des Lipoproteinprofils erschienen für Weißbüschelaffen geeignet und können für zukünftige Untersuchungen empfohlen werden. Bei den Kontrollgruppen fiel auf, dass ältere Tiere u. a. signifikant mehr LDL- und VLDL-CHOL, aber signifikant weniger HDL-TG und n3-FS hatten als jüngere, was auf ein wie beim Menschen mit dem Alter steigendes Herz-Kreislauf-Risiko (CARLSSON et al. 2010) schließen lässt. Sowohl DEX F2 als auch DEX F3 wiesen signifikant höhere Konzentrationen von LDL-CHOL, signifikant niedrigere Werte von HDL-TG, mehr Gesamt-CHOL sowie einen höheren Quotienten CHOL : HDL-CHOL im Blutplasma auf als die Kontrolltiere. Diese Parameter gehören zu den in der humanmedizinischen Diagnostik genutzten Herz-Kreislauf-Risikofaktoren und die Veränderungen weisen auf eine erhöhte Auftrittswahrscheinlichkeit kardiovaskulärer Erkrankungen hin (KANNEL et al. 1994, LUSIS et al. 2004, NCEP 2002). Zusätzlich fielen bei DEX F1, DEX F2 und DEX F3 im Vergleich zu den Kontrollen signifikant erniedrigte Gehalte an n3-FS auf, die u. a. für ihre antiphlogistische und kardioprotektive Wirkung bekannt sind (ALONSO et al. 2003, CALDER 2004, KINSELLA et al. 1990). Pränatale GC-Behandlung rief demzufolge über Veränderungen im Fettstoffwechsel ein erhöhtes Risiko für Herz-Kreislauf-Erkrankungen bei ihren weiblichen Nachkommen F1 bis F3 hervor. Dies lässt auf epigenetische Effekte schließen, welche in weiterführenden Untersuchungen genauer erforscht werden sollten.:Abkürzungsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Fettstoffwechsel 4 2.2 Atherosklerose 7 2.3 Stress in der Schwangerschaft 9 2.4 Der Weißbüschelaffe (Callithrix jacchus) 15 3 Tiere, Material und Methoden 17 3.1 Tiere 17 3.1.1 Vorangegangener Versuch in Göttingen – Pränataler Stress 17 3.1.1.1 Material und Methoden 17 3.1.1.2 Ergebnisse 18 3.1.2 Versuchsgruppen 19 3.1.3 Haltung 20 3.1.4 Ernährung 21 3.1.5 Geburtenkontrolle 22 3.2 Datensammlung 23 3.2.1 Körpergewicht 23 3.2.2 Blutparameter des Fettstoffwechsels 23 3.2.2.1 Probengewinnung 24 3.2.2.2 Lipoproteinanalyse 25 3.2.2.3 Fettsäureanalyse 26 3.3 Statistische Auswertung 27 4 Ergebnisse 28 4.1 Körpergewicht 28 4.2 Blutparameter des Fettstoffwechsels 30 4.2.1 Vergleich der Methoden MU und MD 36 4.2.2 CONTROL YOUNG im Alter von 9 und 19 Monaten 36 4.2.3 Altersabhängigkeit der Parameter bei gesunden Kontrolltieren37 4.2.4 Einfluss pränataler DEX-Gabe auf die Nachkommen F1 bis F3 38 4.2.4.1 Lipoproteine 38 4.2.4.2 Fettsäuren 40 5 Diskussion 41 5.1 Versuchsaufbau 41 5.2 Ergebnisse: Körpergewicht 43 5.3 Ergebnisse: Blutparameter des Fettstoffwechsels 45 5.4 Fazit 47 6 Zusammenfassung 48 7 Summary 50 8 Literaturverzeichnis 52 9 Anhang I 9.1 Buchwald U, Teupser D, Kuehnel F, Grohmann J, Schmieder N, Beindorff N, Schlumbohm C, Fuhrmann H, Einspanier A. Prenatal stress programs lipid metabolism enhancing cardiovascular risk in the female F1, F2, and F3 generation in the primate model common marmoset (Callithrix jacchus). J Med Primatol. 2012;41:231-40. doi: 10.1111/j.1600-0684.2012.00551.x. [Zeitschriftenartikel] I 9.2 Buchwald U, Gassdorf F, Grohmann J, Teupser D, Habla C, Einspanier A. Prenatal dexamethasone application influences parameters of lipid metabolism in the female F2 and F3 generation of common marmoset monkeys (Callithrix jacchus). New Paradigms in Laboratory Animal Science. 2010;33. [Abstract zu einem Vortrag] XXIII 9.3 Buchwald U, Kühnel F, Grohmann J, Teupser D, Einspanier A. Intrauterine Stresshormone beeinflussen den Fettstoffwechsel weiblicher Nachkommen des Weißbüschelaffen (Callithrix jacchus). Leipzig Research Festival for Life Sciences. 2010;220. ISBN 978-3-9810760-6-6. [Abstract zu einem Poster] XXV Danksagung / As for many other civilization diseases, the way for atherosclerosis and hence heart attack and stroke can be paved even before birth. The mechanism by which negative environmental circumstances, first of all the influence of stress hormones, can alter the development of the fetus and cause a predisposition for diseases later in life is called prenatal or fetal programming (SCHWAB 2009, SECKL 2001). The aim of the present study was to investigate the consequences of stress during pregnancy on lipid metabolism of the offspring with special regard to known cardiovascular risk factors. Therefore, 28 common marmosets (F0) were given dexamethasone (DEX) – a synthetic glucocorticoid (GC) with the ability to pass the placenta easily (TEGETHOFF et al. 2009) – orally, once daily for one week during gestation (BEINDORFF et al. 2006, EINSPANIER et al. 2006c). The three female filial generations DEX F1 (n = 5), DEX F2 (n = 6) and DEX F3 (n = 3) of those monkeys were investigated. Only the F0 generation was treated with DEX, while all of the following pregnancies remained undisturbed. At the age of 3.3 up to 5.6 years (DEX F1) and from birth until 1.5 years (DEX F2, DEX F3), respectively, the animals were weighed weekly. Blood samples were analyzed on the one hand for fatty acids (FA), on the other hand for cholesterol (CHOL), triglycerides (TG) and lipoproteins using two different methods – enzymatic analysis after ultracentrifugation and direct assay. All results were compared to those of healthy controls of similar age (n = 12). Body mass of the offspring of dams prenatally treated with DEX was not significantly different from that of the controls at any point of time. Either there was no programming effect on weight or it was masked by individual fluctuations, maybe potentiated by hyperactivity or a higher sensitivity to stress of the DEX offspring (FRENCH et al. 2004, SCHWAB 2009) and hence a tendency to loose weight (KAPLAN and SHELMIDINE 2010). Both methods for lipoprotein analysis seemed to be suitable for the common marmoset and can be recommended for future investigations. In the controls, older animals showed significantly more LDL and VLDL CHOL, but significantly less HDL TG and n3 FA than younger ones, which points out to a cardiovascular risk rising with age as in humans (CARLSSON et al. 2010). DEX F2 and DEX F3 had significantly higher concentrations of LDL CHOL, significantly lower levels of HDL TG, more total CHOL and a higher ratio of CHOL : HDL CHOL in blood plasma than the controls. Those parameters are well-known human medicine cardiovascular risk factors and the aberrations detected indicate a higher probability of developing cardiovascular diseases (KANNEL et al. 1994, LUSIS et al. 2004, NCEP 2002). Additionally, compared to the controls, all DEX generations F1 to F3 showed significantly lower levels of n3 FA, which are known for their antiinflammatory and cardioprotective effects amongst others (ALONSO et al. 2003, CALDER 2004, KINSELLA et al. 1990). Consequently, prenatal treatment with GC caused an increased risk for cardiovascular diseases in the female offspring F1 up to F3 via alteration of lipid metabolism. This points out to epigenetic effects, which require further investigation.:Abkürzungsverzeichnis 1 Einleitung 1 2 Literaturübersicht 4 2.1 Fettstoffwechsel 4 2.2 Atherosklerose 7 2.3 Stress in der Schwangerschaft 9 2.4 Der Weißbüschelaffe (Callithrix jacchus) 15 3 Tiere, Material und Methoden 17 3.1 Tiere 17 3.1.1 Vorangegangener Versuch in Göttingen – Pränataler Stress 17 3.1.1.1 Material und Methoden 17 3.1.1.2 Ergebnisse 18 3.1.2 Versuchsgruppen 19 3.1.3 Haltung 20 3.1.4 Ernährung 21 3.1.5 Geburtenkontrolle 22 3.2 Datensammlung 23 3.2.1 Körpergewicht 23 3.2.2 Blutparameter des Fettstoffwechsels 23 3.2.2.1 Probengewinnung 24 3.2.2.2 Lipoproteinanalyse 25 3.2.2.3 Fettsäureanalyse 26 3.3 Statistische Auswertung 27 4 Ergebnisse 28 4.1 Körpergewicht 28 4.2 Blutparameter des Fettstoffwechsels 30 4.2.1 Vergleich der Methoden MU und MD 36 4.2.2 CONTROL YOUNG im Alter von 9 und 19 Monaten 36 4.2.3 Altersabhängigkeit der Parameter bei gesunden Kontrolltieren37 4.2.4 Einfluss pränataler DEX-Gabe auf die Nachkommen F1 bis F3 38 4.2.4.1 Lipoproteine 38 4.2.4.2 Fettsäuren 40 5 Diskussion 41 5.1 Versuchsaufbau 41 5.2 Ergebnisse: Körpergewicht 43 5.3 Ergebnisse: Blutparameter des Fettstoffwechsels 45 5.4 Fazit 47 6 Zusammenfassung 48 7 Summary 50 8 Literaturverzeichnis 52 9 Anhang I 9.1 Buchwald U, Teupser D, Kuehnel F, Grohmann J, Schmieder N, Beindorff N, Schlumbohm C, Fuhrmann H, Einspanier A. Prenatal stress programs lipid metabolism enhancing cardiovascular risk in the female F1, F2, and F3 generation in the primate model common marmoset (Callithrix jacchus). J Med Primatol. 2012;41:231-40. doi: 10.1111/j.1600-0684.2012.00551.x. [Zeitschriftenartikel] I 9.2 Buchwald U, Gassdorf F, Grohmann J, Teupser D, Habla C, Einspanier A. Prenatal dexamethasone application influences parameters of lipid metabolism in the female F2 and F3 generation of common marmoset monkeys (Callithrix jacchus). New Paradigms in Laboratory Animal Science. 2010;33. [Abstract zu einem Vortrag] XXIII 9.3 Buchwald U, Kühnel F, Grohmann J, Teupser D, Einspanier A. Intrauterine Stresshormone beeinflussen den Fettstoffwechsel weiblicher Nachkommen des Weißbüschelaffen (Callithrix jacchus). Leipzig Research Festival for Life Sciences. 2010;220. ISBN 978-3-9810760-6-6. [Abstract zu einem Poster] XXV Danksagung

Overweight and obesity are associated with Type 2 Diabetes, non-alcoholic fatty liver disease, cardiovascular disease and cancer, but all fat is not equal as storing excess lipid in subcutaneous white adipose tissue (SWAT) is more metabolically favorable than in visceral fat. Here, we uncover a critical role for mTORC2 in setting SWAT lipid handling capacity. We find that subcutaneous white preadipocytes differentiating without the essential mTORC2 subunit Rictorexpress mature adipocyte markers but develop a striking lipid storage defect. In vivo,this results in smaller adipocytes, reduced tissue size, lipid re-distribution to visceral and brown fat, and sex-distinct effects on systemic metabolic fitness. Mechanistically, mTORC2 promotes transcriptional upregulation of select lipid metabolism genes controlled by PPARgand ChREBP. These include genes that control lipid uptake, synthesis, and degradation pathways as well as Akt2, the gene encoding its substrate and insulin effector. Finally, we reveal a potential novel mTORC2 target, ACSS2, which might control intracellular acetyl-CoA availability and regulate metabolic gene expression by altering histone modification in white adipocytes. Exploring this pathway may uncover strategies to promote safe lipid storage and improve insulin sensitivity.

Evidence suggests that pesticide exposure is a risk factor for atherosclerosis, a pathology involving oxidative stress and dysregulated cholesterol metabolism in monocytes and macrophages as vital causative factors. This research focused on understanding two different mechanisms by which organochlorine and organophosphate pesticides may contribute to atherogenesis. First, the ability of organochlorine insecticides to contribute to elevated oxidative stress was investigated. Urinary concentrations of F2-isoprostanes (a systemic oxidative stress biomarker) and serum levels of the persistent organochlorine compounds p,p’-DDE, trans-nonachlor and oxychlordane were quantified in human samples and the association of these factors with diagnosis of atherosclerosis was described in a cross-sectional study. Subsequently, the ability of three bioaccumulative organochlorine insecticides, trans-nonachlor, dieldrin and p,p’-DDE, to induce the production of superoxide radical anion via NADPH oxidase activation in cultured human THP-1 monocytes through a phospholipase A2 (PLA2)-derived arachidonic acid (AA) signaling cascade was investigated. Trans-nonachlor induced NOX-dependent generation of superoxide/ROS (as measured using three distinct assay types) and stimulated the phosphorylation and membrane translocation of the p47phox regulatory subunit (two biomarkers of Nox activation). Measurement of arachidonic acid and eicosanoid release from OC-exposed monocytes by LC-MS/MS analysis subsequently confirmed the role of PLA2 as a central signaling node in the induction of reactive oxygen production in this process. To investigate a separate mechanism by which organophosphate toxicity may contribute to atherosclerosis, the ability of the esterase/lipase carboxylesterase 1 (CES1), a major enzyme target of OP toxicants, to regulate endocannabinoid and cholesterol homeostasis in human macrophages was assessed. Experimental ablation of CES1 activity altered cholesterol uptake, but not efflux in macrophage foam cells in vitro. Numerous genes involved in the cholesterol homeostatic process, including scavenger receptors (SR-A, CD36), cholesterol transporters (ABCA1, ABCG1), nuclear receptors (LXR, PPAR) and oxysterol forming enzymes (CYP27A1), were profoundly downregulated in CES1 knockdown cells. CES1 appears to play a broad central role in both normal macrophage physiology and the homeostatic response to modified LDL, potentially by liberating esterified molecules from lipoprotein particles that serve as ligands for transcription factors such as PPAR and LXR that control the expression of genes critical to the cholesterol metabolic process.

The prevalence of obesity is dramatically increasing and thus constitutes a major risk factor for developing chronic diseases such as type 2 diabetes, dyslipidemia, cardiovascular diseases, and certain forms of cancer. High-caloric nutrition and a lack of physical activity are the main contributing factors for this global epidemic. Estrogen receptors (ERs) are recognized to be involved in many processes related to the control of energy homeostasis. In my studies, I investigated the impact of estrogens (17beta-estradiol (E2)) on energy homeostasis. Special emphasis was given to the effects of two synthetic ER subtype-selective agonists, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), to determine to what extend the two distinct ER subtypes are involved in the underlying molecular mechanisms. Because of its estrogenic activity and also its widespread use as a nutritional supplement the influence of the isoflavone genistein (Gen) was examined. For this purpose two different female rat models were used: Wistar rats with nutrition-induced obesity and leptin resistant Zucker diabetic fatty (ZDF) rats. In both experiments, the animals were ovariectomized (OVX) and treated with vehicle (untreated controls) or the estrogenic compounds. The most important finding was that treatment of OVX animals with Beta enlarges soleus muscle fiber sizes in both animal models compared to untreated OVX animals. This anabolic effect may in turn improve the muscle/fat ratio of the body that enhances muscular uptake and utilization of fuels. By contrast, in the gastrocnemius muscle of OVX ZDF rats substitution with Alpha increased expression and distribution of the insulin-dependent glucose transporter 4 (GLUT4). Consequently, systemic insulin sensitivity in both animal models was improved by treatment with estrogenic compounds compared to untreated OVX animals. The strongest effect was observed in E2-treated rats that indicate an additive effect through activation of both pathways. In all OVX rats, treatment with either ER subtype-selective agonist showed an anti-lipogenic effect in adipose tissue, liver, and skeletal muscle of nutrition-induced obese Wistar rats in comparison to OVX animals without treatment. Decreased visceral fat mass, adipocyte sizes, serum leptin levels, triglyceride accumulation in liver and muscle as well as mRNA expression of genes that are involved in lipo-/adipogenesis reflected this. Therefore, the lower visceral fat mass as well as decreased accumulation of triglycerides in non-adipose tissues such as liver and skeletal muscle most likely contributes to the improved insulin sensitivity in such treated animals. Gen exerted effects similar to those of the ER beta-selective agonist (except on adipose tissue in Wistar rats). Especially, the similar ability to induce anabolic activity in the soleus muscle might be highly relevant. Gen-treated animals might have a more effective utilization of fuels compared to untreated OVX animals because they showed a lower TG content in muscle and liver as well as improved glucose metabolism. In conclusion, because of my studies and the fact that ER beta signaling is not involved in proliferation of uterus and mammary gland, an effective way to treat obesity and co-morbidities in postmenopausal women might be substances that only activate ER beta. A combination with physical activity may support the therapy of obesity and co-morbidities. The isoflavone Gen is able to activate both ER-subtypes. This compound is already placed on the market for treatment of postmenopausal complaints, although adverse effects of Gen cannot be excluded so far (e.g., increased risk of breast cancer). However, Gen might be a natural alternative – not only to the conventional hormone replacement therapy, but also as a strategy for treatment of obesity and co-morbidities – that deserves further research with respect to these new data. / Die dramatisch zunehmende Prävalenz der Adipositas und das damit verbundene Risiko für Folgeerkrankungen wie Diabetes mellitus, Hypertonie, Dyslipidämie und koronare Herzkrankheiten stellt eine große Herausforderung für das Gesundheitswesen dar. Als Hauptursache wird ein chronisches Missverhältnis der Energiehomöostase aufgrund permanenter Überernährung und Bewegungsmangel postuliert. Estrogene beeinflussen den Glukose- und Lipidstoffwechsel und sind somit in die Regulation des Energiehaushaltes involviert. Estrogene vermitteln ihre Effekte über zwei Estrogenrezeptor (ER)-Subtypen, den ER alpha und den ER beta. Ziel der vorliegenden Arbeit war es mittels tierexperimentellen Studien den Einfluss von Estrogenen, speziell 17beta-Estradiol, auf den Energiehaushalt zu untersuchen. Um einen tieferen Einblick in die zugrundeliegenden molekularen Mechanismen zu erhalten, wurden zwei Subtyp-selektive ER-Agonisten, 16alpha-LE2 (Alpha) and 8beta-VE2 (Beta), synthetischer Herkunft eingesetzt. Aufgrund der estrogenen Aktivität und der Verfügbarkeit als Nahrungsergänzungsmittel wurde des Weiteren der Einfluss des Isoflavons Genistein untersucht. Für die Studien wurden zwei Tiermodelle genutzt: zum einen weibliche Wistar-Ratten mit ernährungsinduzierter Adipositas und zum anderen weibliche leptinresistente „Zucker diabetic fatty“ (ZDF)-Ratten. Die Tiere wurden ovarektomiert (OVX) und entweder mit einem Vehikel (unbehandelte Kontrolltiere) oder mit der entsprechenden estrogenen Substanz behandelt. Die interessanteste Erkenntnis war, dass im Vergleich zu unbehandelten OVX-Tieren beider Tiermodelle die Behandlung mit Beta zur Vergrößerung der Faserquerschnitte im Soleusmuskel führte. Dieser anabole Effekt könnte die muskuläre Aufnahme und Verwertung von Brennstoffmolekülen verbessern und sich insgesamt positiv auf die Körperzusammensetzung auswirken. Den stärksten Effekt hinsichtlich einer erhöhten Expression und Translokation des insulinabhängigen Glukosetransporters 4 (GLUT4) in die Zellmembran des Gastrocnemiusmuskels zeigte sich dagegen durch die Behandlung von OVX ZDF-Ratten mit Alpha. Im Endergebnis zeigten die Tiere beider Modelle durch die Behandlung mit estrogenen Substanzen eine verbesserte systemische Insulinsensitivität im Vergleich zu unbehandelten Kontrolltieren. E2-behandelte Tiere tolerierten die Glukose am besten und lassen einen additiven Effekt aufgrund der Aktivierung beider Signalwege vermuten. Im Vergleich zu unbehandelten OVX Wistar-Ratten führte die Behandlung mit E2 oder mit jeweils einem der beiden ER-Subtyp-selektiven Agonisten zu einer geringeren viszeralen Fettmasse, kleineren Fettzellen, niedrigeren Leptinspiegeln im Serum und geringeren Triglyzeridwerten in Leber und Muskel. Auf der Ebene der Genexpression waren zudem geringere mRNA-Spiegel von lipo- und adipogenen Genen messbar. Somit scheinen beide ER-Subtypen in die antilipogene Wirkung von E2 involviert zu sein. Sowohl die reduzierte viszerale Fettmasse als auch die geringere Anreicherung von Triglyzeriden in Leber und Muskel tragen sehr wahrscheinlich ebenfalls zur verbesserten Insulinsensitivität bei. Die Behandlung von OVX Tieren mit Gen führte zu ähnlichen Ergebnissen wie die Behandlung mit Beta. Eine alleinige Ausnahme stellte das Fettgewebe dar, da hier eine Gen-Behandlung keine antilipogenen/-adipogenen Effekte zeigte. Speziell die Fähigkeit von Gen ebenfalls anabol zu wirken, könnte die molekulare Grundlage sein, weshalb Gen-behandelte Tiere im Vergleich zu unbehandelten Tiere eine verbesserte Toleranz gegenüber Glukose und eine geringere Anreicherung von Triglyzeriden in Muskel und Leber zeigten. Der ER beta ist nicht in die estrogenvermittelte Proliferation von Uterus und Brustdrüse involviert. Vor diesem Hintergrund lassen meine Ergebnisse vermuten, dass eine Behandlung mit ER beta-selektiven Substanzen eine effektive Möglichkeit darstellt, um Adipositas und deren Folgeerkrankungen in postmenopausalen Frauen zu behandeln, ohne deren Risiko für estrogenabhängige Krebsformen zu erhöhen. Eine Kombination mit regelmäßiger körperlicher Aktivität könnte die Erfolge bei der Behandlung von Adipositas und deren Folgeerkrankungen noch maximieren bzw. eine geringere Dosierung der verwendeten Substanz bei gleichbleibendem Behandlungserfolg ermöglichen. Das Isoflavon Gen mit seiner Fähigkeit beide ERs zu aktivieren ist eine bereits auf dem Markt befindliche Substanz und wird zur Behandlung von postmenopausalen Beschwerden eingesetzt, obwohl mögliche negative Effekte (z.B. ein erhöhtes Brustkrebsrisiko) noch nicht abschließend geklärt sind. Falls diese Risiken von Gen ausgeräumt werden können, könnte diese Substanz eventuell eine kostengünstige Alternative darstellen, um sowohl postmenopausale Beschwerden als auch Adipositas und deren Folgekrankheiten zu behandeln.

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<p>Phosphatidylinositol (PI) is a phospholipid that constitutes only a minor component of eukaryotic membranes. However, they are critical in many fundamental cellular processes, such as signal transduction pathways, vesicular trafficking and actin cytoskeletal dynamics. PI is highly enriched in specific acyl chains at both the <em>sn-1</em> and <em>sn-2</em> positions, the major species being 1-stearoyl-2-arachidonoyl. Enzymes required for PI synthesis are believed to play a major role in this enrichment through the selective catalysis of specific substrates. We have studied several aspects of two enzymes involved in PI synthesis, Diacylglycerol kinase ε (DGKε) and CDP-Diacylglycerol synthases (CDS). We have studied the role of the ATP-binding motif of DGKε and showed that this enzyme is not only required for enzymatic activity, but substrate specificity and sub-cellular localization. We have also looked at the region adjacent to the catalytic site, containing a cholesterol recognition motif, and determined that this also affects the enzymes activity and substrate specificity. Finally, we have characterized the enzymatic properties of two CDS isoforms <em>in vitro</em> and demonstrated that these isoforms exhibit different substrate specificities. Taken together, our results serve to further our understanding of both DGKε and CDS1/2 and their roles in PI synthesis and enrichment with specific acyl chains.</p> / Master of Science (MSc)

Lipid metabolism is critically important to the normal function of individual cells and for signaling between tissues of the human body. There is a great need to quantify changes in lipid metabolism because of its implications in both normal healthy conditions and during disease development. In our first study, we developed a liquid chromatography mass spectrometry based workflow to assess in vivo metabolism of murine brain lipids. This involved sample preparation, data acquisition, and analysis software development and improvements. Regarding sample preparation, we maintained the mice at 5% body water for the duration of the experiment. As mouse metabolism proceeds, enzymes can add deuterium atoms (D) from D2O into lipid C-H bonds. These newly synthesized D-labeled lipids display shifts in their isotopic envelopes. To observe these shifts, we used mass spectrometry acquisitions to measure the mass spectra of isotopic envelopes. We calculated changes in these isotopic envelope shifts to deduce several metabolic metrics. We used these metabolic metrics to make inferences about metabolism changes. These metrics include n-value, fraction new, rate, and asymptote for each lipid. We deduced n-value by replacing one hydrogen with a deuterium at a time in the lipid's theoretical chemical formula. The number of deuterium atoms in the theoretical D-labeled chemical formula that agrees best with its respective empirical spectrum is the n-value. A large part of this effort was assessing the reproducibility and quality control of n-values that were derived from empirical spectra. We compared these n-values to two sets of ground truth n-values that we generated. We generated one set of ground truth n-values by referencing biochemical pathways and published n-values. We used a linear algebra approach to deduce the other set of ground truth n-values. We compared both sets of ground truth n-values to n-values derived from empirical D-labeled lipid spectra. We found that both sets of ground truth n-values correlate well with n-values from empirical spectra. Using these n-values, we calculated fraction new for each lipid. This fraction new indicates what percentage of a lipid's pool is newly synthesized at a given time. For a given lipid, we calculated the fraction new for each time point and biological replicate. We plotted these fraction values together against time in days. From this fraction new vs time plot for each lipid, we deduced its asymptote and rate constant. In our second study, we added the additional dimension of drift time to the data acquisition and analysis using ion mobility spectrometry. We added this additional dimension so that we could further separate lipid isomers and prevent spectral convolution. Preliminary results suggest that lipid isomers may have distinct metabolic regulation.