The mammalian brain is the most cholesterol-rich organ of the body, requiring in situ de novo cholesterol synthesis to maintain its cholesterol requirement. Defects in brain cholesterol homeostasis are implicated in cognitive deficits related to aging and in neurodegenerative diseases such as Alzheimer's Disease (AD). Oxysterol-binding protein (OSBP) - related proteins are highly conserved cytosolic proteins that coordinate lipid homeostasis by regulating cell signaling, inter-organelle membrane contact sites and non-vesicular transport of cholesterol. Previously, ORP6, a poorly characterized member of this family, was found to be part of complex transcriptional cascade coordinated by SBREP2 and emerged as a novel regulator of intracellular cholesterol trafficking in hepatocytes and macrophages. Yet how ORP6 regulates these pathways and its function in the brain where it is most highly expressed is unknown. Here, we show that ORP6 is highly expressed in the brain, where it exhibits spatial and cell-type specific expression. ORP6 expression is enriched in the hippocampus and caudal-putamen brain regions, specifically within neurons and astrocytes. ORP6 knockdown in astrocytes altered the expression of cholesterol biosynthesis, cholesterol efflux and cholesterol esterification genes, resulting in the accumulation of esterified cholesterol within cytoplasmic lipid droplets and reduced cholesterol efflux highlighting a role for ORP6 in astrocytic cholesterol metabolism. We also present in this thesis, the newly generated second viable ORP family member knockout mouse. ORP6 ablation in mice results in the dysregulation of brain and whole-body lipid homeostasis, increased Aβ deposition in the brain and neuroanatomical alterations. Together, our findings highlight a critical role for cholesterol trafficking proteins in brain cholesterol homeostasis and identify ORP6 as a potential novel target for AD.

L'aquaculture a connu un développement considérable au cours des dernières décennies. De par leurs disponibilités limitées, la farine de poisson et l'huile de poisson, considérées comme des ingrédients traditionnels de l'alimentation des poissons d’élevage, ont été largement remplacées par des matières premières végétales afin de soutenir le développement durable de l'aquaculture. Cette évolution de la composition des aliments aquacoles a entraîné une réduction importante de la teneur en cholestérol des aliments aquacoles. Dans ce contexte, l'objectif de la thèse était d’analyser les modifications du métabolisme du cholestérol chez la truite arc-en-ciel en réponse à une alimentation strictement végétale. Nous avons porté une attention particulière aux mécanismes sous-jacents à la régulation du métabolisme du cholestérol, en nous concentrant sur les facteurs de transcription et les microARNs. Enfin, nous avons évalué la possibilité d'utiliser les microARNs comme biomarqueurs non invasifs. Nos études ont montré qu’une alimentation végétale induit chez la truite une hypocholestérolémie associée à une modification de l’expression des gènes impliqués dans la synthèse et dans l'efflux du cholestérol mais aussi des facteurs de transcription SREBP-2 et LXRα. Nous avons également observé une diminution de l’expression du miR-223 dans le foie des truites nourries avec un régime à base de plantes ce qui suggère l'implication de mécanismes post-transcriptionnels dans la régulation de la synthèse du cholestérol chez la truite arc-en-ciel. Nous avons ensuite effectué une étude de la régulation du métabolisme du cholestérol chez une lignée de truite arc-en-ciel sélectionnée pour sa meilleure capacité de croissance sur aliment végétal. Nous avons observé une augmentation de l'expression du miR-33a chez les truites nourries avec un régime à base de plantes et une expression plus importante des miR-122 et 128 chez les truites de la lignée sélectionnée, quel que soit le régime. En analysant l’expression de gènes cibles potentiels des miR-33a, 122 et 128, nous avons pu mettre en évidence une régulation cohérente entre les miR-33a et 128 et leurs cibles potentielles que sont respectivement la caspase 6 apoptosis-related cysteine peptidase like 2 (casp6l2) et la phosphodiesterase 4B cAMP-specific a (pde4ba). Ces résultats mettent en évidence deux nouvelles voies moléculaires affectées par l’alimentation végétale chez la truite. Grâce à la combinaison d’approches in vivo et in vitro, nous avons constaté que l'expression des gènes impliqués dans la synthèse de cholestérol et celle du facteur de transcription SREBP-2 et du miR-33a augmentent chez les truites nourris avec l’aliment végétal et inversement diminuent en culture primaire d’hépatocytes de truite stimulés par du 25-hydroxycholestérol. Il semble donc SREBP-2 et miR-33a agissent en synergie pour réguler la synthèse du cholestérol. Enfin, nous avons détecté avec succès miR-1, miR-33a, miR-122, miR-128 et miR-223 dans le plasma de truite. Nous avons observé que les taux plasmatiques de miR-128 et de miR-223 sont soumis à des variations postprandiales similaires à celles observées pour leurs homologues hépatiques. Des corrélations significatives ont été observées entre les taux hépatiques et plasmatiques des miR-128 et miR-223. D’autres corrélations apparaissent entre les miR-122, 128 et 223 et l'expression de gènes liés à la synthèse et à l'efflux du cholestérol. Ces résultats indiquent que les micrARNs plasmatiques 122, 128 et 223 constituent de potentiels biomarqueurs non invasifs du métabolisme du cholestérol chez la truite arc-en-ciel. / Aquaculture has been subjected to a huge development during the last decades. Due to limiting availabilities, fishmeal and fish oil, the traditional ingredients of fish feed, have been widely replaced with vegetable ingredients in order to support the sustainable development of aquaculture. This evolution of aquafeeds has resulted in a reduction of the cholesterol content of the diets. In this context, the major objective of the thesis was to decipher the physiological changes related to cholesterol metabolism occurring in fish fed the plant-based diet. We furthermore analyzed the mechanisms underlying the regulation of cholesterol metabolism, focusing on transcription factors and microRNAs. Additionally, the possibility of utilizing miRNA as potential noninvasive biomarker was also investigated in the thesis. Our studies have shown that utilization of plant-based diet resulted in hypocholesterolemia and modified the expression of genes involved in cholesterol synthesis and efflux as well as that of their corresponding transcriptional factors SREBP-2 and LXRα. We also observed a lower expression of miR-223 in the liver of trout fed the plant-based diet suggesting the involvement of posttranscriptional mechanisms in the regulation of cholesterol synthesis. We then carried out another experiment using a line of rainbow trout selected for better growth performance on plant-based diet. We found that the hepatic expression of miR-33a increased in trout fed the plant-based diet, while the expression of miR-122 and miR-128 was much higher in the selected line regardless of the diet. By analyzing the expression of putative target genes of miR-33a, 122 and 128, we noticed consistent regulations between miR-33a and 128 and their respective putative targets, which were caspase 6 apoptosis-related cysteine peptidase like 2 (casp612) and cAMP-specific phosphodiesterase 4B a (pde4ba). These results highlighted two new molecular pathways affected by the plant-based diet. Through the combination in vivo and in vitro approaches, we demonstrated that the expression of the genes involved in cholesterol synthesis and that of the transcription factor SREBP-2 and the miR-33a increased in trout fed plant-based diet devoid of cholesterol while conversely decreased in rainbow trout primary cell culture of hepatocytes stimulated by 25-hydroxycholesterol. These data indicate that SREBP-2 and miR-33a act synergistically to control cholesterol synthesis. Finally, we successfully detected miR-1, miR-33a, miR-122, miR-128 and miR-223 in trout plasma. We observed similar postprandial changes between plasma levels of miR-128 and miR-223 and their hepatic counterparts. Significant correlations were observed between hepatic and plasma levels of miR-128 and miR-223. Other correlations appeared between miR-122, 128 and 223 and the expression of genes related to synthesis and efflux of cholesterol. Altogether, these results indicate that plasma microRNAs 122, 128 and 223 are potential non-invasive biomarkers of cholesterol metabolism in rainbow trout.

A síndrome metabólica (SM) e o diabete melito (DM) caracterizam-se por uma série de alterações no metabolismo de lipoproteínas, entre elas a hipertrigliceridemia e a redução nas concentrações de HDL-colesterol (HDL-C). Em estudo prévio demonstramos que indivíduos saudáveis, não obesos, com concentração de HDL-C abaixo de 40mg/dL, quando comparados àqueles com concentração de HDL-C acima de 60mg/dL, apresentam, no plasma, esteróis marcadores de absorção intestinal de colesterol alimentar diminuídos, e de síntese de colesterol aumentados. Achados semelhantes foram descritos por outros autores em portadores de SM e DM, sugerindo que a resistência à insulina participa na origem do distúrbio, embora não se saiba por quais mecanismos. Considerando nossos achados prévios, os objetivos deste estudo são investigar quais os mecanismos moleculares envolvidos nas alterações do metabolismo de colesterol presentes em indivíduos com concentrações alta (HIPERALFA, HDL-C > 60mg/dL, n = 36) ou baixa (HIPOALFA, HDL-C < 40mg/dL, n = 37) de HDL-C por meio de medida de: 1) conteúdo celular de colesterol e expressão gênica de enzimas e receptores críticos para a regulação intracelular de colesterol, tendo como modelo células linfomononucleares de sangue periférico; 2) parâmetros que regulam o metabolismo de lipoproteínas no plasma e que são influenciados pela insulina, tais como lecitina-colesterol aciltransferase (LCAT), lipoproteína lipase (LPL) e lipase hepática (LH) pós-heparina, proteína de transferência de colesterol esterificado (CETP), proteína de transferência de fosfolípides (PLTP) e pré- beta1 HDL. Os critérios de exclusão foram: diabete melito, IMC 30Kg/m2, tabagismo, consumo elevado de álcool e uso de fármacos capazes de interferir no metabolismo de lipoproteínas. Mostramos que, quando comparado ao grupo HIPERALFA, o grupo HIPOALFA apresentou maiores concentrações de insulina, triglicérides, ALT(TGP), índice HOMA, atividade de LCAT e LH, e menor atividade de LPL e concentração de pré-beta1 HDL. Não houve diferença entre os grupos com relação ao conteúdo celular de colesterol, à expressão dos genes estudados (ABCA1, ABCG1, SR-BI, LDLR, HMG CoA redutase, SREBP-1c e LXR alfa), às atividades de CETP e PLTP no plasma e à ultrassonografia de carótidas. Nossos resultados mostram que indivíduos com concentração alta ou baixa de HDL-C no plasma diferem com relação à sensibilidade à insulina, além de parâmetros envolvidos na regulação do metabolismo de lipoproteínas. Estes achados não se relacionaram com o metabolismo celular de colesterol no modelo estudado, mas sugerem que este quadro metabólico, cuja origem é desconhecida, precede o aparecimento, no decorrer da idade, de outras alterações típicas da síndrome metabólica no grupo com baixas concentrações de HDL-C no plasma / The metabolic syndrome (MS) and the diabetes mellitus (DM) are characterized for a series of alterations in lipoprotein metabolism as hypertriglyceridemia and reduced HDL-cholesterol (HDL-C) concentration. In previous study we demonstrated that healthy non obese subjects with HDL-C concentration below 40mg/dL, when compared with those with HDL-C above 60mg/dL, present low plasma sterol markers of alimentary cholesterol intestinal absorption and high plasma sterol markers of cholesterol synthesis. Similar findings have been described by others in subjects with MS and DM, suggesting that insulin resistance participates in the origin of the disorder, although by unknown mechanisms. Considering our previous findings, the objectives of this study are to investigate the molecular mechanisms involved in alterations of cholesterol metabolism in subjects with high HDL-C (HYPERALPHA, HDL-C > 60mg/dL, n = 36) or low HDL-C (HYPOALPHA, HDL-C < 40mg/dL, n = 37) concentration by means of measuring: 1) cellular cholesterol content and gene expression of critical enzymes and receptors involved in the intracellular cholesterol regulation, having peripheral blood mononuclear cells as model; 2) parameters that regulate the plasma lipoproteins metabolism and that are influenced by insulin, such as lecithin: cholesterol acyltransferase (LCAT), post-heparin hepatic (HL) and lipoprotein lipase (LPL), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP) and pre-beta1 HDL. The exclusion criteria were: diabetes mellitus, body mass index 30Kg/m2, smoking, heavy drinking and use of medications that interfere with lipoprotein metabolism. We demonstrated that, as compared with HYPERALPHA, the HYPOALPHA group presented higher insulin, triglycerides and alanine aminotransferase concentrations, HOMA index, LCAT and HL activities and lower LPL activity and pre-beta1 HDL concentration. There was no difference between the groups in the cellular cholesterol content, expression of genes (ABCA1, ABCG1, SR-BI, LDLR, HMG CoA reductase, SREBP-1c and LXR alpha), plasma CETP and PLTP activities and carotid ultrasonography. Our results show that subjects with high or low plasma HDL-C concentration differ in relation to insulin sensitivity and in parameters involved in lipoprotein metabolism regulation. These findings were not related to the cellular cholesterol metabolism in the studied model, but they suggest that this metabolic disturbance, whose origin is unknown, precedes the appearance, in the course of the human life of other typical alterations of metabolic syndrome in the low HDL-C concentration group

A síndrome metabólica (SM) e o diabete melito (DM) caracterizam-se por uma série de alterações no metabolismo de lipoproteínas, entre elas a hipertrigliceridemia e a redução nas concentrações de HDL-colesterol (HDL-C). Em estudo prévio demonstramos que indivíduos saudáveis, não obesos, com concentração de HDL-C abaixo de 40mg/dL, quando comparados àqueles com concentração de HDL-C acima de 60mg/dL, apresentam, no plasma, esteróis marcadores de absorção intestinal de colesterol alimentar diminuídos, e de síntese de colesterol aumentados. Achados semelhantes foram descritos por outros autores em portadores de SM e DM, sugerindo que a resistência à insulina participa na origem do distúrbio, embora não se saiba por quais mecanismos. Considerando nossos achados prévios, os objetivos deste estudo são investigar quais os mecanismos moleculares envolvidos nas alterações do metabolismo de colesterol presentes em indivíduos com concentrações alta (HIPERALFA, HDL-C > 60mg/dL, n = 36) ou baixa (HIPOALFA, HDL-C < 40mg/dL, n = 37) de HDL-C por meio de medida de: 1) conteúdo celular de colesterol e expressão gênica de enzimas e receptores críticos para a regulação intracelular de colesterol, tendo como modelo células linfomononucleares de sangue periférico; 2) parâmetros que regulam o metabolismo de lipoproteínas no plasma e que são influenciados pela insulina, tais como lecitina-colesterol aciltransferase (LCAT), lipoproteína lipase (LPL) e lipase hepática (LH) pós-heparina, proteína de transferência de colesterol esterificado (CETP), proteína de transferência de fosfolípides (PLTP) e pré- beta1 HDL. Os critérios de exclusão foram: diabete melito, IMC 30Kg/m2, tabagismo, consumo elevado de álcool e uso de fármacos capazes de interferir no metabolismo de lipoproteínas. Mostramos que, quando comparado ao grupo HIPERALFA, o grupo HIPOALFA apresentou maiores concentrações de insulina, triglicérides, ALT(TGP), índice HOMA, atividade de LCAT e LH, e menor atividade de LPL e concentração de pré-beta1 HDL. Não houve diferença entre os grupos com relação ao conteúdo celular de colesterol, à expressão dos genes estudados (ABCA1, ABCG1, SR-BI, LDLR, HMG CoA redutase, SREBP-1c e LXR alfa), às atividades de CETP e PLTP no plasma e à ultrassonografia de carótidas. Nossos resultados mostram que indivíduos com concentração alta ou baixa de HDL-C no plasma diferem com relação à sensibilidade à insulina, além de parâmetros envolvidos na regulação do metabolismo de lipoproteínas. Estes achados não se relacionaram com o metabolismo celular de colesterol no modelo estudado, mas sugerem que este quadro metabólico, cuja origem é desconhecida, precede o aparecimento, no decorrer da idade, de outras alterações típicas da síndrome metabólica no grupo com baixas concentrações de HDL-C no plasma / The metabolic syndrome (MS) and the diabetes mellitus (DM) are characterized for a series of alterations in lipoprotein metabolism as hypertriglyceridemia and reduced HDL-cholesterol (HDL-C) concentration. In previous study we demonstrated that healthy non obese subjects with HDL-C concentration below 40mg/dL, when compared with those with HDL-C above 60mg/dL, present low plasma sterol markers of alimentary cholesterol intestinal absorption and high plasma sterol markers of cholesterol synthesis. Similar findings have been described by others in subjects with MS and DM, suggesting that insulin resistance participates in the origin of the disorder, although by unknown mechanisms. Considering our previous findings, the objectives of this study are to investigate the molecular mechanisms involved in alterations of cholesterol metabolism in subjects with high HDL-C (HYPERALPHA, HDL-C > 60mg/dL, n = 36) or low HDL-C (HYPOALPHA, HDL-C < 40mg/dL, n = 37) concentration by means of measuring: 1) cellular cholesterol content and gene expression of critical enzymes and receptors involved in the intracellular cholesterol regulation, having peripheral blood mononuclear cells as model; 2) parameters that regulate the plasma lipoproteins metabolism and that are influenced by insulin, such as lecithin: cholesterol acyltransferase (LCAT), post-heparin hepatic (HL) and lipoprotein lipase (LPL), cholesteryl ester transfer protein (CETP), phospholipid transfer protein (PLTP) and pre-beta1 HDL. The exclusion criteria were: diabetes mellitus, body mass index 30Kg/m2, smoking, heavy drinking and use of medications that interfere with lipoprotein metabolism. We demonstrated that, as compared with HYPERALPHA, the HYPOALPHA group presented higher insulin, triglycerides and alanine aminotransferase concentrations, HOMA index, LCAT and HL activities and lower LPL activity and pre-beta1 HDL concentration. There was no difference between the groups in the cellular cholesterol content, expression of genes (ABCA1, ABCG1, SR-BI, LDLR, HMG CoA reductase, SREBP-1c and LXR alpha), plasma CETP and PLTP activities and carotid ultrasonography. Our results show that subjects with high or low plasma HDL-C concentration differ in relation to insulin sensitivity and in parameters involved in lipoprotein metabolism regulation. These findings were not related to the cellular cholesterol metabolism in the studied model, but they suggest that this metabolic disturbance, whose origin is unknown, precedes the appearance, in the course of the human life of other typical alterations of metabolic syndrome in the low HDL-C concentration group

La modélisation de maladies métaboliques hépatiques et les approches de thérapie cellulaire nécessitent de disposer d’une source fiable et illimitée d’hépatocytes. Grâce à leurs propriétés spécifiques, les cellules souches pluripotentes peuvent représenter une telle source. Nous avons tout d’abord mis au point une approche originale pour différencier une lignée de cellules souches embryonnaires humaines européenne, générée en conditions GMP-compatibles, en hépatocytes fonctionnels in vitro et in vivo après transplantation dans un modèle murin d'insuffisance hépatique aiguë. Nous avons ensuite utilisé les cellules souches pluripotentes induites (iPSC) spécifiques d’un patient homozygote pour une mutation entrainant une absence de récepteur des lipoprotéines de basse densité (RLDL) afin de modéliser l'hypercholestérolémie familiale (HF) in vitro. Nous avons amélioré notre approche pour différencier ces iPSC en hépatocytes plus matures et polarisés car les hépatocytes sont les seules cellules capables de dégrader le cholestérol via la bile. Nous avons montré que ce modèle reproduit la physiopathologie de l'HF et établit la preuve de concept de la correction génétique ciblée par la technologie CRISPR/Cas au locus AAVS1 par la restauration de l’expression, inductible par les statines, et de la fonctionnalité du récepteur. Nous avons également mis en évidence que le RLDL ne semble pas impliqué dans l'entrée du virus de l'hépatite C (VHC) mais plutôt dans les étapes tardives de la morphogénèse virale. Ce modèle pourra désormais servir à l’étude physiopathologique de différents patients HF, au criblage de nouvelles drogues hypocholestérolémiantes et antivirales ainsi qu'à de nouvelles approches thérapeutiques. / Liver metabolic diseases modeling and cell therapy approaches require a a reliable and well-characterized cell source. Due to their specific properties, pluripotent stem cells represent a credible alternative to primary human hepatocytes. Thus, we have defined a new approach to differentiate a European human embryonic stem cell line, generated in GMP-compatible conditions, into hepatocytes that are functional in vitro and in vivo after transplantation into a murine model of acute liver failure. We have then used induced pluripotent stem cells from a homozygous patient with a mutation leading to an absence of the low-density lipoproteins receptor (LDLR) to model familial hypercholesterolemia type IIA (FH) in vitro. As hepatocytes are the only cells able to metabolize cholesterol into bile acids, we have improved our approach to differentiate these iPSC into hepatocytes displaying cell functional organization and polarization. We have shown that our model reproduced FH physiopathology and have also restored, by the genetic targeted correction at the AAVS1 locus using CRISPR/Cas technology and subsequent hepatocyte differentiation, the LDLR expression – inducible by statins - and functionality. Moreover, we have demonstrated that the LDLR does not seem to be involved in hepatitis C virus entry or replication but rather in viral morphogenesis steps. This model will be useful to develop new cholesterol-lowering and antiviral drugs as well as new cell therapy options. Furthermore, it can be applied to similar studies for other liver metabolic disorders.

No description available.

Cette maitrise a été fait en co-direction : Jean-Marc Lavoie (UdeM) et David St-Pierre (UQAM). / Mise en contexte : La présente étude a pour but de tester l’hypothèse selon laquelle l’accumulation excessive de lipides au foie perturbe le métabolisme du cholestérol. La stéatose hépatique perturberait ainsi principalement les voies métaboliques qui impliquent les récepteurs de LDL au foie. Méthodologie: Des rats Wistar (n/groupe = 10) ont été soumis soit à une diète standard (SD), une diète enrichie en lipides (HFD : High Fat Diet) ou à une diète occidentale (WD : Western Diet) pour une durée de 2 ou 6 semaines. Au niveau de la composition des diètes, 60% de l’apport calorique de la diète enrichie en lipides provient des lipides tandis que la diète occidentale est composée à 40% de lipides et 35% de sucrose dont 17,5% de fructose. Résultats: Comparativement aux animaux traités pendant 2 semaines, le poids des tissus adipeux était environ trois fois plus élevé (~ 20 vs 7 g) chez les animaux soumis à 6 semaines de diètes obésogènes. Une augmentation significative du gain de poids (~ 40g) a été observée uniquement après 6 semaines chez les groupes soumis à la HFD ou la WD (P < 0.01). Comparativement aux animaux soumis à la diète conventionnelle, les niveaux de triglycérides (TG) hépatiques étaient significativement supérieurs chez les rats nourris avec la HFD et WD (P < 0.01) et ce, indépendamment de la durée du traitement. Après deux semaines, des concentrations de TG hépatiques significativement plus élevées (P < 0.05) ont été observés chez les animaux avec la WD comparativement à celles des rats avec la HFD. Des niveaux de cholestérol plasmatiques significativement plus élevés (P < 0.05) ont été mesurés chez les animaux avec la WD par rapport à la SD et la HFD et ce indépendamment de la durée. Après 2 et 6 semaines de diètes, l’expression génique au foie de LDL-R, PCSK9 et SREBP2, qui sont impliqués dans la captation des LDL-cholestérol, a significativement diminué chez les animaux soumis à la WD comparativement à ceux nourris avec la diète SD ou HFD (P < 0.01). De la même manière, des niveaux d’ARNm de LRP1 et ACAT2 significativement diminués (P < 0.01) ont été mesurés chez les animaux nourris avec WD comparativement ceux du groupe SD. L’expression de l’HMGCoAR, l’enzyme limitante impliquée dans la régulation de la synthèse endogène de cholestérol, a été significativement 6 diminuée chez les animaux soumis à la WD comparativement à ceux traités avec la SD ou la HFD après 2 (P < 0.001) et 6 semaines (P < 0.05). Dû au fait que la diète soit enrichie en sucrose et conséquemment en fructose, la WD a fortement favorisé l’expression de ChREBP et ACC, deux régulateurs majeurs dans la voie de la lipogenèse de novo. Conclusion: Ces résultats suggèrent que la diète de type occidentale augmenterait les niveaux de TG en favorisant simultanément la captation exogène de lipides ainsi que leur production endogène par l’activation de la lipogenèse de novo. L’altération de la voie de la captation du cholestérol par les LDL-R favoriserait une augmentation rapide des taux plasmatiques de cholestérol. / Background: The present study was designed to test the hypothesis that excessive fat accumulations impair cholesterol metabolism mainly through alterations in the LDL-receptor (LDL-R) pathway in liver. Method: Rats were either submitted to standard (SD), high fat (HFD; 60% kcal) or western (WD; 40% fat + 35% sucrose (17.5% fructose)) diets for 2 or 6 weeks. Results: Weight gain (~ 40g) was observed only following 6 weeks of the obesogenic diets (P < 0.01). Compared to the 2-week treatment, obesogenic diets tripled fat pad weight (~ 20 vs 7 g) after 6 weeks. Hepatic triglyceride (TG) levels were greater in response to both the WD and HFD compared to the SD (P < 0.01) at 2 and 6 weeks and their concentrations were greater (P < 0.05) in WD than HFD at 2 weeks. Plasma cholesterol levels were higher (P < 0.05) in animals submitted to WD compare to SD and HFD. After 2 and 6 weeks, liver expression of LDL-R, PCSK9 and SREBP2, involved in LDL-cholesterol uptake, was lower in animals submitted to WD than in others treated with HFD or SD (P < 0.01). Similarly, LRP1 and ACAT2 mRNA levels were lower (P < 0.01) among WD compared to SD-fed rats. Expression of the gene coding the main regulator of endogenous cholesterol synthesis, HMGCoAR was reduced in response to WD compared to SD and HFD at 2 (P < 0.001) and 6 (P < 0.05) weeks. Being enriched in fructose, the WD strongly promoted the expression of ChREBP and ACC, two key regulators of de novo lipogenesis. Conclusion: These results show that the WD promptly increased TG levels in the liver by potentiating dietary fat storage and de novo lipogenesis. This effect impaired hepatic cholesterol uptake via the LDL-R axis and promoted a rapid increase in plasma cholesterol levels.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Excess caloric intake and/or obesity currently remain the largest predisposing risk factors for the development of type 2 diabetes. Discerning the cellular and molecular mechanisms responsible and amendable to therapy represents a growing challenge in medicine. At a cellular level, increased activity of the hexosamine biosynthesis pathway (HBP), a sensor of excess energy status, has been suggested to promote the exacerbation of insulin resistance through increasing adipose tissue and skeletal muscle membrane cholesterol content. This in turn compromises cortical filamentous actin structure necessary for proper incorporation of the insulin-sensitive glucose transporter GLUT4 into the plasma membrane. The current studies attempted to elucidate the mechanism by which hexosamines provoke membrane cholesterol toxicity and insulin resistance. In 3T3-L1 adipocytes cultured with pathophysiologic hyperinsulinemia to induce insulin resistance, increased HBP flux was observed. This occurred concomitant with gains in the mRNA and protein levels of HMG-CoA reductase (HMGR), the rate limiting enzyme in cholesterol synthesis. Mechanistically, immunoprecipitation demonstrated increased HBP-induced N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), a regulator of HMGR synthesis. This was associated with increased affinity toward and activity of Hmgcr, the gene encoding HMGR. Global HBP inhibition or Sp1 binding to DNA prevented membrane cholesterol accrual, filamentous actin loss, and glucose transport dysfunction. Furthermore, hyperinsulinemia and HBP activation impaired cholesterol efflux in adipocytes, exacerbating cholesterol toxicity and potentially contributing to cardiovascular disease. In this regard, chromium picolinate (CrPic), known to have beneficial effects on glucose and lipoprotein metabolism, improved cholesterol efflux and restored membrane cholesterol content. To test the role of membrane cholesterol accumulation in vivo, studies were conducted on C57Bl/6J mice fed a low or high fat diet. High fat feeding promoted increased HBP activity, membrane cholesterol accumulation, and insulin resistance. Supplementation of mice with CrPic in their drinking water (8µg/kg/day) countered these derangements and improved insulin sensitivity. Together, these data provide mechanistic insight for the role of membrane cholesterol stress in the development of insulin resistance, as well as cardiovascular disease, and highlight a novel therapeutic action of chromium entailing inhibition of the HBP pathway.