The effect of exercise training on cholesterol and bile acid metabolism in ovariectomized rats

Il existe un nombre grandissant de preuves au cours des dernières années que la diminution de la sécrétion des œstrogènes chez les animaux ovariectomisés (Ovx) et chez les femmes ménopausées conduit à une accumulation importante de triglycérides (TG) dans le foie. Cependant, les évidences de perturbations dans le métabolisme du cholestérol, en lien avec la diminution des œstrogènes, sont limitées à des observations de niveaux élevés de cholestérol total dans le plasma trouvés chez la femme ainsi que chez les animaux. En fait, l'impact de la suppression des œstrogènes sur le métabolisme du cholestérol dans le foie a reçu peu d'attention et montre quelques controverses. Par conséquent, les trois études présentées dans cette thèse ont été réalisées chez des rats Ovx, comme modèle animal de femmes post-ménopausées, afin de documenter les effets du retrait des œstrogènes sur les marqueurs moléculaires clés du métabolisme du cholestérol et des acides biliaires dans le foie et dans l'intestin et des effets potentiels de l’entraînement physique. Il a été en effet démontré que l'entraînement physique peut réduire le niveau plasmatique de cholestérol. Une amélioration du transport du cholestérol en périphérie vers le foie pour sa sécrétion subséquente dans la bile et pour son l'excrétion de l'organisme a été suggérée, bien que les mécanismes sous-jacents ne soient pas entièrement compris. Dans la première étude, nous avons démontré que les rattes Ovx nourris avec une diète standard et une diète standard + cholestérol avait un taux de cholestérol total dans le foie plus élevé (P <0,05) que les rattes avec une ovariectomie simulée (Sham) nourris avec ces deux derniers types de diète, tandis que la teneur en triglycérides du foie était plus élevée chez les rattes Ovx que chez les rattes Sham nourris avec une diète standard, une diète standard + cholestérol et aussi une diète riche en grasses + cholestérol. Étonnement, la diète standard + cholestérol a été associée à un niveau plasmatique plus faible (P <0,001) de cholestérol total et de triglycérides chez les rats Ovx que les rats Sham, ce qui suggère une diminution de la sécrétion de lipoprotéines à très basses densités (VLDL). Par conséquent, la transcription de plusieurs marqueurs clés de la synthèse des VLDL, y compris la microsomal triglyceride transfer Protein (MTP) et apoB-100, ont été réduites (P <0,05) chez les rattes Ovx par rapport aux rattes Sham nourris avec tous les trois types diètes et cette diminution de MTP et apoB-100 était plus prononcée chez les rats nourris avec la diète standard + cholestérol. Pour aller un peu plus loin, dans la deuxième étude, nous avons déterminé les effets de l'entraînement physique sur les marqueurs clés hépatiques de la voie farnesoid X receptor (FXR) - small heterodimer partner (SHP) - de cholestérol 7 alpha-hydroxylase (CYP7A1) (FXR-SHP-CYP7A1) impliquée dans la conversion de cholestérol en acides biliaires et de leur excrétion chez les rat Ovx nourris avec une diète standard + cholestérol. Notre groupe expérimental principal comprenait des rats Ovx nourris avec une diète riche en cholestérol (Ovx-Chol). Ce groupe a été comparé à un groupe de rats Ovx nourris avec une diète standard (Ovx-SD) et un groupe de rats Sham nourris avec une diète riche en cholestérol (Sham-Chol) pour observer, respectivement, l'effet de l'alimentation et l’effet du retrait de l'œstrogène. Les résultats de cette étude ont démontré que les niveaux de cholestérol total dans le plasma et dans le foie ne sont pas affectés par l'entraînement physique dans aucune des conditions expérimentales. L'alimentation en cholestérol a induit une accumulation plus importante chez les rats Sham et Ovx a mené à une accumulation du cholestérol dans le foie significativement plus élevée (P <0,001) que chez les rats Ovx-SD. Un effet principal d'entraînement physique (P <0,05) a été trouvée dans l’expression génique du SHP et de CYP7A1. Ce dernier gène est reconnu pour son implication majeure sur le contrôle de la biosynthèse des acides biliaires à partir du cholestérol. De plus, cette étude a montré que le récepteurs des LDL (LDL-R) et proprotein convertase subtilisin/kexin type 9 (PSCK9) au foie, qui sont impliqués dans l'absorption du cholestérol de la circulation, ne sont pas influencés par l’entraînement physique. Ces résultats suggèrent que l'entraînement physique module le métabolisme du cholestérol chez les animaux Ovx par un réglage positif de la formation des acides biliaires. Un nombre croissant de preuves récentes suggèrent que le transport inverse du cholestérol (RCT) peut également passer par une voie non-biliaire connue sous le nom « transintestinal cholesterol excretion » (TICE). En effet, le foie et l'intestin sont impliqués dans l'excrétion du cholestérol excédentaire du corps. Dans cette optique, dans la troisième étude, nous avons élargi nos recherches afin de déterminer si l'entraînement physique module l’expression des récepteurs de cholestérol de la membrane intestinale qui sont impliqués dans TICE chez les rats intacts et Ovx nourris avec une diète standard et une diète riche en cholestérol. Les résultats de cette étude ont montré que l'entraînement physique a augmenté (P <0,01) l’expression génique intestinale de LDL-R et de PCSK9 impliquées dans la captation du cholestérol intestinal de la circulation et de leur récepteur nucléaire, « sterol regulatory element-binding protein 2 » (SREBP2) (P <0,05) chez les rats Sham et Ovx par rapport aux rats sédentaires (Sed). D'autre part, l’expression des gènes hépatiques de LDL-R et de PCSK9 ont été supprimées (P <0,01) par l’alimentation riche en cholestérol, mais pas affectée par l'entraînement physique. L'expression du gène « flavin monooxygénase 3 » (FMO3), en tant que régulateur de l'équilibre du cholestérol dans le foie, a été diminuée de façon significative (P <0,01) par le cholestérol alimentaire chez les rats Sham et Ovx par rapport aux rats nourris avec la diète standard, mais demeure inchangée suite à l'entraînement physique et le retrait des œstrogènes. Un réglage positif de l'expression de gènes du LDL-R et PCSK9 intestinale par l'entraînement physique chez les rats intacts et Ovx suggère que l'entraînement physique peut contribuer à l’accroissement de l'élimination de cholestérol par la voie TICE. Dans l'ensemble, nos résultats indiquent qu'une combinaison d’une diète riche en cholestérol et un retrait des œstrogènes a mené à une diminution de l'expression des gènes des marqueurs essentiels de la synthèse de VLDL, ce qui implique une réduction de l'excrétion du cholestérol du foie. Il semble que la réduction de LDL-R hépatique pourrait être due à l'accumulation du cholestérol dans le foie. De plus, nos résultats ont présenté l’entraînement physique comme une intervention non pharmacologique appropriée pour stimuler l'excrétion du cholestérol excédentaire de l'organisme par le réglage positif des gènes impliqués dans la biosynthèse des acides biliaires dans le foie et les récepteurs intestinaux de cholestérol dans la voie TICE.There has been accumulating evidence in recent years that the estrogen deficient state in ovariectomized (Ovx) animals and in postmenopausal women results in substantial liver triglyceride (TG) accumulation. However, evidence of disturbances in cholesterol metabolism in link with estrogen deficiency is limited to observations of higher plasma total cholesterol levels found in human as well as in animals. In fact, the impact of estrogen withdrawal on liver cholesterol metabolism has received little attention and shows some controversies. Therefore, the three studies presented in this thesis have been conducted in Ovx rats, as an animal model of post-menopausal women, to investigate the effects of estrogen withdrawal on key molecular markers of cholesterol and bile acid metabolism in liver and in transintestinal cholesterol excretion (TICE), and also to determine the potential role of exercise training as a positive alternative intervention. It has been shown that exercise training can improve plasma cholesterol levels. An enhanced transport of peripheral cholesterol toward the liver for subsequent secretion into bile and excretion from the body has been suggested; however, the underlying mechanism for this action is not fully understood. In the first study, we showed that estrogen withdrawal was associated with higher (P < 0.05) liver total cholesterol under the standard diet and the standard diet + cholesterol diet, while liver triglyceride (TG) content was higher in Ovx than in Sham rats in all three dietary conditions which are the standard diet, the standard diet + cholesterol and the high fat diet + cholesterol. Surprisingly, the standard diet + cholesterol was associated with lower (P < 0.001) plasma total cholesterol and TG levels in Ovx than in Sham rats, suggesting a decrease in very low-density lipoprotein (VLDL) secretion. Accordingly, several transcripts of key markers of VLDL synthesis including microsomal triglyceride transfer protein (MTP) and apoB-100 were decreased (P < 0.05) in Ovx compared to Sham rats under the three dietary conditions and even more so for MTP and apoB-100 when rats were fed the standard diet + cholesterol. To go one step further, in the second study we determined the effects of exercise training on hepatic key markers of farnesoid X receptor (FXR)-small heterodimer partner (SHP)-cholesterol 7 alpha-hydroxylase (CYP7A1) (FXR-SHP-CYP7A1) pathway, involved in cholesterol conversion into bile acid and excretion from the body, in Ovx cholesterol fed rats. Our main experimental group was Ovx rats fed a high cholesterol diet (Ovx-Chol) that was compared, on one hand, to a group of Ovx rats fed a standard diet (Ovx-SD) to observe the effects of the diet and, on the other hand, compared to a group of Sham operated rats fed the cholesterol diet (Sham-Chol) to observe the effect of estrogen withdrawal. Results of this study showed that plasma and liver total cholesterol levels were not affected by exercise training in any of the experimental conditions. Cholesterol feeding in both Sham and Ovx rats resulted in significantly (P<0.001) higher hepatic cholesterol accumulation than in Ovx-SD rats. A main effect of training (P< 0.05) was, however, found for transcripts of SHP and CYP7A1. The SHP and CYP7A1 transcripts were increased by training. These results suggest that exercise training through up-regulation of genes involved in bile acid formation may modulate cholesterol metabolism in Ovx animals. Finally, a recent growing body of evidence suggests that reverse cholesterol transport (RCT) can also proceed through a non-biliary pathway known as transintestinal cholesterol excretion (TICE). Indeed, both liver and intestine are involved in excretion of the excess cholesterol from the body. Based on this concept, we expanded our research to determine whether exercise training has an effect on intestinal membrane cholesterol receptors involved in TICE pathway in intact and Ovx rats fed a normal and a high cholesterol diet. Results of the third study showed that exercise training increased (P< 0.01) transcripts of intestinal LDL-R and PCSK9, which are involved in intestinal cholesterol uptake from circulation, and their nuclear transcription factor, intestinal sterol regulatory element-binding protein 2 (SREBP2) (P< 0.05) in both Sham and Ovx rats compared to rats remaining sedentary (Sed). On the other hand, hepatic LDL-R and PCSK9 gene expression was suppressed (P< 0.01) by cholesterol feeding but not affected by exercise training. Flavin monooxygenase 3 (FMO3) gene expression, as a cholesterol balance regulator in liver, was significantly decreased (P<0.01) by cholesterol feeding in both Sham and Ovx rats compared to rats were fed the SD diet but unchanged following exercise training and estrogen withdrawal. An up-regulation of intestinal gene expression of LDL-R and PCSK9 following voluntary wheel running in intact and Ovx rats suggests that exercise training may contribute to increased cholesterol elimination through the TICE pathway. Overall, our results indicate that a high cholesterol diet and ovariectomy combine to decrease the gene expression of key markers of VLDL synthesis suggesting a reduction in cholesterol excretion from the liver. Alternatively, it seems that reduced hepatic LDL-R transcript found in Ovx animals might be due to hepatic cholesterol accumulation. Moreover, our findings introduced exercise training as an appropriate non-pharmacological intervention to stimulate the excretion of the excess cholesterol from the body through upregulation of genes involved in bile acid biosynthesis in liver and intestinal basolateral cholesterol transporters in TICE

In Vitro Study of Mebendazole (Anthelmintic drug) Effects on the Aspartate Aminotransferase (AST) Enzyme Activity of Hydatid Cyst Parasite

Hydatid disease is caused by the larva of Echinococcus granulosus parasite. Mebendazole (MBZ) is used as an alternative choice for the treatment of the disease. Aspartate aminotransferase (AST) is an essential enzyme in amino acid metabolism. The aim of the present study is to evaluate the effect of MBZ on AST activity of hydatid cyst parasite in order to detect enzymatic parameter for drug efficiency. In the present study, AST activity was estimated in the extracts of untreated parasite (hydatid cyst protoscolices) and treated samples by MBZ (100 µg final concentration). Samples’ protein quantity and quality were detected by Bradford and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods respectively. For the purpose of determining the significant difference between the two independent samples, t-test was conducted. The values of the assayed AST specific activities of treated and untreated parasite samples were measured as 0.18 and 1.53U/ml/mg protein respectively. The difference between AST activities mean values of the two groups proved to be significant (P&lt;0.05). SDS-PAGE demonstrated protein band of 50 kDa for AST enzyme. Considering the effect of the MBZ drug on AST activity in parasite, it can be concluded that this enzyme is useful for improving the drug efficiency

FREQUENCY OF NEPHROPATHY AND ITS RELATION TO METABOL-IC CONTROL IN PATIENTS WITH DIABETES MELLITUS

Abstract &nbsp;&nbsp; INTRODUCTION: Diabetic nephropathy is a serious complication of both type 1 and type 2 diabetes, and unless arrested, leads to end-stage renal disease. The aim of this study was to find the prevalence of kidney dysfunction in patients with diabetes mellitus and to determine its correlation with metabolic control. &nbsp;&nbsp; METHODS: We randomly selected 1203 cases with diabetes mellitus presenting to the Institute of Endocrinology and Metabolism. Urinary protein and creatinine were determined in a sample of 24-hour urine collection by the enzymatic methods and spot urine dipstick blood urea nitrogen, serum creatinine, HbA1c and fasting blood glucose were assessed. &nbsp;&nbsp; RESULTS: In this study, 1203 patients (438 patients with type 1 diabetes mellitus [T1DM] and 777 with type 2 diabetes mellitus [T2DM]) were randomly selected. They consisted of 512 females and 721 males. Mean&nbsp;&plusmn;&nbsp;SD (standard deviation) of HbA1c was 7.9&plusmn;3.4 in T1DM and 7.4&plusmn;3.5 in T2DM. Based on HbA1c levels, good control was detected in 50.2% of patients, fair control in 20.4% and bad control in 29.4%. Of 1022 patients who were evaluated for proteinuria, 201 (19.7%) had albuminuria or clinical proteinuria. Of 931 patients, 19% had high levels of blood urea nitrogen and serum creatinine. End-stage renal disease (ESRD) was seen in 10 (0.8%) of all the cases. A statistically significant positive correlation was found between duration of DM, serum creatinine and 24-hour urinary protein (P&lt; 0.001). &nbsp;&nbsp; CONCLUSION: We found a high prevalence of clinical proteinuria in diabetic patients. Duration of diabetes and poor metabolic control were identified as a strong predictors of kidney damage in patients with diabetes. &nbsp; &nbsp;&nbsp; Keywords: Diabetic nephropathy, albuminuria, end-stage renal disease. &nbsp;</p

COMPARING SERUM LEPTIN AND ADIPONECTIN LEVELS IN CONTROLLED AND NON-CONTROLLED TYPE 2 DIABETES

Abstract &nbsp;&nbsp; INTRODUCTION: It has been demonstrated in recent studies that abnormal levels of adipocytokines may contribute to insulin resistance and type 2 diabetes. The aim of the present study was to compare serum leptin and adiponectin levels in controlled and non-controlled type 2 diabetes.&nbsp;&nbsp;&nbsp; &nbsp;&nbsp; METHODS: 117 patients with controlled and non-controlled type 2 diabetes were studied. Patient, were divided into two groups based on their serum HbA1c level; there were 62 patients in the controlled group (6 %&lt; HbA1c&le; 8%) and 55 patients in uncontrolled group (HbA1c&gt;8%). Parameters like age, sex, duration of diabetes and biochemical indicators such as fasting blood sugar, HbA1c, insulin resistance, leptin and adiponectin were determined. &nbsp;&nbsp; RESULTS: Higher leptin and lower adiponectin levels were observed in non-controlled type 2 diabetes. The levels of fasting blood sugar and insulin resistance were significantly higher in the non-controlled group (P&lt;0.05). &nbsp;&nbsp; CONCLUSION: Leptin and adiponectin may play an important role in the regulation of insulin sensitivity and control of type 2 diabetes. &nbsp;&nbsp; &nbsp; &nbsp;&nbsp; Keywords: Type 2 Diabetes, Insulin resistance, Leptin, Adiponectin.</p

The effect of exercise training on upregulation of molecular markers of bile acid metabolism in the liver of ovariectomized rats fed a cholesterol-rich diet

BACKGROUND: Small heterodimer partner (SHP) is an important transcriptional factor involved in the regulation of glucose, lipid, and bile acid metabolism in the liver. SHP has been reported to be down-regulated in ovariectomized (Ovx) mice and up-regulated by estrogens suggesting a link between estrogens and SHP. The aim of the present study was to determine the effects of exercise training on SHP and key molecular markers of cholesterol and bile acid homeostasis in Ovx rats under cholesterol feeding. METHODS: Our main experimental group was composed of Ovx rats fed a high-cholesterol diet (Ovx-Chol) that was compared to a group of Ovx rats fed a standard diet (Ovx-SD) and a group of sham operated rats fed the cholesterol diet (Sham-Chol). These three groups of Ovx and sham rats were subdivided into either voluntary wheel running (Tr) or sedentary (Sed) groups for 5 weeks. The mRNA expression of all genes was measured by quantitative real-time polymerase chain reaction. RESULTS: Liver total cholesterol levels were not affected by exercise training in any of the experimental conditions. Cholesterol feeding in both sham and Ovx rats resulted in significantly higher hepatic cholesterol accumulation than in Ovx-SD (P &lt; 0.001). Hepatic low-density lipoprotein-receptor (LDL-R) involved in cholesterol uptake from circulation was not influenced by training. A main effect of training &nbsp;was, however, found for transcripts of SHP and cholesterol 7 alpha-hydroxylase (CYP7A1, P &lt; 0.05). CYP7A1 is the main gene involved in bile acid biosynthesis from cholesterol. CONCLUSION: These results suggest that voluntary wheel running modulates cholesterol metabolism in Ovx animals through up-regulation of SHP and bile acid formation.&nbsp;&nbsp;</p

Pegylation of nanoliposomal paclitaxel enhances its efficacy in breast cancer

Purpose: To encapsulate paclitaxel into nanoliposomes, followed by pegylatation, in order to improve its therapeutic index and reduce side effects in breast cancer. Methods: In order to prepare nanoliposomal paclitaxel, varying ratios of phosphatidylcholine, cholesterol and paclitaxel were mixed and the formulations pegylated with poly-ethylene glycol 2000 (PEG 2000) to enhance stability, efficiency, as well as solubility. The mean diameter of nanoliposomal paclitaxel and pegylated nanoliposomal paclitaxel were measured by Zeta sizer device and release of paclitaxel from both formulations was determined within 28 h by dialysis method. The cytotoxicity of nanoliposomal and pegylated nanoliposomal paclitaxel was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The mean diameter of nanoliposomal paclitaxel and pegylated nanoliposomal paclitaxel was 421.4 and 369.1 nm, respectively, while encapsulation efficiency was 91.3 ± 5.7 and 95.2 ± 6.3 %, respectively. Paclitaxel released from both formulations in 28 h was 5.53 and 5.02 %, respectively. The cytotoxicity of pegylated nanoliposomal paclitaxel was significantly (p < 0.05) greater than that of nanoliposomal paclitaxel (their IC = 79.8±2.9 and 86.25±3.4 μg/ml, respectively). Conclusion: The release pattern and cytotoxicity of pegylated nanoliposomal paclitaxel show that the formulation is superior to nanoliposomal paclitaxel. Furthermore, the mean particle size of pegylated nanoliposome is smaller than that of the non-pegylated preparation

An investigation into the parameters affecting preparation of polybutyl cyanoacrylate nanoparticles by emulsion polymerization

Emulsion polymerization was used to synthesize poly butyl cyanoacrylate nanoparticles in presence of steric stabilizer dextran 70. Nanoparticles were characterized by particle size analysis, scanning electron microscopy and light microscopy. Polymerization factors affecting particle size and distribution such as dextran 70, polysorbate 80 (PS 80) and H concentration, polymerization time and temperature, and sonication were studied. Distinct concentrations of stabilizer were needed to produce proper nanoparticles. In this case, the appropriate value was 2 % of total volume. At pH 4 significant decrease in production efficiency demonstrated the substantial effect of H concentration on nanoparticles. Furthermore significant increases in particle size and distribution was observed at 50 °C compared to room temperature. 0.001 % (v/v) PS 80 represented notable influence on size and distribution. In addition, shaped nanoparticles were obtained by altering polymerization time from 5.5 h to 18 h. Finally, nanoparticle features were influenced by different factors. Appropriate manipulating of such factors can lead to obtaining desirable nanoparticles

Lipid peroxidation and antioxidant enzymes activity in controlled and uncontrolled Type 2 diabetic patients

BACKGROUND: This study was designed to compare lipid peroxidation and antioxidant enzymes activity in Type 2 diabetes patients with good or weak glycemic control. METHODS: In this case-control study, 62 Type 2 diabetic patients with glycated hemoglobin (HbA1c) between 6 and 8 were enrolled as the controlled group and 55 patients with HbA1c &gt; 8 were selected as an uncontrolled group. Patients were all referred to Iranian Diabetes Association in Tehran, Iran, from 2010 onward. Groups were chosen by convenience sampling and were matched based on age, sex and duration of disease. Demographic questionnaire, two 24-hour food recall, HbA1c, insulin, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase were measured in blood samples. Data were analyzed by Food Processor II and SPSS software. RESULTS: A mean daily consumption of energy, carbohydrate, protein, and fat was not significantly different between two groups. MDA in the uncontrolled group was significantly higher than controlled group (2.03 &plusmn; 0.88 vs. 1.65 &plusmn; 1.01 nmol/ml; P = 0.030). A mean SOD was slightly higher in the uncontrolled group comparing to the control group (843.3 &plusmn; 101.9 vs. 828.0 &plusmn; 127.3 U/g Hb; P = 0.400). CONCLUSION: These data suggest that MDA as a lipid peroxidation indicator is higher in uncontrolled diabetes probably due to chronic high blood sugar followed by higher oxidative stress. &nbsp;&nbsp;</p