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Interaction between adipocytes and immune cells in pathogenesis of obesity related pro-inflammatory state of adipose tissue
Mališová, Lucia ; Rossmeislová, Lenka (advisor) ; Flachs, Pavel (referee) ; Kazdová, Ludmila (referee)
Obesity is considered to be a worldwide epidemic disease characterized by an accumulation of AT. Increased adiposity can perturb normal metabolic functions and lead to the development of diseases like insulin resistance and other metabolic disorders. A large amount of clinical studies have been shown that changes in inflammatory signaling in adipose tissue cells, increased infiltration of immune cells into AT as well as stress of endoplasmic reticulum belong to the key molecular steps leading to the development of metabolic disturbances associated with this disease. Adverse metabolic effects of AT accumulation can be diminished by calorie restriction resulting in weight loss. In addition, stress of endoplasmic reticulum could be alleviated by chemical chaperones including bile acids. These two approaches for the treatment of obesity or the obesity-associated disturbances were basis for this PhD thesis. In the first part of this work, we studied inflammation status of gluteal in comparison with abdominal AT and differentiation and secretory capacity of adipocytes after weight loss in obese patients. We revealed that inflammatory profile of gluteal AT, estimated by mRNA level of macrophages and cytokines as markers of inflammatory status of the body, did not explain the different clinical impact of...
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Molecular mechanisms affected by n-3 polyunsaturated fatty acids
Hensler, Michal ; Flachs, Pavel (advisor) ; Rossmeislová, Lenka (referee) ; Dlouhý, Pavel (referee)
Adipose tissue and its hormones have an irreplaceable role in the physiology of mammals. The imbalance between energy intake and energy expenditure leads to the expansion of adipose tissue and changes in its secretion profile. With obesity are associated diseases including cardiovascular diseases, dyslipidemia, hypertension and insulin resistance, one of the major public health issues. Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) from marine origin, mainly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids exert numerous beneficial effects, such as improvements of lipid metabolism and prevention of obesity and diabetes. Studies with obesity-prone model mice (C57BL/6) provide us important knowledge regarding their effect on mammalian tissues and to test potential therapeutic interventions. The thesis is based on five published studies (A-E). Three studies are focused on white adipose tissue. In these works we proved that adipose tissue is a flexible organ and LC n- 3 PUFAs are potent regulators of adipose tissue biology. Our results document that LC n- 3 PUFAs affect adipose tissue mass by a mechanism, which depends on counteraction of both, differentiation and proliferation of adipose cell (publication A). The anti-obesity effect of EPA and DHA could be magnified by mild calorie...
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Effect of fatty acids on lipid metabolism and weight reduction
Hlavatý, Petr ; Kunešová, Marie (advisor) ; Müllerová, Dana (referee) ; Flachs, Pavel (referee)
Fat tissue plays a main role in the development of the metabolic syndrome. Many components of the metabolic syndrome may be improved by dietary arrangements, including an increased intake of n-3 PUFA. In addition to the positive effect of n-3 PUFA, a possible effect of a higher calcium intake on influencing weight loss and energy metabolism has also been discussed for a long time. In addition to nutritional factors, genetic factors significantly contribute to influencing weight and lipid and glucose metabolism. In study A, 40 obese women were observed during three weeks of weight reduction management. Women were randomly divided into two groups. The first group received yogurt enriched with n-3 PUFA, and the second group consumed yoghurt without the supplementation. The results show that low-dose supplementation with n-3 PUFA in yogurt in combination with a reduced energy intake increases n-3 PUFA content in serum lipids and prevents adverse changes in the composition of FA in serum after a short-term low-calorie diet. In study B, we monitored the influence of n-3 PUFA supplementation on short-term weight management with VLCD in 20 women with severe obesity, who were randomly divided into two groups. The first group received VLCD enriched with n-3 PUFA, the second group VLCD with a placebo. The addition of...
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New regulatory metabolic factors in patients with obesity and type 2 diabetes mellitus
Matějková, Mirka ; Haluzík, Martin (advisor) ; Flachs, Pavel (referee)
Fibroblast growth factors are proteins with diverse biological function in development, tissue repair, and metabolism. The human FGF gene family consists of 22 members. FGF 19 subfamily includes FGF 19, FGF 21, and FGF 23. They act as systemic factors in an endocrine manner. FGF 19 subfamily requires klotho protein as a cofactor for its action. FGF 19 produced by intestine acts mainly in the liver through FGFR4, where it inhibits bile acid and fatty acid synthesis. FGF 21 is produced by the liver and contributes to the regulation of carbohydrate and lipid metabolism through modulation of glucose uptake in adipocytes. Serum FGF 21 levels are increased in patients with obesity and type 2 diabetes mellitus. Serum FGF 19 levels are on the contrary decreased in patients with obesity and type 2 diabetes mellitus and more probably depend on the nutritional status of the organism than on the glucose metabolism and insulin sensitivity. Key words: type 2 diabetes mellitus, FGF 19, FGF 21, obesity, adipose tissue
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Relation between n-3 polyunsaturated fatty acids and cellular sensors of energetic state
Zouhar, Petr ; Pecina, Petr (referee) ; Flachs, Pavel (advisor)
The important factor in regulation of metabolic processes is regulatory proteins, which are able to react by feed-back to energetic state of the cell. Big attention is focused on the AMP activated kinase (AMPK) and NAD+ activated deacetylase SIRT1. These enzymes interact together and their stimulation increases mitochondrial biogenesis and fatty acid oxidation. Due to this it functions beneficially against the onset of obesity, insulin resistance and ageing. Fasting, exercise and some antidiabetogenic drugs act by these regulators. n-3 polyunsaturated fatty acids (PUFA) are also known because of their stimulative effects on mitochondrial biogenesis and -oxidation. Previous work of our group have showed that intake of higher dose of n-3 polyunsaturated fatty acids (PUFA) in diet lead to increase in activity of AMPK in white adipose tissue. New results presented in this thesis show that SIRT1 is essential for increase in expression of stimulators of -oxidation (PPAR etc) in response to n-3 PUFA in diet. n-3 PUFA futher improve the metabolic profile synergistically with calorie restriction probably through SIRT1.
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Distribution of mitochondrial uncoupling proteins in selected tissues from mice and rat
Alán, Lukáš ; Flachs, Pavel (referee) ; Ježek, Petr (advisor)
Mitochondrial uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carriers. The longest known is UCP1, predominantly expressed in brown adipose tissue, where it takes part in nonshivering thermogenesis. In the late 1990s were discovered other sequence homologs of UCP1 with tissue specific distribution. The Function of these "new" uncoupling proteins is still uncertain. It is assumed that each of the isoforms has a specific function depending on the type of tissue. This thesis showed differences in tissue transcription pattern between rat and mice using RT-PCR absolute quantification. Significant differences in pattern were found in lungs, brain and muscle. In each case UCP expression was higher in mice tissues. Mice lungs express mainly UCP2. The difference in mice brain is caused by ucp4 and ucp5 genes transcription and finally in muscle is highest content of UCP3 mRNA. We investigated whether any of ucp transcript can complement ucp2 transcripton in spleen or lungs of ucp2 -/- mice. We did not find any difference which can explain, that in isolated lung mitochondria of fasted ucp2-/- mice were uncoupled in state 4. In the last project, we found relationship between ucp2 transcription in insulinoma INS-1E cells and oxygen levels of the cultivation atmosphere.
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The influence of deep hypothermia on the endocrine function of adipose tissue
Drápalová, Jana ; Haluzík, Martin (advisor) ; Flachs, Pavel (referee)
Introduction and aims: Deep hypothermia is a medical method used in a variety of car- diological operations. Adipose tissue is one of the main endocrine organs and therefore its en- docrine function significantly influences the state of a patient. The aim of this study was to explore the influence of deep hypothermia on the expression of selected proteins in adipose tissue, which has yet to be described in scientific literature. Material and methods: Samples of subcutaneous and visceral fat were taken from 10 pa- tients, who underwent planned surgery for pulmonary hypertension. The samples were taken at the beginning and the end of hypothermia, and at the end of the operation, respctively. Real-time PCR method was used to determine relative gene expression of selected adipocy- tokines (BCL-2, GLUT1, GPX1, HIF1- , IL-6, IRS1, MCP1, MIF, MT3, TNF-) from total fat and isolated adipocytes. Results: Gene expression of GLUT1 (total subcutaneous fat), HIF1- (total subcutaneous and visceral fat) and IL-6 a MCP-1 (in all tissues) at the end of the operation was significantly higher compared to the gene expression at the beginning and end of hypothermia. Gene ex- pression of TNF- in total subcutaneous fat increased significantly at the end of the operation compared to the beginning of hypothermia. Gene...
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Molecular adaptations of adipose tissue in relation to dietary treatment of obesity in human
Tencerová, Michaela ; Štich, Vladimír (advisor) ; Flachs, Pavel (referee) ; Kazdová, Ludmila (referee) ; Kunešová, Marie (referee)
The general goal of this work was to investigate the molecular adaptations of human AT in relation to DIs with respect to its secretory activity as well as cellular composition focused on macrophages population. Specifically, we studied the role of novel adipokines, such as visfatin and RBP4, related to insulin resistance and AT metabolism. Furthermore, we wanted to characterize the effect of the dietary-induced changes on the content of ATM together with metabolic amelioration. In our studies, we found that lifestyle modifications had a beneficial effect on metabolic and biochemical parameters depending on the duration and type of DI. Regarding both of the investigated adipokines, visfatin and RBP4, we revealed modifications at the transcriptional and cirulating levels during DI. However, we did not find any association with the pattern of evolution of insulin resistance. Our findings do not support a clear hypothesis on the role of these adipokines in the diet-induced improvement of insulin sensitivity and other obesity-related metabolic disturbances. In respect to the changes of ATM content during long-term DI, we demonstrated using flow cytometry that the AT composition was changed at the end of the whole dietary intervention (i.e. after WM phase). This diet-induced AT remodelling was expressed by a...
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Use of RNA Interference for Targeted Inhibition of Gene Expression on the mRNA Level
Radilová, Hana ; Kvasničková, Eva (advisor) ; Šmarda, Jan (referee) ; Flachs, Pavel (referee)
Cyklooxygenáza (COX) je klíčovým enzymem v metabolismu arachidonové kyseliny (AA). Přeměňuje AA na prostaglandin H2, který je dále metabolizován různými enzymy na biologicky aktivní prostanoidy. Tato práce byla zaměřena na studium vztahů mezi geny souvisejícími s metabolismem AA. Pomocí RNA interference byla specificky snižována exprese vždy jedné z izoforem COX v buněčné linii rakoviny děložního čípku (Hep2). Cyklooxygenáza 1 (COX-1) byla úspěšně inhibována jak pomocí siRNA ("small interfering RNA"), tak shRNA ("short hairpin RNA") sekvencí. Pro snížení exprese cyklooxygenázy 2 (COX-2) se nepodařilo získat výrazně účinnou sekvenci shRNA. Bylo připraveno několik klonů buněčné linie Hep2 se specificky a stabilně inhibovanou expresí COX-1 (Hep2_shRNA1), které byly využity pro další studie. Snížení COX-1 bylo potvrzeno na úrovni mRNA pomocí real-time PCR, na úrovni proteinů pomocí Westernu blotu a na funkční úrovni metodou ELISA. Specifická a stabilní inhibice exprese COX-1 způsobila výrazné snížení mikrozomální prostaglandin E syntázy 1 (mPGES-1). Dále bylo v linii Hep2_shRNA1 nalezeno významné zvýšení hladiny mRNA genu ABCC4 ("ATP-binding cassette, sub-family C, member 4"). Tento gen kóduje transportér MRP4 ("Multidrug resistance protein 4"), který je přenašečem některých cytostatik, antivirotik a...
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